The regulatory role of mixed lineage kinase 4 beta in MAPK

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Theses and Dissertations
2013
The regulatory role of mixed lineage kinase 4 beta
in MAPK signaling and ovarian cancer cell invasion
Widian F. Abi Saab
The University of Toledo
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Abi Saab, Widian F., "The regulatory role of mixed lineage kinase 4 beta in MAPK signaling and ovarian cancer cell invasion" (2013).
Theses and Dissertations. Paper 2.
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A Dissertation
entitled
The Regulatory Role of Mixed Lineage Kinase 4 Beta in MAPK Signaling and Ovarian
Cancer Cell Invasion
by
Widian F. Abi Saab
Submitted to the Graduate Faculty as partial fulfillment of the requirements for the
Doctor of Philosophy Degree in Biology
_________________________________________
Dr. Deborah Chadee, Committee Chair
_________________________________________
Dr. Douglas Leaman, Committee Member
_________________________________________
Dr. Fan Dong, Committee Member
_________________________________________
Dr. John Bellizzi, Committee Member
_________________________________________
Dr. Max Funk, Committee Member
_________________________________________
Dr. Robert Steven, Committee Member
_________________________________________
Dr. William Taylor, Committee Member
_________________________________________
Dr. Patricia R. Komuniecki, Dean
College of Graduate Studies
The University of Toledo
May 2013
Copyright 2013, Widian Fouad Abi Saab
This document is copyrighted material. Under copyright law, no parts of this document
may be reproduced without the expressed permission of the author.
An Abstract of
The Regulatory Role of Mixed Lineage Kinase 4 Beta in MAPK Signaling and Ovarian
Cancer Cell Invasion
by
Widian F. Abi Saab
Submitted to the Graduate Faculty as partial fulfillment of the requirements for the
Doctor of Philosophy Degree in Biology
The University of Toledo
May 2013
Mixed lineage kinase 4 (MLK4) is a member of the MLK family of mitogenactivated protein kinase kinase kinases (MAP3Ks). As components of a three-tiered
signaling cascade, MAP3Ks promote activation of mitogen-activated protein kinase
(MAPK), which in turn regulates different cellular processes including proliferation and
invasion. Here, we show that the beta form of MLK4 (MLK4β), unlike its close relative,
MLK3, and other known MAP3Ks, negatively regulates the activities of the MAPKs,
p38, ERK and JNK, even in response to stimuli such as sorbitol or TNFα. MLK4β also
negatively regulates basal, but not TNFα-induced, NF-κB activity. Moreover, MLK4β
undergoes autophosphorylation and has kinase activity towards histone H2A, but has no
kinase activity towards the MAP2K, MEK4/SEK1, a known substrate for MLK3 and
other MAP3Ks. Furthermore, MLK4β interacts with MLK3 and inhibits MLK3
activation. In addition, MLK4 blocks matrix metalloproteinase-9 gelatinase activity and
invasion in SKOV3 ovarian cancer cells, both of which are cellular responses that require
MLK3. Collectively, our data establish MLK4β as a novel suppressor of MLK3
activation, MAPK signaling and cell invasion.
iii
This work is dedicated to my dad, Fouad Abi Saab, and mom, Nabila Abi Saab,
who sacrificed a lot to provide a good education for my brother and me. I most certainly
would not be where I am today if it wasn’t for them.
I also dedicate this work to my brother (Rawad), my grandmas (Fayza and
Samia), my aunts (Thouraya, Feryal, Noha and Sonia) and all my cousins (Yara, Ziad,
Lama, Wahid, Tamara and Faisal). However, a special dedication goes to my beloved
Grandma, Fayza Darweesh, who is my role model. She is my inspiration and the source
of my strength and has always been my number one supporter. Her words and constant
encouragement are my driving force to move forward in life.
I would also like to grab this opportunity to thank my dearest friends (Alexis, Ani,
Celia, Hadil, Hashem, Meenakshi, Mirella, Nancy and Natalya) who’ve been extremely
encouraging and supportive throughout my Ph.D. program.
Acknowledgements
First, I would like to thank my advisor, Dr. Chadee, who had given me the chance
to be here and who taught me most of what I currently know in this field. Dr. Chadee is a
very supportive and positive person and creates a very amiable environment for her
students. In addition to being successful in her field, she is also extremely compassionate
and understanding. She was very supportive especially during hard times and for that I’ll
be forever grateful. Not only is Dr. Chadee successful in her career, but she also has an
exemplary sense of humanity which makes her a great role model for me.
I would also like to thank my committee members Dr. Douglas Leaman, Dr. Fan
Dong, Dr. John Bellizzi, Dr. Max Funk, Dr. Robert Steven and Dr. William Taylor for
their constant input and guidance. I especially thank Dr. Taylor, Dr. Leaman and Dr.
Dong, for their technical support in a number of experiments.
I would like to especially thank Cathy (Dr. Yu Zhan) for teaching me most of the
techniques in lab and for being a good friend. Special thanks to Natalya Blessing for
being a wonderful lab mate and friend. I would also like to thank Meenakshi Bhansali for
her amazing friendship and support. Last but not least, I would like to thank Dr. Leah
Rider, Jenny, Alan, Peter, Sneha, April and Kyoung for being such good friends and for
adding a joyful and pleasant atmosphere to our working environment.
v
Table of Contents
Abstract .............................................................................................................................. iii
Acknowledgements ..............................................................................................................v
Table of Contents ............................................................................................................... vi
List of Figures .................................................................................................................... ix
List of Abbreviations ......................................................................................................... xi
1
Introduction………………………………………………………………………..1
1.1 The Mitogen-activated protein kinase signaling cascade ...............................1
1.2 Characteristics and functions of MAPK proteins…………………………….3
1.2.1 The ERK1/2 pathway………………………………………………..3
1.2.2 The JNK pathway…………………………………………………...7
1.2.3 The p38 pathway…………………………………………………...11
1.2.4 The ERK5 pathway………………………………………………...14
1.3 The matrix metalloproteinases……………………………………………….15
1.4 The MAP2Ks………………………………………………………………...18
1.5 The MAP3Ks………………………………………………………………...20
1.5.1 The MEKK group………………………………………………….21
1.5.2 The Raf MAP3Ks………………………………………………….23
1.5.3 The TAK1 MAP3K group…………………………………………25
1.5.4 The TAO/Tpl2 and Mos MAP3K groups………………………….27
1.5.5 The MLK family of MAP3Ks……………………………………..27
vi
1.5.5.1 The DLK subgroup ...........................................................28
1.5.5.2 The ZAK subgroup………………………………………30
1.6 The MLK subfamily…………………………………………………………31
1.6.1 MLK1 and MLK2………………………………………………….32
1.6.2 MLK3 activation…………………………………………………...33
1.7 MLK3 signaling……………………………………………………………...36
1.7.1 MLK3 signaling in cancer…………………………………………38
1.8 MLK4: characteristics and function………………………………………….39
1.9 Significance…………………………………………………………………..40
2
Materials and Methods ...........................................................................................42
2.1 Cell culture…………………………………………………………………...42
2.2 Expression vectors…………………………………………………………...43
2.3 Plasmids and siRNA transfections…………………………………………...43
2.4 Immunoblotting………………………………………………………………45
2.5 Preparation of whole cell extracts and treatments…………………………...47
2.6 Immunoprecipitation…………………………………………………………47
2.7 MLK4β kinase assay…………………………………………………………48
2.8 Cell proliferation assay………………………………………………………49
2.9 Luciferase assay……………………………………………………………...50
2.10 Invasion assay………………………………………………………………50
2.11 Gelatin zymography………………………………………………………...51
3
Results……………………………………………………………………………52
3.1 The role of MLK4β in p38 signaling………………………………………...52
vii
3.1.1 The effect of ectopic expression of MLK4β on p38 activation…...52
3.1.2 The effect of endogenous MLK4 on the activation of p38. ………54
3.2 The effect of MLK4 on MEK3/MEK6 activation….………………………..56
3.3 The role of MLK4β in NF-κB signaling……………………………………..57
3.4 Comparison of the effects of MLK4β and MLK3 on p38 activation ……….60
3.5 The effects of MLK3 and MLK4 on ERK and JNK activation……………..62
3.6 MLK4β is not an upstream activator of MEK4……………………………...65
3.7 MLK4β kinase activity……………………………...……………………….67
3.8 The effect of MLK4β on MLK3 activation………………………………….69
3.9 The correlation between MLK4β expression and active MLK3 in different cell
lines……………………………………………………………...……………….72
3.10 MLK4β associates with MLK3……………………………………………..75
3.11 The effect of MLK4 on cell proliferation…………………………………..77
3.12 MLK3 is required for cell invasion in ovarian cancer cells………………...79
3.13 MLK4β inhibits SKOV3 cell invasion……………………………………..81
3.14 MLK3 regulates MMP-2 and MMP-9 enzyme activity…………………….82
3.15 MLK4β reduces MMP-9 activity in SKOV3 cells…………………………85
4
Discussion………………………………………………………………………..86
References……………………………………………………………………………….95
viii
List of Figures
1-1
The MAPK signaling cascade ..................................................................................2
1-2
The Ras/Raf/ERK1/2 signaling pathway .................................................................6
1-3
JNK-mediated apoptosis…………………………………………………………10
1-4
The p38 MAPK signaling pathway………………………………………………13
1-5
MMP-2 and -9: structure and activation…………………………………………17
1-6
The NF-κB pathway……………………………………………………………...26
1-7
Signaling of the DLK family of MAP3Ks……………………………………….29
1-8
The structural domains of MLKs………………………………………………...31
1-9
Model mechanism of MLK3 activation by Cdc42………………………………35
3-10
MLK4β expression inhibits basal and stimulus-induced p38 activation ...............54
3-11
Elevated active p38 in MLK4 knockdown cells…………………………………55
3-12
MLK4 negatively regulates MEK3/MEK6 activation…………………………...57
3-13
MLK4β negatively regulates basal NF-κB activation but has no effect on TNFα-
induced NF-κB signaling……………………………………..………………………….59
3-14
MLK4β, unlike MLK3, inhibits activation of p38 ………………………………61
3-15
MLK3 promotes the activation of ERK and JNK in SKOV3 and HEY1B cells...62
3-16
MLK4 negatively regulates ERK and JNK activation…………………………...64
3-17
MLK4β does not phosphorylate Thr261 on GST-SEK1-KR……………………66
3-18
MLK4β: autophosphorylation and substrate specificity…………………………68
3-19
MLK4β inhibits induced MLK3 activation……………………………………...69
ix
3-20
MLK4 inhibits the basal activation of MLK3 in SKOV3 cells………………….71
3-21
Correlation between MLK4β expression and active MLK3.. …………...............74
3-22
MLK4β associates with MLK3………………………………………………......76
3-23
MLK4 has no effect on HCT116 cell proliferation……………………………...78
3-24
MLK3 is essential for SKOV3 and HEY1B cell invasion……………………….80
3-25
MLK4β reduces the invasion of SKOV3 cells..…………………………………82
3-26
MLK3 mediates MMP-2 and MMP-9 activation in SKOV3 and HEY1B cells by a
mechanism that involves ERK and JNK………………….……………………………..84
3-27
MLK4β reduces MMP-9 activity in SKOV3 cells………………………………85
4-28
Schematic diagram illustrating the role of MLK4β in MAPK signaling………..94
x
List of Abbreviations
Akt1............................Rac-alpha serine/threonine kinase
APS ............................Ammonium persulfate
AP-1 ...........................Activator protein 1
ASK............................Apoptosis signal-regulating kinase
ATF2 ..........................Activating transcription factor 2
ATM………………..Ataxia telangiectasia
ATP ...........................Adenosine triphosphate
Bax .............................Bcl2-associated X
Bcl-2 ...........................B-cell lymphoma 2
BSA ............................Bovine serum albumin
CBD ...........................Collagen binding domain
CR ..............................Conserved region
CRIB ..........................Cdc42/Rac interactive binding protein
DTT ............................Dithiothreitol
DLK ...........................Dual leucine zipper-bearing kinase
DNA ...........................Deoxyribonucleic acid
DSP ............................Dual specificity phosphatases
ECM ...........................Extracellular matrix
EMT ...........................Epithelial-mesenchymal transition
EGF ............................Epidermal growth factor
ERK ...........................Extracellular signal-regulated kinase
FADD.……….……..Fas-associated death domain protein
FasL............................Fas ligand
FBS ............................Fetal bovine serum
FFA…………………Free fatty acids
FGD1……………….FYVE, RhoGEF and PH domain-containing protein 1
GADD45 ....................Growth arrest and DNA damage-inducible 45
GDP………………...Guanosine diphosphate
GEF ............................Guanosine nucleotide exchange factor
xi
Grb2 ...........................Growth factor receptor binding protein 2
GST ............................Glutathione S-transferase
GTP ............................Guanosine triphosphate
h……………………..hours
HBx…………………Hepatitis B x antigen
HPK1..........................Hematopoietic protein kinase 1
IκBα ...........................Inhibitor of kappa B alpha
IKK ............................IκB kinase
IKKK..........................IκB kinase kinase
IP ................................Immunoprecipitation
JIP1 ............................JNK-interacting protein 1
JNK ...........................c-Jun N-terminal kinase
LPS .............................Lipopolysaccharide
LZ ...............................Leucine zipper
KSR ............................Kinase suppressor of ras
MAPK ........................Mitogen activated protein kinase
MAPKAP-K...............MAPK-activated protein kinase
MAP2K ......................MAPK kinase
MAP3K ......................MAPK kinase kinase
MEK...........................MAPK/ERK kinase
MEKK ........................MEK kinase
Met .............................Methionine
MLK ...........................Mixed lineage kinase
MLTKα ......................Mitogen-activated protein triple kinase alpha
MKP ...........................MAPK phosphatase
MMP ..........................Matrix metalloproteinase
MNK ..........................MAPK interacting kinase
MP-1 ..........................MEK partner 1
MSK ...........................Mitogen and stress activated kinase
ND………………….Neuro-D
NF ..............................Neurofibromatosis
NF-κB ........................Nuclear factor kappa-light-chain enhancer of activated B cells
NGF............................Neuron growth factor
PAK1..........................p21-GTPase activated kinase 1
PARP……………….poly (ADP-ribose) polymerase (PARP)
PB1.............................Phox/Bem1P
PBS ............................Phosphate buffered saline
PHD………………...Plextrin-homology domain
xii
PMSF .........................Phenylmethylsulphonyl fluoride
PP2A ..........................Serine/threonine protein phosphatase 2A
PP5 .............................Protein phosphatase 5
Pro ..............................Proline
PTP............................. protein tyrosine phosphatase
PVDF .........................Immobilon-P Polyvinylidene Flouride
ROS………………...Reactive oxygen species
RSK ............................p90 ribosomal S6 kinase
RTK............................Receptor tyrosine kinase
SAP1………………..Sodium-associated protein 1
SAM ...........................Sterile-alpha-motif
SAPK .........................Stress-activated protein kinase
SCG………………...Superior cervical ganglion
Ser ..............................Serine
SH ..............................Src homology
siRNA ........................small interfering RNA
SOS ............................Son of sevenless
STAT3........................Signal transducer and activator of transcription 3
TAB1..........................TAK1-binding protein 1
TAK1 .........................Transforming growth factor β-activted protein 1
TAO ...........................Thousand and one amino acid
TCR…………………T cell antigen receptor (TCR)
TGFβ………………..Transforming growth factor beta
TIMP ..........................Tissue inhibitor of metalloproteinases
Thr ..............................Threonine
TNFα ..........................Tumor necrosis factor alpha
TLR4 ..........................Toll-like receptor 4
Tpl2 ............................Tumor progression locus 2
TRAF4 .......................TNF receptor-associated factor 4
Tyr ..............................Tyrosine
ZAK ...........................Zipper-sterile-alpha motif kinase
xiii
Chapter 1
Introduction
1.1 The Mitogen-activated protein kinase signaling cascade
The mitogen-activated protein kinase (MAPK) signaling pathway is a three-tiered
signaling cascade that is conserved from yeast to higher mammals including humans
(Widmann, et al., 1999). The MAPK pathway is activated by a wide range of stimuli such
as stress, cytokines and growth factors and leads to different cellular responses including
proliferation, inflammation, invasion and apoptosis (Figure 1) (Kyriakis and Avruch,
2001; Pearson, et al., 2001; Uhlik, et al., 2004). The MAPK kinase kinases, or MAP3Ks,
form the top tier of the signaling cascade (Dhanasekaran and Johnson, 2007). Once
MAP3Ks are activated, they phosphorylate and activate their immediate downstream
targets, the MAPK kinases (MAP2Ks or MEKs) that in turn phosphorylate and activate
MAPKs, the cascade’s executor kinases (Figure 1) (Johnson and Lapadat, 2002; Kyriakis
and Avruch, 2001; Lawler, et al., 1998; Raingeaud, et al., 1996). The mammalian
extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK),
p38 kinase and ERK5 are four major MAPKs involved in this signaling cascade, which
upon stimulation, activate cytosolic or nuclear-localized effectors and thereby translate
1
the stimulus into a corresponding cellular response (Figure 1) (Ben-Levy, et al., 1998;
Raingeaud, et al., 1996; Uhlik, et al., 2004).
Figure 1. The MAPK signaling cascade. MAP3Ks are activated in response to stress,
cytokines or growth factors. Active MAP3Ks phosphorylate and activate MAP2Ks that in
turn phosphorylate and activate MAPKs. Active MAPKs activate cytosolic targets or
activate transcription factors that regulate the expression of genes that control different
cellular processes like proliferation, invasion, apoptosis and inflammation.
2
1.2 Characteristics and functions of MAPK proteins
MAPKs are proteins that are ubiquitously expressed in all eukaryotic cell types
but yet regulate different cellular responses in a stimulus- and cell type-specific manner
(Dhanasekaran and Johnson, 2007; Uhlik, et al., 2004; Widmann, et al., 1999). MAPKs
are proline directed serine/threonine kinases that activate nuclear or cytosolic substrates,
by phosphorylation at serine or threonine residues found within the Pro-X-Ser/Thr-Pro
consensus sequence (Alvarez, et al., 1991; Maeda and Firtel, 1997). MAPK proteins are
activated upon dual phosphorylation, by specific MAP2Ks, on both the threonine and
tyrosine residues of the Thr-X-Tyr motif present in the activation loop, where the amino
acid X varies with different MAPKs (Ahn, et al., 1991; D'Mello, et al., 1993; DiDonato,
et al., 1996; Estus, et al., 1994; Faris, et al., 1998; Frandsen and Schousboe, 1990).
MAPKs undergo an ordered phosphorylation mechanism, whereby the tyrosine residue of
the Thr-X-Tyr motif is phosphorylated first resulting in an increase in the affinity
between MAPKs and their specific MAP2Ks, a step that allows the subsequent
phosphorylation of the threonine residue and full MAPK activation (Haystead, et al.,
1992). Dual phosphorylation of MAPKs triggers a series of conformational changes in
the activation loop and surrounding sequences that ultimately result in the activation of
these proteins (Canagarajah, et al., 1997). Of the MAPK pathways, ERK, JNK and p38
signaling pathways are the best characterized.
1.2.1 The ERK1/2 pathway
ERK1 (44 kDa) and ERK2 (42 kDa), often referred to as ERK1/2, are two main
ubiquitously expressed isoforms of ERK that share more than 85% sequence identity
3
(Boulton, et al., 1991; Chen, et al., 2001; Seger and Krebs, 1995). Activation of ERK1/2
occurs upon specific recognition and subsequent phosphorylation of the Thr and Tyr
residues in the Thr-X-Tyr motif (Thr183 and Tyr185 in human ERK2 and Thr202 and
Tyr204 in human ERK1) by the upstream MAP2Ks, MEK1 and MEK2 (Crews, et al.,
1992; Zheng and Guan, 1993). Activity of ERK1/2 is also regulated by phosphatases,
including the MAPK phosphatases or MKPs which are dual-specificity phosphatases
(DSPs) that dephosphorylate both phospho-tyrosine and phospho-threonine residues
(Owens and Keyse, 2007; Raman, et al., 2007). Of the different MKPs, MKP3 shows
higher specificity towards ERK1/2 than other MAPKs (Zhang, et al., 2003). The
serine/threonine protein phosphatase 2A, or PP2A, also functions as a regulator of
ERK1/2 activity by dephosphorylating the threonine residue of the Thr-X-Tyr motif in
ERK1/2 (Anderson, et al., 1990).
Growth factors and mitogens are the primary activators of the ERK1/2 pathway,
however, cytokines, activators of G protein-coupled receptors and different stresses have
also been reported to activate this pathway (Johnson and Lapadat, 2002; Yoon and Seger,
2006). Early studies revealed a key role for Ras GTPases and B-Raf in ERK activation
(depicted in Figure 2 below). Briefly, receptor tyrosine kinases (RTKs), upon binding to
their ligands such as growth factors, undergo dimerization and cytoplasmic domain
transphosphorylation. Another transphosphorylation process then follows on specific
tyrosine residues in the cytoplasmic region of the RTK, which leads to full activation of
the receptor. The phospho-tyrosines create docking sites for the Src homology 2 (SH2)
domain of adaptor proteins, such as the growth factor receptor binding protein 2, or Grb2.
Grb2 then recruits the guanine nucleotide exchange factor (GEF), son of sevenless (SOS),
4
via its Src homology 3 (SH3) domain. SOS then activates Ras by promoting the switch
from an inactive GDP-bound to an active GTP-bound form (Buday and Downward,
2008; Downward, 1996; Wittinghofer, et al., 1997). Upon activation, Ras interacts with
and promotes the activation of members of the Raf family of MAP3Ks, Raf-1, B-Raf and
A-Raf. Once activated, Rafs phosphorylate and activate MEK1 and MEK2 that in turn
activate ERK1/2 (Chadee and Kyriakis, 2004; Dhillon, et al., 2007; Dunn, et al., 2005).
Active ERK1/2 will then undergo dimerization and either activate cytoplasmic substrates
such as p90 ribosomal S6 kinases (RSKs), mitogen and stress activated kinases (MSKs)
and MAPK interacting kinase (MNK), or translocate to the nucleus and regulate the
expression of certain genes by directly activating several transcription factors including
AP-1, c-Myc and c-Fos (Buday and Downward, 2008; Chen, et al., 2001; Dunn, et al.,
2005; Raman, et al., 2007).
5
Figure 2. The Ras/Raf/ERK1/2 signaling pathway. In response to interaction with
growth factors (GF), RTKs undergo dimerization and activation. Grb2 binds to the active
RTK and recruits SOS which in turn activates Ras. Active Ras interacts with and
activates the Raf members. Active Rafs phosphorylate and activate MEK1/2 that in turn
phosphorylate and activate ERK1/2. Once activated, ERK1/2 can trigger a cellular
response either by activating cytoplasmic targets or by inducing transcriptional activation
by translocating to the nucleus and activating transcription factors.
6
ERK1/2 is one of the main regulators of cell proliferation and transformation and
the ERK signaling pathway is deregulated
at a high frequency in human cancers
(Dhillon, et al., 2007). ERK1/2, however, is implicated in numerous other functions
including differentiation, cell motility, cell migration, cytoskeletal polymerization and
golgi fragmentation (Ishibe, et al., 2004; Jesch, et al., 2001; Reszka, et al., 1995; Yoon
and Seger, 2006). The ERKs have different subcellular localizations. In addition to the
cytoplasm and nucleus, ERK1/2 were found to associate with membrane receptors and
transporters, intracellular membrane compartments, microtubules, adherens junctions and
focal adhesions (Furuchi and Anderson, 1998; Ishibe, et al., 2004; Jesch, et al., 2001;
Reszka, et al., 1995). Moreover, continuous ERK1/2 nucleo-cytoplasmic trafficking is
required for proper signaling of ERK1/2 (Costa, et al., 2006; Marchi, et al., 2008).
1.2.2 The JNK pathway
JNK is a proline-directed kinase that is activated by stresses and TNFα (Kyriakis,
et al., 1994). JNK1/SAPKβ, JNK2/SAPKα and JNK3/SAPKγ (ranging between 46 and
54kDa) are three JNK isoforms that share more than 85% sequence identity (Johnson and
Lapadat, 2002; Kyriakis and Avruch, 2001). While JNK3 expression is mostly restricted
to the brain and heart, JNK1 and JNK2 are ubiquitously expressed (Dhillon, et al., 2007).
JNK is activated upon the dual phosphorylation of the threonine and tyrosine residues of
the Thr-X-Tyr motif by the MAP2Ks, MEK4 and MEK7 (Lawler, et al., 1998). The dual
specificity phosphatases MKP7 and VH5 also function to regulate JNK activity by
dephosphorylating
the
active
JNK
sites
(Alonso,
et
al.,
2004).
The
phosphoserine/threonine protein phosphatase 5 (PP5) is yet another phosphatase that
7
deregulates the JNK pathway. PP5, however, does so by acting upstream of JNK,
primarily by inactivating MAP3Ks such as the apoptosis signal-regulating kinase 1, or
ASK1 (Chinkers, 2001; Zhou, et al., 2004).
The JNK pathway is mainly activated by cytokines and stresses but is also
activated to a lesser extent by growth factors and inhibition of DNA and protein synthesis
(Kyriakis and Avruch, 2001). Once activated, JNK translocates to the nucleus where it
activates numerous transcription factors, including c-Jun. Activation of c-Jun by JNK
was found to mediate Ras-induced tumorigenesis and cellular transformation in vitro
(Johnson, et al., 1996; Smeal, et al., 1991). Other transcription factors activated by JNK
proteins include p53, STAT3, ATF2, ELK1 and nuclear factor of activated T cells (Chen,
et al., 2001; Ip and Davis, 1998). Through gene expression driven by one or more of
these transcription factors, JNK can mediate differentiation, cytokine production, actin
reorganization, inflammatory responses and apoptosis (Chen, et al., 2001). Of these
different cellular responses, the ability of JNK to promote apoptosis is one of the best
characterized.
Although the exact mechanism by which JNK triggers cell death is not well
known, several lines of evidence support a role for JNK in mitochondrial apoptotic cell
death (Dhanasekaran and Reddy, 2008). Mitochondrial apoptosis or the intrinsic
apoptotic pathway is characterized by the permeabilization of the mitochondrial
membrane, the release of cytochrome c, and the activation of caspases 9 and 3 (Baliga
and Kumar, 2003). This process is regulated by the Bcl-2 family of proteins (Kim, 2005).
JNK can activate mitochondrial cell death through phosphorylation and inhibition of the
anti-apoptotic protein, Bcl-2 (Figure 3) (Dhanasekaran and Reddy, 2008). Bcl-2 normally
8
sequesters and inactivates the pro-apoptotic protein Bax (Breckenridge and Xue, 2004).
Upon phosphorylation, Bcl-2 dissociates from Bax rendering Bax active. Bax then
undergoes multimer formation that inserts in the mitochondrial membrane, forming
channels that allow the release of cytochrome c and other apoptotic proteins leading to
caspase-3 dependent cell death (Figure 3) (Breckenridge and Xue, 2004). JNK can also
induce apoptosis in a p53-dependent manner. p53, upon phosphorylation and activation
by JNK, can induce apoptosis by mediating the expression of pro-apoptotic genes such as
Bax (Figure 3) (Miyashita and Reed, 1995). p53 can also induce apoptosis in a
transcription-independent manner (Figure 3) (Caelles, et al., 1994). JNK can also trigger
apoptosis by the induction of the extrinsic apoptotic pathway (Figure 3) (Dhanasekaran
and Reddy, 2008; Tang, et al., 2012). The extrinsic apoptotic pathway is mediated by
FasL that upon binding to Fas and the adaptor protein FADD, activates caspase 8 which
then triggers apoptosis in a caspase 3-dependent manner (Guicciardi and Gores, 2009;
Strasser, et al., 2009). JNK was shown to activate this pathway by inducing the
expression of FasL (Figure 3) (Dhanasekaran and Reddy, 2008). For instance, JNK
upregulates FasL protein expression in HepG2 cells in response to the hepatitis B virus X
protein (HBx) in a mechanism that depends on MLK3 and MEK7 (Tang, et al., 2012).
9
JNK
Bcl-2
Bax
P
Cyt c
Cyt c
P
p53
P
Bcl-2
Bax
Expression of FasL
Bax
Bax
Cyt c
Cyt c
Extrinsic pathway
Bax
Bax
Bax
Cyt c
Activation of
caspase 8
Transcription of proapoptotic genes
Cyt c
Cyt c
Casp 9
Cyt c
Apaf- 1
Activation of caspase 3
Activation of caspase 3
APOPTOSIS
Figure 3. JNK-mediated apoptosis. JNK can induce mitochondrial apoptotic cell death
through the phosphorylation and inactivation of Bcl-2 either directly or through Bad and
Bim. This leads to the activation of Bax which forms mitochondrial channels through
which cytochrome c is released. Cytochrome c then activates the caspase cascade. JNK
can also trigger the expression of pro-apoptotic genes by activating transcription factors
such as p53. JNK activates the extrinsic apoptotic pathway by increasing the expression
of the pro-apoptotic FasL gene.
10
1.2.3 The p38 pathway
p38 is a family of MAPK proteins that consists of four main members in
vertebrates (α, β, γ and δ). The p38 isoforms are encoded by different genes with
variations in the sequence homology (Jiang, et al., 1996; Lechner, et al., 1996). The
variations in the sequence homology and in the interactions with other molecules could
explain the diverse functions of p38 (Cuenda and Rousseau, 2007). Moreover, of the four
p38 isoforms, only p38α and p38β are ubiquitously expressed while the other forms show
more limited expression, with p38γ mainly found in skeletal muscle and p38δ in
pancreas, kidney and small intestine (Goedert, et al., 1997; Raman, et al., 2007).
Thr-Gly-Tyr is the conserved motif in the activation loop of the p38 family, and
dual phosphorylation of the threonine and tyrosine residues lead to p38 activation
(Kyriakis and Avruch, 2001). MEK3 and MEK6 are the MAP2Ks that selectively
activate the p38 family of MAPKs (Cuenda, et al., 1997; Raingeaud, et al., 1996). The
JNK activator MEK4 can also activate p38 (Brancho, et al., 2003). Although p38 is
mainly activated by MEKs, activation of p38α can also occur in a MEK-independent
manner. In this respect, p38α, upon binding to TAB1 (Transforming growth factor βactivated protein 1 (TAK1)-binding protein 1), undergoes autophosphorylation at Thr180
and Tyr182 and subsequent activation (Ge, et al., 2002). Moreover, in T cells, p38α was
proposed to be activated by undergoing conformational change and autophosphorylation
at the activation sites (Thr180 and Try182) following the phosphorylation of Tyr323 by
tyrosine kinases in a mechanism that is mediated by the T cell antigen receptor (TCR)
(Salvador, et al., 2005). Similar to other MAPKs, the activity of p38 is also regulated by
multiple phosphatases. Some of these phospatases that interact with and inhibit p38
11
include MKP1, MKP5, MKP7 and MKP8. These phosphatases can also act on JNK but
show low affinity towards ERK1/2 (Keyse, 2000; Raman, et al., 2007; Tanoue and
Nishida, 2003). MKP1-null mice show enhanced activity of p38 and production of
proinflammatory cytokines in response to low exposures to LPS as compared to wild
types, suggesting MKP1 as an important regulator of LPS-induced p38 activity (Chi, et
al., 2006). The tyrosine phosphatase PTP and the serine/threonine phosphatases PP2C,
PP2A and PP2B have also been shown to inactivate p38 (Raman, et al., 2007; Takekawa,
et al., 2000; Takekawa, et al., 1998).
Similar to JNK, p38 is activated by stresses and cytokines, but can also be
activated to a much lesser extent by growth factors (Zarubin and Han, 2005). Activated
p38 then either activates cytoplasmic substrates or translocates to the nucleus where it
activates transcription factors that regulate the expression of genes involved in a number
of cellular responses such as apoptosis, growth, migration, differentiation and
inflammation, the deregulation of which can lead to certain pathologies (Figure 4) (BenLevy, et al., 1998; Krens, et al., 2006; Zarubin and Han, 2005). The wide range of p38
substrates is responsible for the induction of these different biological functions.
Although all p38 isoforms have many substrates in common including ATF2, ELK-1, and
SAP1, differences in substrate specificity exist between p38α and p38β and between p38γ
and p38δ. For instance, while MAPKAP-K2 and MAPKAP-K3 are substrates for p38α
and p38β, they cannot be activated by p38γ and p38δ (Cuenda, et al., 1997; Goedert, et
al., 1997). Some substrates are also specific to either p38γ or p38δ. p38γ binds to and
phosphorylates many PDZ domain-containing proteins via its KETXL C-terminal
sequence that is unique among the p38 isoforms (Hasegawa, et al., 1999; Sabio, et al.,
12
2005; Sabio, et al., 2004). p38δ has stathmin and tau as substrates, both of which play a
role in regulating microtubule dynamics (Feijoo, et al., 2005; Parker, et al., 1998).
Figure 4. The p38 MAPK signaling pathway (adapted from (Cuenda and Rousseau,
2007)). p38 in response to stimulating agents, is activated by MEK3/MEK6, also known
as MKK3/MKK6. Once activated, p38 phosphorylates and activates numerous cytosolic
proteins and transcription factors to regulate many biological functions. Deregulation of
p38 signaling can contribute to the development of several pathologies.
13
p38 has many targets in different subcellular localizations. In some cell lines, p38
was found in the nucleus in resting cells and in response to stimuli, p38 was
phosphorylated and formed a complex with MAPKAP-K2 or MAPKAP-K5 (Ben-Levy,
et al., 1998; Seternes, et al., 2002). p38-MAPKAP-K2 or p38-MAPKAP-K5 complexes
then are exported to the cytoplasm where p38 further activates cytosolic substrtaes (BenLevy, et al., 1998; Seternes, et al., 2002). The intracellular localization of p38α can also
be regulated by TAK1 and TAB1 (Lu, et al., 2006). The localization of p38 can also vary
with isoforms. For instance, in cardiac myocytes, p38α and p38β shuttle between the
nucleus and the cytosol and are retained in the nucleus in response to the nuclear export
inhibitor, leptomycin B. This inhibitor, however, did not affect the localization of p38γ
which was mainly associated with cytosolic foci (Court, et al., 2002).
1.2.4 The ERK5 pathway
The ERK5 pathway is the least characterized among the different MAPK
pathways. ERK5 signaling is triggered in response to EGF and TNFα (Kato, et al., 1998;
Weldon, et al., 2002). ERK5 is activated upon phosphorylation of Thr218 and Tyr220 of
the Thr-X-Tyr motif by the MAP2K, MEK5 (Weldon, et al., 2002). MEKK2 and
MEKK3 are the only MAP3Ks shown to activate MEK5 and hence ERK5 (Sun, et al.,
2001). A role for ERK5 in cell proliferation, transformation and metastasis has been
demonstrated (Buschbeck, et al., 2005; Kato, et al., 1998; Kesavan, et al., 2004). For
instance, ERK5 promoted cell proliferation in response to EGF (Kato, et al., 1998).
Moreover, ERK5 was shown to mediate transformation downstream of the oncogene,
BCR/Abl (Buschbeck, et al., 2005). In addition to regulating MMP-9 and IL-6, ERK5
14
was found to be overexpressed in human breast cancer patients with poor prognosis.
These findings support a role for ERK5 in cancer (Kesavan, et al., 2004; Mehta, et al.,
2003; Montero, et al., 2009).
1.3 The matrix metalloproteinases
Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes that
can function as downstream targets of MAPKs (Ispanovic and Haas, 2006; Spallarossa, et
al., 2006; Westermarck and Kahari, 1999). MMPs play a key role in cancer cell invasion
but are also involved in other biological functions including development,
morphogenesis, angiogenesis and inflammation (Hua, et al., 2011). Of the 28 different
MMPs, members of the gelatinase group (MMP-2 and MMP-9) are implicated the most
in cancer (Bauvois, 2012). The expression and activation of these MMPs can be mediated
by p38, ERK1/2 and/or JNK (Cho, et al., 2000; Ispanovic and Haas, 2006; Spallarossa, et
al., 2006). MMPs can thus execute signals triggered by MAP3Ks. MMP-2 and MMP-9
consist of the following structural domains: the N-terminal signal peptide domain, the
propeptide region, the catalytic domain containing the highly conserved zinc binding
region, and the C-terminus that has a hemopexin-like domain that is connected to the
catalytic domain by a small hinge region (Figure 5) (Bauvois, 2012; Nagase and
Woessner, 1999). The catalytic domain of gelatinases is characterized by the presence of
a collagen-binding domain (CBD), composed of three fibronectin type II modules that
function in enhancing the efficiency of collagen and gelatin degradation (Figure 5)
(Morgunova, et al., 1999).
15
MMPs are first synthesized as inactive pre-proenzymes that undergo two main
steps of activation (Benjamin and Khalil, 2012; Overall and Lopez-Otin, 2002). The first
step involves the cleavage of the signal peptide during translation to yield proMMPs
(Figure 5) (Benjamin and Khalil, 2012). ProMMPs are then maintained inactive by a
conserved cysteine residue in the prodomain that acts as a “cysteine switch”, whereby, it
forms a coordination bond with the active site zinc ion in the catalytic domain (Figure 5)
(Springman, et al., 1990). Full activation of MMPs occurs upon disruption of this bond
often by the removal of the propeptide domain by an autoproteolytic event that can also
be induced by chemicals (Figure 5) (Springman, et al., 1990).
MMP-2 and -9 are considered as biomarkers for malignant tumors with poor
prognosis (Bauvois, 2012). MMP-2 and/or -9 were reported to be overexpressed in a
number of cancers including, breast, colorectal, ovarian, prostate and lung (Roy, et al.,
2009; Turpeenniemi-Hujanen, 2005). MMP-2 and -9, through their ability to degrade the
basement membrane and different other extracellular-matrix (ECM) components, were
found to promote cell invasion in several types of cancer (Hua, et al., 2011). MMP-2 and
MMP-9 can also mediate the epithelilal-mesenchymal transition (EMT), a process
required for tumor invasion, which further supports a role for these MMPs in cell
invasion (Tester, et al., 2007; Thiery, et al., 2009; Xu, et al., 2009).
16
Figure 5. MMP-2 and -9: structure and activation. MMP-2 and MMP-9 generally
contain several structural domains that sequentially are: the signal peptide domain (SPD),
the propeptide domain (Pro), the catalytic domain containing the Zn2+-binding region and
the collagen-binding domain (CBD), a hemopexin-like C-terminus connected to the
catalytic domain by a hinge linker. Activation of these MMPs is a two-step process that
first involves cleavage of the SPD then followed by the proteolytic cleavage of the propepetide domain. C stands for cysteine and Zn2+ for zinc.
17
1.4 The MAP2Ks
MAP2Ks, as mentioned earlier, represent the second tier of kinases in the MAPK
signaling cascade. The MAP2Ks are dual specificity kinases where they act as both
tyrosine and serine/threonine kinases, and phosphorylate the tyrosine and threonine
residues of the Thr-X-Tyr motif within the MAPKs (Ashworth, et al., 1992). In addition
to the Thr-X-Tyr sequence containing the motif, the tertiary structure of the MAPK
protein is also very important for MAP2K binding (Widmann, et al., 1999). Thus, each
MAP2K interacts with and regulates a limited number of MAPKs (Widmann, et al.,
1999). MAP2Ks are much fewer in number than MAP3Ks (Widmann, et al., 1999). Since
numerous MAP3Ks are activated in response to each stimuli, the multiple signals
triggered are thus funneled down by the MAP2Ks, which specifically direct those signals
to one or more of the MAPK pathways (Widmann, et al., 1999).
MAP2Ks have two domains, a docking (D) domain and a domain for versatile
docking (DVD), that are essential for binding to MAPKs and MAP3Ks, respectively
(Sharrocks, et al., 2000; Takekawa, et al., 2005). The DVD domain of MAP2Ks lies 20
residues downstream of the catalytic core (Takekawa, et al., 2005). In addition to the D
and DVD domains, MEK5, an activator of ERK5, contains a Phox/Bem1P (PB1) domain
that plays a role in protein-protein interactions and associates with other PB1-containing
proteins such as the MAP3Ks, MEKK2/MEKK3 (Lamark, et al., 2003; Nakamura, et al.,
2006). The N-terminal and C-terminal regions of the PB1 domain of MEK5 also bind to
ERK5, allowing MEK5 to act as a scaffold protein that couples MEKK2/MEKK3 to
ERK5 (Nakamura, et al., 2006).
18
Seven members of MAP2Ks, MEK1-7, exist with MEK1 and MEK2 being
specific activators of ERK1/2, MEK3 and MEK6 of p38, MEK4 and MEK7 of JNK and
MEK5 of ERK5 (Dhanasekaran and Johnson, 2007; Dhillon, et al., 2007; Raman, et al.,
2007). MEK4, however, also phosphorylates and activates p38 (Brancho, et al., 2003).
While MEK7 was suggested to be a stronger activator of JNK than MEK4, full activation
of JNK was reported to require both MEK4 and MEK7 that preferentially phosphorylate
the tyrosine and threonine residues within the Thr-X-Tyr motif of JNK, respectively
(Lawler, et al., 1998). Another study reported that both of these residues can be
phosphorylated by either of the two MAP2Ks (Chen, et al., 2002). However, JNK
activation in the absence of either MEK4 or MEK7 was found to be lower than that in the
presence of both MAP2Ks (Chen, et al., 2002). MEK4 and MEK7 were both shown to be
activated by stress to trigger the JNK pathway (Wang, et al., 2007). Pro-inflammatory
cytokines on the other hand, induce JNK signaling primarily by activating MEK7
(Moriguchi, et al., 1997). MEK4 and MEK7 also exhibit some variations in the MAP3Ks
that activate them (Wang, et al., 2007). For instance, while MEKK4 activates MEK4,
MEKK1 activates both MEK4 and MEK7 (Takekawa, et al., 2005).
The p38 activators, MEK3, MEK4 and MEK6 are activated in response to
stresses, proinflammatory cytokines and UV irradiation (Brancho, et al., 2003). p38α is
activated by all three MEKs (Brancho, et al., 2003). p38γ is activated in response to
environmental stresses by both MEK3 and MEK6, while activation by TNFα is mainly
mediated by MEK6 (Remy, et al., 2010). Similar to p38γ, stress-induced p38β activation
is mediated by both MEK3 and MEK6 (Remy, et al., 2010). The activity of the p38δ, in
19
response to numerous stimuli, including UV radiation, stress and TNFα is primarily
regulated by MEK3 (Remy, et al., 2010).
Mutations in MAP2Ks have also been implicated in cancers. For instance, MEK4,
though at a low frequency, was reported to be inactivated in numerous types of cancer,
including breast, colon and lung cancer (Cunningham, et al., 2006). This might suggest a
suppressive role for MEK4 in cancer. In contrast, overexpressing MEK4 in breast and
pancreatic cancer cells promoted proliferation and invasion which implicates a prooncogenic role for MEK4 (Wang, et al., 2004). Moreover, although, knocking out MEK4
in human pancreatic cancer cell lines did not yield any effect on proliferation, metastasis
was suppressed (Cunningham, et al., 2006). The differences observed in the cellular
responses triggered by MEK4 could be attributed to the degree of activation in response
to stresses and environmental context of cells that favor the induction of either
proliferation or apoptosis (Dhillon, et al., 2007).
1.5 The MAP3Ks
The MAP3K are serine/threonine kinases that are encoded by at least 20 genes,
and represent the largest and most diverse components of the MAPK cascade. MAP3Ks
function to direct signals generated by a wide range of stimuli to the MAPKs that
ultimately lead to specific functional responses (Cuevas, et al., 2007; Uhlik, et al., 2004).
The differences in the domains and motifs between MAP3Ks, allow individual MAP3Ks
to selectively regulate the localization and activation of specific MAP2Ks and MAPKs
(Dhanasekaran and Johnson, 2007). Many of the MAP3Ks however, can activate more
than one MAP2K and thereby regulate multiple MAPK pathways (Cuevas, et al., 2007).
20
Based on protein homology, the different MAP3Ks are distributed into several main
clusters: the mitogen-activated protein/ERK kinase kinase (MEKKs), Rafs, thousand and
one amino acid (TAOs), tumor progression locus 2 (Tpl2) and apoptosis signal regulating
kinases (ASKs), TGF-β-activated kinase 1 (TAK1) with MOS, dual leucine zipper
kinases (DLKs) and zipper sterile alpha kinase (ZAKs), and the mixed lineage kinases
(MLK1-4), (Craig, et al., 2008; Cuevas, et al., 2007). However, this cluster distribution
is different if only the homology in the kinase domain is considered, with ASKs being
grouped with MEKKs, Tpl2 with the TAOs, and the DLKs and ZAKs with MLKs (Craig,
et al., 2008). Moreover, MAP3Ks, also have kinase-independent functions that are
mediated by protein-protein interactions (Mita, et al., 2002). In addition, MAP3Ks, can
regulate other signaling pathways as well, including the NF-κB and Akt/MTOR pathways
(Hirano, et al., 1996).
1.5.1 The MEKK group
The MEKK family includes six members, MEKK (1-4) as well as ASK1 and
ASK2. Each of the MEKK members can regulate multiple biological functions, some of
which are not redundant, as evidenced by the lethal effects of knocking out MEKK3 or
MEKK4, but not MEKK1 or MEKK2, on mice embryos (Abell and Johnson, 2005;
Cuevas, et al., 2007; Yang, et al., 2000). MEKK1 was the first to be identified and is one
of the best characterized MEKK members. MEKK1 promotes cell survival by activating
ERK1/2 and JNK pathways in response to cytokines and growth factors (Xia, et al., 2000;
Yujiri, et al., 1998). Moreover, MEKK1, upon association with and activation by small G
proteins, including RhoA, regulates ERK1/2-mediated cell migration (Cuevas, et al.,
21
2003; Fanger, et al., 1997). Although MEKK1 is a potent activator of ERK1/2, it can also
act to negatively regulate ERK1/2 via ubiquitination and subsequent degradation of
ERK1/2 (Lu, et al., 2002). ERK1/2 ubiquitination is mediated by the plextrin homology
(PH) domain of MEKK1 that functions as an E3 ubiquitin ligase (Lu, et al., 2002).
MEKK1 also regulates AP-1-induced gene expression (Cuevas, et al., 2005). MEKK2
and MEKK3 are the only MAP3Ks known to regulate the unique MAPK, ERK5, by
heterodimerization with and subsequent activation of MEK5 (Sun, et al., 2001).
Dimerization between MEKK2 or MEKK3 and MEK5 requires the PB1 domain found in
these three kinases and is a prerequisite for the regulation of ERK5 signaling (Nakamura
and Johnson, 2003).
MEKK2 and MEKK3 share around 95% sequence homology in the kinase
domain, however, the overall sequence identity is only approximately 55% (Blank, et al.,
1996). Although, MEKK2 and MEKK3 both activate ERK5 and induce AP-1-mediated
expression of inflammatory cytokines, MEKK3 also plays a role in regulating the p38
and the NF-κB pathways, while MEKK2 functions to activate the JNK pathway
(Garrington, et al., 2000; Huang, et al., 2004; Kesavan, et al., 2004; Uhlik, et al., 2003;
Xu, et al., 2004).
MEKK4, one of the least characterized MEKK, was shown to activate both JNK
and p38 via the direct phosphorylation of MEK4/MEK7 and MEK3/MEK6, respectively
(Abell, et al., 2005; Chi, et al., 2005). The activity of MEKK4 seems to be modulated by
numerous upstream proteins. Such proteins include Rac, Cdc42 and TRAF4, all of which
trigger JNK signaling by binding to MEKK4 (Abell and Johnson, 2005; Fanger, et al.,
1997; Gerwins, et al., 1997). Other proteins also known to bind to and activate MEKK4
22
are the isoforms α, β, and γ of the small growth arrest and DNA damage-inducible
(GADD45) proteins (Takekawa and Saito, 1998).
ASK1, also known as MEKK5, is activated by numerous stimuli that can result in
elevated intracellular levels of reactive oxygen species (ROS) such as stresses, cytokines
and LPS (Hayakawa, et al., 2006). One of the first characterized functions of ASK1 is the
regulation of stress-induced apoptosis (Tobiume, et al., 1997). ASK1 however, similar to
MEKK4, activates MEK3, MEK6, MEK4 and MEK7 that in turn activate p38 and JNK
(Ichijo, et al., 1997). Moreover, studies focused on the activation of ASK1 by LPS
revealed an important role for ASK1 in regulation of the innate immune system
(Matsuzawa, et al., 2005). A role for ASK2, or MEKK6, in apoptosis has also been
demonstrated, whereby ASK2 can form a heterodimer with ASK1 to trigger apoptosis
(Ortner and Moelling, 2007; Takeda, et al., 2007).
1.5.2 The Raf MAP3Ks
As mentioned earlier, the Rafs are MAP3Ks that mainly mediate Ras-induced
activation of ERK1/2 by phosphorylating MEK1/MEK2 (Dunn, et al., 2005). Three main
Raf proteins, A-Raf, B-Raf and Raf-1 (or c-Raf), have three highly conserved regions
(CR1-3). The CR1, which is localized in the N-terminus and harbors the Ras-binding
domain and the cysteine rich domain, mediates the binding between the Rafs and Ras.
The CR2 is serine/threonine rich and regulates Raf activity by undergoing
phosphorylation, and the CR3 harbors the C-terminal kinase domain (Mott, et al., 1996;
Vojtek, et al., 1993; Wellbrock, et al., 2004). The Raf proteins have distinct expression
patterns and are not functionally redundant (Cuevas, et al., 2007). Raf-1, but not A-Raf or
23
B-Raf, is ubiquitously expressed (Craig, et al., 2008). Both B-Raf and A-Raf are widely
expressed, but A-Raf is mainly found in urogenital and gastrointestinal tissues (Luckett,
et al., 2000). The Raf isoforms can also display differences in function. For instance,
while B-Raf and Raf-1 phosphorylate and activate both MEK1 and MEK2, A-Raf only
activates MEK1 (Sridhar, et al., 2005; Wu, et al., 1996). Moreover, B-Raf appears to be
involved in cellular differentiation through activation of the MAPK, ERK3 (Coulombe, et
al., 2003; Hoeflich, et al., 2006). Raf-1 phosphorylates and inactivates Bad, which
suggests that it has a role in regulating apoptosis (Kebache, et al., 2007). A role for Raf-1
in neuronal and cardiac apoptosis was also demonstrated in mice with cardiac-specific
deletion of Raf-1 (Kanamoto, et al., 2000; Yamaguchi, et al., 2004). A role for Raf-1 in
suppressing apoptosis was also demonstrated in Raf-1 deficient mice, whereby Raf-1-/mice die at the embryonic stage (Mikula, et al., 2001). Moreover, Raf-1-/- fibroblasts were
shown to undergo apoptosis more readily than the wild-type cells, which further supports
an anti-apoptotic function for Raf-1 (Mikula, et al., 2001). Activation of ERK1/2 by Raf
proteins can be facilitated by several scaffold proteins including β-arrestin, and the
kinase suppressor of ras (KSR) (Morrison and Davis, 2003).
B-Raf is a human oncogene that is mutated at a high frequency (more than 60%)
in melanomas (Davies, et al., 2002). b-raf gene mutations are found in a large number of
cancers, including colon cancer and ovarian cancer (Dhillon, et al., 2007; Wan, et al.,
2004). The V600E point mutation in the activation loop of B-Raf is the most common
among b-raf mutations (Wan, et al., 2004). This mutation induces constitutive activation
of B-Raf and ERK signaling (Garnett and Marais, 2004). Interestingly, B-Raf, with
mutations that don’t enhance activity, also increases ERK activity (Wan, et al., 2004).
24
Such mutations were proposed to indirectly promote ERK signaling by activating Raf-1,
a heterodimerization partner of B-Raf (Garnett and Marais, 2004; Garnett, et al., 2005;
Rushworth, et al., 2006).
1.5.3 The TAK1 MAP3K group
TAK1 is mainly activated by TGFβ but can also be activated by other
proinflammatory stimuli such as IL-1β, TNFα and LPS (Irie, et al., 2000; Takaesu, et al.,
2003; Wagner and Siddiqui, 2007). Three isoforms of TAK1, TAK1a, TAK1b and
TAK1c, exist with TAK1a being the most predominant (Craig, et al., 2008). TAK1
triggers JNK and p38 signaling by activating MEK4 and MEK6, respectively (Shim, et
al., 2005). p38 however, when present in high levels, functions as part of a negative
feedback loop to regulate TAK1 activity (Cheung, et al., 2003). TAK1 is essential for
vesicular development, most likely by inducing the expression of JNK- and p38dependent genes involved in angiogenesis, and TAK1 deficiency is lethal to mouse
embryos (Jadrich, et al., 2006). The most distinctive role of TAK1 that has been
identified is the one involving the activation of the NF-κB pathway to regulate cellular
responses including inflammation, apoptosis and cell cycle regulation (Ghosh, 1999;
Sakurai, et al., 1998). Activation of the NF-κB by TAK1 requires the formation of a
complex between TAK1, TAB2, TAB3 and the E3 ubiquitin ligase/TRAF6 (Wang, et al.,
2001). The complex then phosphorylates and activates IKK in a ubiquitin-dependent
manner, a step that is essential for IκBα degradation (Wang, et al., 2001). Briefly, the
transcription factor NF-κB is sequestered in the cytosol in an inactive form by the
inhibitor IκBα (Karin, et al., 2002). Stimulation with proinflammatory cytokines like
25
TNFα and IL-1, activates the IκB kinase (IKK) kinase (IKKK) that in turn
phosphorylates and activates IKK (Figure 6) (Karin, et al., 2002). Once activated, IKK
phosphorylates IκBα on two serine residues, Ser32 and Ser36 (Ghosh and Karin, 2002).
Phospho-IκBα is then targeted for ubiquitination and proteasomal degradation, thereby
allowing NF-κB to translocate to the nucleus and activate the expression of genes
involved in numerous cellular responses (Figure 6) (Karin, et al., 2002).
Figure 6. The NF-κB pathway. NF-κB is maintained in the cytoplasm by its inhibitor
IκBα. Proinflammatory stimuli activate the IKK activator, IKKK, which in turn
phosphorylates IKK. Active IKK then phosphorylates IκBα on Ser32 and Ser36.
Phosphorylation on these residues leads to the ubiquitination and subsequent proteasomal
degradation of IκBα which leads to the release of NF-κB. NF-κB then translocates to the
nucleus where it activates gene expression.
26
1.5.4 The TAO/Tpl2 and Mos MAP3K groups
The TAO kinases are one of the least characterized MAP3Ks. Three TAO
members TAO1, TAO2 and TAO3 have so far been identified (Craig, et al., 2008).
TAO1, in response to stress, mainly activates the p38 pathway by associating with and
activating MEK3 (Hutchison, et al., 1998). DNA damage is another factor that acts as an
activator of TAO (Raman, et al., 2007). This process is mediated by Ataxia telangiectasia
mutated (ATM), a cell-cycle protein that binds to and phosphorylates TAO resulting in
p38-dependent DNA repair (Raman, et al., 2007). TAO2, upon activation by osmotic
stress, also functions as a negative regulator of TAK1-induced NF-κB activation
(Huangfu, et al., 2006).
Tpl2 is a MAP3K that activates ERK and JNK signaling in response to LPS,
CD40 and TNF (Dumitru, et al., 2000). In response to these stimuli, Tpl2 is
phosphorylated and activated by IKKβ, an upstream activator of NF-κB, suggesting that
Tpl2 plays a role to coordinate both MAPK and NF-κB signaling (Cho, et al., 2005;
Eliopoulos, et al., 2003). As for c-Mos, it was found to activate ERK1/2 signaling via
direct association and activation of MEK1 (Chen and Cooper, 1995; Okazaki and Sagata,
1995). C-Mos-mediated activation of ERK1/2 stabilizes c-Fos, a main component of the
AP-1 transcription factor, and thus drives AP-1-dependent gene expression (Okazaki and
Sagata, 1995).
1.5.5 The MLK family of MAP3Ks
MLKs, or mixed-lineage kinases, are so named because they exhibit
characteristics of both serine/threonine kinases and tyrosine kinases. Out of the eleven
27
protein kinase conserved regions in MLKs, the first seven have sequence similarity to
serine/threonine kinases while the remaining four are more similar to tyrosine kinases
(Gallo and Johnson, 2002). However, since early studies on several members of the
MLKs have demonstrated autophosphorylation on serine and threonine residues and also
phosphorylation of downstream MAP2Ks on serine residues, MLKs are considered to be
serine/threonine kinases (Gallo and Johnson, 2002; Gallo, et al., 1994). The MLK family
is further subdivided into the dual leucine zipper-bearing kinases (DLKs), the zippersterile-α motif kinase (ZAK) and the mixed-lineage kinases (MLKs) subfamilies (Gallo
and Johnson, 2002).
1.5.5.1 The DLK subgroup
The DLK subfamily consists of two members, DLK and the leucine zipper kinase
(LZK) that are approximately 87% identical in the catalytic domains (Gallo and Johnson,
2002). This subfamily is characterized by the presence of two leucine zipper domains that
are 31 amino acids apart and localized downstream of the kinase domain (Gallo and
Johnson, 2002). The DLK members both undergo homodimerization via the leucine
zipper domains, a step that is required for autophosphorylation and subsequent activation
of these proteins (Nihalani, et al., 2000). It is noteworthy that, homodimerization of DLK
and LZK occurs independently of both protein phosphorylation and kinase activity (Mata,
et al., 1996). Heterodimerization between DLK and LZK is also possible but involves the
amino terminal ends of both proteins rather than the leucine zipper domain (Nihalani, et
al., 2000). However, the association between DLK and LZK is indirect and is thought to
be mediated by the scaffold protein JNK-interacting protein 1 (JIP1) (Nihalani, et al.,
28
2000). JIP1 also complexes with and regulates the activities of DLK and LZK to induce
JNK signaling (Ikeda, et al., 2001; Nihalani, et al., 2001). DLK signaling is important in
the neuronal system, where it triggers JNK-dependent synaptic formation and migration
in the cerebral cortex development but induces neuronal cell apoptosis when
overexpressed or when cells are deprived of trophic factors (Figure 7) (Harris, et al.,
2002; Hirai, et al., 2006; Mota, et al., 2001; Xu, et al., 2001). In addition to JNK, DLK
can also mediate differentiation and apoptosis by activating p38 (Figure 7) (Craig, et al.,
2008; Gallo and Johnson, 2002).
Figure 7. Signaling of the DLK subfamily of MAP3Ks (adapted from (Craig, et al.,
2008). While LZK mainly induces JNK signaling by phosphorylating MEK4/7, DLK can
trigger migration, apoptosis and differentiation through the activation of both
MEK4/7/JNK and MEK3/6/p38 pathways.
29
1.5.5.2 The ZAK subgroup
The ZAK MAP3K, also known as MLTKα, is characterized by the presence of
the sterile-α-motif or (SAM) domain in the middle of the protein sequence just
downstream of the leucine zipper domain (Gallo and Johnson, 2002). A shorter splice
variant of ZAKα, ZAKβ, also commonly known as MLK7, that is highly expressed in
cardiomyocytes, was identified (Gotoh, et al., 2001). This isoform is identical to ZAKα
from the N-terminus to the leucine zipper domain but lacks the SAM domain (Gallo and
Johnson, 2002). Similar to other MAP3Ks, ZAKα activates p38, JNK and ERK
pathways. In lung cancer cells, overexpression of ZAKα suppresses proliferation and AP1 transcriptional activity in a JNK- and ERK-dependent manner (Yang, et al., 2010).
ZAKα was shown to be required for doxorubicin-induced pro-inflammatory and proapoptotic responses in HaCaT cells, a pseudo-normal keratinocyte cell line, in a
mechanism that is mediated by p38 and JNK signaling (Sauter, et al., 2010). However, an
oncogenic function for ZAKα was also demonstrated (Cho, et al., 2004). For instance,
injecting athymic nude mice with cells stably expressing ZAKα results in the formation
of tumors (Cho, et al., 2004). In addition, ZAKα induces transformation of neoplastic
cells (Cho, et al., 2004). Thus, ZAKα, through the activation of MAPKs, can either
induce tumorigensis or apoptosis. Similar to ZAKα, ZAKβ can induce p38- and JNKdependent apoptosis upon activation by stresses such as UV irradiation and anisomycin
(Wang, et al., 2005). Moreover, a role for ZAKβ in regulating cardiac function has also
been demonstrated. For instance, in transgenic mice that specifically overexpress ZAKβ
in the cardiac tissue, stimulation of ZAKβ by beta-adrenergic stimuli activates both p38
and JNK and leads to cardiac myocyte hypertrophy as well as mice mortality (Christe, et
30
al., 2004). ZAKβ can also induce JNK activation in response to saturated free fatty acids
or FFA (Jaeschke and Davis, 2007).
1.6 The MLK subfamily
Similar to other MAP3Ks, several members of the MLK subfamily activate
multiple MAPK signaling pathways. The four MLK members (MLK1-MLK4) all share
similar domain structures that consist of an N-terminus Src-homology-3 (SH3) domain, a
kinase domain, a leucine zipper (LZ) domain, a Cdc42/Rac-interacting binding (CRIB)
domain and a proline rich C-terminus (Figure 8) (Gallo and Johnson, 2002). In addition
to these domains, MLK3 is also characterized by a Gly-Pro-rich N-terminus (Gallo and
Johnson, 2002). Unlike the different structural domains that have more than 60%
sequence homology between the four members, the C-terminal domain is highly diverse
(Gallo and Johnson, 2002). The kinase domain of all MLK subfamily members contains a
putative activation region with a TTXXS motif (Leung and Lassam, 2001).
H2N SH3
Kinase
Zipper
CRIB
COOH
Figure 8. The structural domains of MLKs. The four MLK members share
sequentially an N-terminal SH3 domain, a kinase domain, a leucine zipper domain, a
CRIB domain and a proline-rich C-terminal region.
31
1.6.1 MLK1 and MLK2
MLK1 and MLK2 show a more restricted expression than other MLKs, with
MLK1 being mainly found in epithelial cells and MLK2 in the brain as well as in skeletal
tissue (Gallo and Johnson, 2002).
MLK2 strongly activates JNK by directly
phosphorylating and activating MEK4 (Hirai, et al., 1997). Moreover, MLK2 when
overexpressed in COS-1 cells slightly activates the p38 and ERK pathways as well (Hirai,
et al., 1997). In Xenopus, MLK2-induced JNK activation can be inhibited by the p21GTPase activated kinases, PAK1 (Poitras, et al., 2003). MLK2 was also implicated in
neuronal development and survival since it phosphorylates and activates NeuroD (ND), a
transcription factor that functions as a key regulator of these processes (Marcora, et al.,
2003). In a more recent study, MLK2 was found to directly bind to, phosphorylate and
inactivate the transcription factor E47 in a mechanism that is JNK-dependent (Pedraza, et
al., 2009). This leads to a reduction in the protein levels of the TrkB receptor, whose
expression is regulated by E47 (Pedraza, et al., 2009). As for MLK1, very little is known
except for a study that addressed the physiological functions of MLK1 and MLK2
subfamily members in vivo. In this aspect, disrupting the gene encoding MLK1 or MLK2
did not produce any phenotypical or developmental defects in mice (Bisson, et al., 2008).
Moreover, mice with null mutations in both MLK1 and MLK2 were also viable, fertile
and healthy (Bisson, et al., 2008). These results suggest that MLK1 and MLK2 do not
have a significant role in development and may have functions that are compensated for
by other MAP3Ks.
32
1.6.2 MLK3 activation
MLK3 is the best characterized MLK subfamily member. This kinase is
ubiquitously expressed (Gallo and Johnson, 2002). In MLK3, the TTXXS motif (residues
276-281) undergoes autophosphorylation on T277 and S281, a step that is required for
MLK3 activity (Leung and Lassam, 2001). Phosphorylation of these sites by
hematopoietic protein kinase 1 (HPK1), leads to the activation of MLK3 (Leung and
Lassam, 2001). This motif is conserved in the other MLK subfamily members as well,
and it is believed that the kinase activity of other MLKs is also regulated by
autophosphorylation. MLK3 dimerizes through the LZ domain (Leung and Lassam,
2001;
Vacratsis
and
Gallo,
2000).
Dimerization
is
not
a prerequisite for
autophosphorylation but it is required for MLK3-mediated activation of JNK signaling
(Gallo and Johnson, 2002). In the absence of a stimulant, the N-terminal SH3 domain of
MLK3 binds to a single proline residue that lies between the LZ and the CRIB domain,
and maintains MLK3 in an inactive state by preventing dimerization and
autophosphorylation (Figure 9) (Zhang and Gallo, 2001). Members of the Rho family of
GTPases, such as Cdc42, interact with and activate MLK3 (Bock, et al., 2000; Teramoto,
et al., 1996). Co-expressing active Cdc42 with MLK3 results in oligomerization and
enhanced activation of MLK3 (Bock, et al., 2000; Leung and Lassam, 2001; Teramoto, et
al., 1996). Moreover, both active Cdc42 and Rac were found to bind to the CRIB motif of
MLK3 (Burbelo, et al., 1995). Although the exact mechanism by which Cdc42 or Rac
activate MLK3 is not well understood, the proposed model suggests that upon binding of
active Cdc42 or Rac to the CRIB domain of MLK3, the autoinhibitory effect of the SH3
domain is relieved, thereby promoting MLK3 dimerization, autophosphorylation and
33
subsequent activation (Figure 9) (Gallo and Johnson, 2002). Active Cdc42 and Rac also
function to localize MLK3 to the plasma membrane and other cellular membrane
compartments where it mediates spatial MAPK signaling (Du, et al., 2005; Gallo and
Johnson, 2002). In addition to Cdc42 and Rac, the protein kinase B or Akt1 also plays a
role in regulating MLK3 activity by phosphorylation of MLK3 on Ser674, which inhibits
MLK3 kinase activity (Barthwal, et al., 2003). Another physiological inhibitor of MLK3
is the tumor suppressor Neurofibromatosis-2 (NF-2) protein, merlin (Zhan, et al., 2011).
The proline-rich MLK C-terminus has a cluster of phosphorylation sites that are
potential targets for the SH3-containing kinases, such as MAPKs and cyclin-dependent
kinases (Phelan, et al., 2001; Vacratsis, et al., 2002). A consensus sequence for MAPK
phosphorylation (PXS/TP) is present in the MLK3 C-terminus and is phosphorylated by
JNK (Schachter, et al., 2006). This phosphorylation triggers a positive feedback effect
that leads to MLK3 activation and hence sustained JNK signaling (Schachter, et al.,
2006). Other than MLK3, JNK was also found to associate with and phosphorylate
MLK2 (Sakuma, et al., 1997).
34
Figure 9. Model mechanism of MLK3 activation by Cdc42 (adapted from (Gallo
and Johnson, 2002)). The N-terminal SH3 domain of MLK3 has an autoinhibitory effect
and maintains MLK3 in an inactive form. Binding of GTP-bound Cdc42 to the CRIB
domain relieves this autoinhibitory effect which subsequently allows MLK3
dimerization, autophosphorylation and activation. P = Proline residue.
35
1.7 MLK3 signaling
Of the four MLK subfamily members, MLK3 has been best characterized in terms
of its biological and biochemical function. MLK3, like other MAP3Ks, is activated by
stress, cytokines, and ceramides, but, how these different stimuli activate MLK3 is still
unclear (Kyriakis and Avruch, 2001). MLK3, once activated by dimerization and
autophosphorylation, phosphorylates and activates the MAP2Ks, MEK4/7 and MEK3/6,
that in turn phosphorylate and activate JNK and p38, respectively (Tibbles, et al., 1996).
The activated MAPKs then activate various transcription factors involved in mediating
different cellular responses such as proliferation, inflammation and apoptosis (Gallo and
Johnson, 2002). For instance, MLK3 activates MEK7 and triggers JNK3-dependent cell
death in the rat hippocampus (Pan, et al., 2005). MLK3 overexpression induces JNKdependent apoptosis in superior cervical ganglion (SCG) neurons, and withdrawal of
neuron growth factor (NGF) activates and induces apoptosis (Mota, et al., 2001). In
accordance with such a role, transient expression of wild-type MLK3 (MLK3-WT) or
kinase dead MLK3 (MLK3-KD) induced poly (ADP-ribose) polymerase (PARP)
cleavage and enhanced etoposide-induced cell death in HEK293 cells (Cole, et al., 2009).
However, MLK3-WT enhances etoposide-induced cell death to a greater extent than
MLK3-KD, suggesting an important function for the kinase activity of MLK3 in this
mechanism (Cole, et al., 2009). Moreover, knocking down MLK3 elevated the level of
active IKK in NIH3T3 and HEK293 cells, demonstrating an inhibitory role for MLK3 on
IKK activity (Cole, et al., 2009). A recent study also established a role for MLK3 in
intestinal epithelial cell migration (Kovalenko, et al., 2012). This was demonstrated in the
epithelial intestinal Caco-2 cells, whereby, treating the cells with the MLK inhibitors,
36
CEP-1347 and CEP-11004, reduced ERK and JNK signaling and migration of cells
across collagen substrates (Kovalenko, et al., 2012). Thus, both the stimulus and cell type
dictate the signaling pathway activated by MLK3 and the resulting cellular response.
A few studies have also addressed the role of MLK3 in vivo. Similar to MLK1-//MLK2-/- mice, mice deficient in MLK3 are viable and had a similar life span to wild-type
mice and only showed minor morphological defects indicated by a reduction in the
thickness of the dorsal epidermal tissues (Brancho, et al., 2005). MLK3-/- mice also
displayed defects only in TNF-induced JNK activation suggesting a role for MLK3 in
mediating this pathway (Brancho, et al., 2005). More recently, targeted deletion of MLK3
in mice resulted in reduced bone mass and several other skeletal defects (Zou, et al.,
2011). Similar effects were observed in mice expressing a mutant allele of mlk3 that is
incapable of activation by FYVE, RhoGEF, and PH domain-containing 1 (FGD1), which
was shown in vitro to act as an upstream activator of MLK3 (Zou, et al., 2011). These
results demonstrate a role for MLK3 in mediating FGD1 regulatory functions in bone
development (Zou, et al., 2011). Furthermore, MLK3 knockout mice revealed reduced
jejunal mucosal ulcer healing, as compared to wild-type mice, that was also associated
with a decrease in ERK and JNK signaling (Kovalenko, et al., 2012). These observations
also implicate a role for MLK3 in regulating intestinal ulcer healing in vivo (Kovalenko,
et al., 2012). In addition to all these functions, MLK3 was also shown to play important
roles in cancer.
37
1.7.1 MLK3 signaling in cancer
Cancer is a multistep process that is initiated by alterations in one or more
essential genes such as tumor suppressor genes or oncogenes leading to uncontrolled cell
proliferation and formation of tumors (Hanahan and Weinberg, 2011). Additional
genetic/epigenetic modifications promote tumor cells to undergo different processes such
as transformation, migration and angiogenesis all of which allow tumors to gain
metastatic potential (Hanahan and Weinberg, 2011; Umar, et al., 2002). Interestingly, a
role for MLK3 in the induction of apoptosis was characterized in several tumor cell lines.
In FaO rat and Hep3B human hepatoma cells, MLK3 activates the p38 but not the JNK
apoptotic pathway (Kim, et al., 2004). In the human ovarian cancer epithelial cells,
SKOV3, silencing MLK3 reduced the protein levels of the NF-κB inhibitor, the inhibitor
of kappa B alpha (IκBα), implicating MLK3 as a negative regulator of the NF-κB cell
survival pathway in these cells (Cole, et al., 2009).
A role for MLK3 in cell proliferation has also been documented in fibroblasts and
tumor cells with mutations in the oncogene K-ras or loss of function mutations in
neurofibromatosis-1 (NF1) or NF2, through activation of JNK or p38 and ERK,
respectively (Brancho, et al., 2005; Chadee and Kyriakis, 2004; Hartkamp, et al., 1999) .
A hallmark of malignant tumors is their invasive capacity (Hanahan and Weinberg,
2011). A role for MLK3 in cell invasion was also implicated. In this respect, MLK3 was
shown to be essential for JNK/AP-1-mediated breast cancer cell migration and invasion
(Chen, et al., 2010). A requirement for MLK3 in cell migration was also demonstrated in
gastric tumor cells and the lung cell line, A459 (Mishra, et al., 2010; Swenson-Fields, et
al., 2008). Recently, MLK3 was also found to promote tumorigenesis in vivo (Cronan, et
38
al., 2012). Orthotopic xenograft models generated from stable MLK3 shRNA-knockdown
MDA-MB-231 cells, exhibited a reduction in tumor growth and metastasis as compared
to control models (Cronan, et al., 2012). Thus, active MLK3, through activation of JNK,
p38 or ERK can induce proliferation, invasion and cell survival pathways in a stimulus
and cell type-specific manner.
1.8 MLK4: characteristics and function
MLK4 represents one of the least characterized members of the MLK family. In
one report, MLK4 was found to be mutated in 6.8% of the colorectal cancers analyzed
(Bardelli, et al., 2003). In half of these cases, the mutations were found in exon 1 or exon
2 that encode for the kinase domain (Bardelli, et al., 2003). However, another study
reported no observed MLK4 mutations in any of the 46 colorectal cancer samples tested
and out of the 24 different colorectal cancer cell lines screened, the mutation (R470C)
was present in only two of the cell lines, LoVo and CaR1 (Shao, et al., 2007). A recent
study showed that while wild-type MLK4 exhibits low kinase activity, the R470C
mutation along with the H261Y, G291E and R555Stp mutations significantly enhanced
the kinase activity (Martini et al. 2013). The frequency of these mutations, however, is
very low (Martini et al. 2013). Moreover, no MLK4 kinase domain mutations were
detected in gastric and hepatocellular carcinomas (Soung, et al., 2006). Therefore, the
relationship between MLK4 mutation and cancer development remains elusive.
MLK4 exists in two forms, α (580 aa) and β (1036 aa), that are generated by
alternative splicing. The two isoforms are identical in all structural domains except for
the C-terminal region (Kashuba, et al. 2011). Expression patterns of MLK4α and MLK4β
39
revealed that although the mRNA expression of both forms was detected in the pancreas,
kidney, liver, lung and brain, MLK4α exhibited a lower level of expression in comparison
to MLK4β (Kashuba, et al. 2011). However, skeletal muscle only expressed MLK4β
(Kashuba, et al. 2011). Overexpression experiments revealed differences in the
intracellular localization between the two isoforms with MLK4α being found only in the
cytoplasm and MLK4β in both the cytoplasm and the nucleus (Kashuba, et al. 2011).
Although transgenic mice expressing MLK4β showed no phenotypic abnormalities,
MCF7 cells with ectopic expression of either MLK4α or MLK4β exhibited reduced
colony formation and cell proliferation (Kashuba, et al. 2011). Nonetheless, the
biochemical and biological function of MLK4 remains largely unidentified.
MLK4
shares >70% sequence homology with MLK3 in the kinase catalytic domain (Kashuba, et
al. 2011). This might suggest that MLK4, like MLK3, functions as an upstream activator
of MAPK signaling. However, in a recent study, MLK4 was found to negatively regulate
Toll-like receptor 4 (TLR4) signaling by inhibiting LPS-induced JNK and ERK
activation (Seit-Nebi, et al., 2012). Therefore, further elucidating the signaling specificity
of MLK4 is critical in unraveling its biological significance and determining whether it
adopts similar functions to MLK3 and other MAP3Ks.
1.9 Significance
Many MAP3Ks, being activators of MAPK signaling, have pivotal roles in cancer
and other pathologies. Increasing evidence implicates a role for MLK3 in different stages
of cancer, yet very little is known about the other members of the MLK subfamily
40
members. Unraveling the biological functions of the other MLKs is essential to reveal
any contributions to cancer and other diseases and can thus provide insights into new
therapeutic approaches. Elucidating the role of MLK4, one of the least characterized
MAP3Ks, is in line with this goal. Therefore, this study is directed at identifying the role
of MLK4β in MAPK signaling and ovarian cancer.
41
Chapter 2
Materials and Methods
2.1 Cell culture
Human colon cancer (HCT116 and HT-29), ovarian cancer (SKOV3) cells,
human embryonic kidney (HEK293) and human glioblastoma (T98G) cells were obtained
from the American Type Culture Collection, Manassas, VA, USA. The ovarian cancer
cell line, HEY1B, was a gift from Dr. Douglas Leaman (The University of Toledo). T80
cells are immortalized human ovarian epithelial cells (Liu, et al., 2004). HCT116,
SKOV3 and HEY1B cells were cultured in growth media made from DMEM
(Mediatech, Herndon, VA, USA) and supplemented with 10% FBS (fetal bovine serum)
(Hyclone, Logan, UT, USA). T80 cells were cultured in medium 199 (Mediatech) with
10% MCDB 105 (Sigma-Aldrich, St. Louis, MO, USA) and 10% FBS. All tissue culture
media were supplemented with 25 µg/ml streptomycin and 25 I.U.
penicillin
(Mediatech). Cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C. For
treatments, HCT116 cells were treated with 0.5 M sorbitol (Fisher Scientific, Fairlawn,
NJ, USA).
42
2.2 Expression Vectors
The mammalian expression vectors used in this study are as follows:
pCMV5-FLAG empty vector, cDNA constructs for expression of human MAPKs:
pCMV-GST-MLK3,
pCMV5-FLAG-MLK3,
pCMV-FLAG-MLK4β,
pCMV-HA-
MLK4β, pCDNA3-HA-ASK1, and pEBG-SEK-1-KR (GST fusion protein).
2.3 Plasmids and siRNA transfections
Transient transfections were performed using either Lipofectamine (Invitrogen,
Frederick, MD, USA) or PolyJetTM In Vitro DNA Transfection Reagent (SignaGen
Laboratories, Rockville, MD, USA). Cells were plated in 6 cm dishes and transiently
transfected when they reached 50-60% confluency. For Lipofectamine transfection,
depending on the plasmid used, (0.7-2) μg of DNA was added to 800 μl of serum-free
media. 8 μl of Lipofectamine was then mixed with the DNA suspension and incubated for
15 min. The cells were washed once with serum-free media and then 1.7 ml of serum-free
media was added to dish. The DNA-Lipofectamine mixture was then added to the cells.
The cells were returned to the incubator for 4-5 hours (h). After the incubation, 2.5 ml of
media containing 20% FBS was added to each dish and the cells were incubated for at
least 16 hours before harvesting. For 10 cm dishes, (1.4-4) μg of DNA and 16 μl of
Lipofectamine in 1.6 ml were used. Transfection using PolyJet was performed as
suggested by the manufacturer. Briefly, 0.7 – 2 μg and 1.4 – 4 μg of DNA was added to
100 μl (for 6 cm dishes) and 250 μl (for 10 cm dishes) of serum-free media, respectively.
In a separate tube containing these same volumes of serum-free media, 5 μl and 10 μl of
Polyjet was added, respectively. The Polyjet-media mix was then added to the DNA
43
suspension, mixed and incubated for 15 min. 2.8 ml and 5 ml of growth media were
added to 6 cm and 10 cm plates, respectively. Two hundred μl (6 cm dish), or 500 μl (10
cm dish) of the Polyjet-DNA complex solution was then added dropwise to each dish.
Transfected cells were incubated for at 18 hours before harvesting.
SiRNA transfection was carried out using either Lipofectamine 2000 (Invitrogen,
Frederick, MD, USA) or GeneMute (SignaGen Laboratories, Rockville, MD, USA). For
Lipofectamine 2000 transfection, 100 nM of siRNA and 8 μl of Lipofectamine 2000 were
used and transfection was performed as described earlier. For GeneMute transfection,
100 nM siRNA and 9 μl of the GeneMute reagent were added to 300 μl of 1X
transfection buffer; For 10 cm dishes, 100 nM siRNA and 24 μl of GeneMute were added
to 800 μl of the transfection buffer. The transfection mixtures were mixed well and
incubated for 15 min. Cells were washed with 1X PBS and 3 ml and 8 ml of growth
media were added to 6 cm and 10 cm plates, respectively. The transfection solution was
then added to the cells and the cells were placed in the incubator for 15 min after which
the transfection media was replaced with fresh full growth media. The cells were then
incubated for at least 16 hours before harvesting. SiRNA in both transfection techniques
was added to a final concentration of 100 nM. The siRNA oligonucleotide sequences are
as follows: human MLK4 siRNA1: 5’-GGGCAGTGATGACTGAGAT-3’ corresponding
to nucleotides 1469-1487; human MLK4 siRNA2 (Ambion, Life Technologies, Grand
Island, NY, USA): 5’-GGACCACCAAAATGAGCAC-3’ corresponding to nucleotides
903-923;
human
MLK3
siRNA1
(Chadee
and
Kyriakis,
2004):
5’-
GGGCAGTGACGTCTGGAGTTT-3’ corresponding to nucleotides 1198-1218; human
MLK3 siRNA2: 5’-AAGCGCGAGATCCAGGGTCTC-3’ corresponding to nucleotides
44
1154-1172; non-targeting siRNA that is used as a control (Dharmacon, Lafayette, CO,
USA): 5’-AATCCGGTTGCATAGTTCATC-3’.
2.4 Immunoblotting
Protein expression was studied by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblotting. For this purpose, 40 ml of 15%
polyacrylamide running gel solution consisting of 20 ml of bis acrylamide (29.8 g
acrylamide and 0.2 g bisacrylamide in 100 ml H2O), 10 ml of 4X Tris-Cl/SDS, pH 8.8
(0.5 M Tris base, 13.87 mM SDS), 160 μl of 50% ammonium persulfate (APS), 16 μl of
TEMED and 10 ml of H2O were prepared. The stacking gels were prepared using 1.3 ml
of 30% acrylamide (29.2 g acrylamide and 0.8 g bisacrylamide in 100 ml H2O), 2.5 ml of
4X Tris-Cl/SDS, pH 6.8, 70 μl of 50% APS, 20 μl of TEMED and 12.2 ml of H2O.
The gels were placed in running buffer (25 mM Tris base, 192 mM glycine and
6.94 mM SDS) and electrophoresis was carried out overnight at 60 volts. Proteins were
then transferred to Immobilon-P Polyvinylidene Fluoride (PVDF) membranes at 57 V for
2 hours using transfer buffer (25 mM Tris base, 192 mM glycine and 20% methanol).
After transfer, membranes were stained with Brilliant Blue R250 solution (40% (v/v)
methanol, 10% (v/v) acetic acid and 0.4 g Brilliant Blue R250) and destained with 70%
(v/v) methanol solution. The membranes were then blocked for an hour on a rotator with
5% (w/v) nonfat dry milk in 1X PBS (prepared from 10X PBS: 137 mM NaCl, 27 mM
KCl, 43 mM KH2PO4 and 14 mM Na2HPO4 pH 7.4). Following blocking, the membranes
were incubated with primary antibodies prepared at a 1:1000 dilution in antibody buffer
that consists of 1X PBS, 0.05% (v/v) (Tween 20, and 5% (w/v) nonfat dry milk. For
45
phosphospecific antibodies, 5% bovine serum albumin (BSA) was used instead of milk in
the antibody buffer. The membranes were then rotated overnight at 4 °C. The next
morning, the membranes were washed in 1X PBS containing 0.05% (v/v) Tween 20, 4
times each for 15 min. Membranes were then incubated in secondary antibody for 2 hours
at room temperature with rotation. The secondary antibody was prepared at a 1:5000
dilution in 5% (w/v) nonfat dry milk dissolved in 1X PBS with 0.05% (v/v) Tween 20
(PBST). The membranes were then washed 4 times in PBST. Protein expression was
detected by developing the membranes with Immobilon-P development solution
(Millipore, Billerica, MA, USA) or with Enhanced Chemiluminescence (ECL) solution.
Immunoblotting was performed with the following antibodies: MLK3 (C-20), p38
(C-20), ERK2 (C-14), JNK (C-17), GST (Z-5), IκBα (C-21), β-Actin (C-4) (Santa Cruz
Biotechnology, Santa Cruz, CA, USA). The MLK4 antibody specifically recognizes the
C-terminus of MLK4β and does not cross-react with MLK4α (Novus Biologicals,
Littleton, CO, USA). Activation-state p-p38 (Thr180/Tyr182), p-ERK (Thr202/Tyr204),
p-JNK (Thr183/Tyr185), p-MLK3 (Thr277/Ser281), p-MEK3/MEK6 (Ser189/207) and
p-SEK1 (Thr261) antibodies were from Cell Signaling Technology (Beverly, MA, USA).
FLAG antibody was from Stratagene (La Jolla, CA, USA). Anti-MEK6 antibody was
from Stressgen Bioreagents Corporation (Victoria, British Columbia, Canada). Anti-HA
monoclonal antibody was prepared from 12CA5 B-cells. The secondary antibodies were
Immun-Star Goat Anti-Mouse (GAM) – Horseradish Peroxidase (HRP), Immun-Star
Goat Anti-Rabbit (GAR) – HRP Conjugate (Bio-Rad, Alfred Nobel Drive, CA, USA)
and Peroxidase-IgG fraction monoclonal mouse Anti-rabbit IgG, light chain specific
(MAR) (Jackson Immunoresearch Laboratories Inc., Baltimore, PA, USA).
46
2.5 Preparation of whole cell extracts and treatments
Whole cell extracts were prepared by first washing the cells with 1X PBS and
then scraping them into 6X SDS sample buffer (300 μl and 600 μl for 6 cm and 10 cm
dishes, respectively) and transferring them to 1.5 ml microcentrifuge tubes. The sample
buffer consisted of 70% (v/v) of 1M Tris-Cl, pH 6.8, 30% (v/v) glycerol, 346.8 mM SDS
and 5% (v/v) β-mercaptoethanol.
Some experiments involved treating the cells with TNFα (Biosource, Camarillo,
CA, USA) or the ERK or JNK inhibitors. TNFα, treatments were performed at a final
concentration of 10 nM or 10 ng/ml and depending on the experiment, the cells were
treated for 10 min, 20 min or 6 h. For inhibiting ERK and JNK activities, U0126 and
SP600125 were used, respectively. The inhibitors were added to the cells at a final
concentration of 10 μM for U0126 (LC Laboratories, Woburn, MA, USA) and 50 μM for
SP600125 (ENZO Life Sciences, Farmingdale, NY, USA), and cells were incubated with
the inhibitors for 4 h. The same volume of the vehicle, DMSO, was added to one dish as
a negative control.
2.6 Immunoprecipitation
Immunoprecipitation of endogenous or overexpressed proteins was prepared by
lysing the cells on ice with 1 ml of lysis buffer that consists of 20 mM Tris, pH 7.4, 2
mM EGTA, 10 mM MgCl2, 0.1% β-mercaptoethanol, 1% Triton X-100, 100 μM
phenylmethane sulphonyl fluoride (PMSF), 2 μM leupeptin and 2 μM pepstatin, 1 μM
aprotinin. The lysed cells were collected in 1.5 ml microcentrifuge tubes and centrifuged
for 10 min at 13,000 rpm. 350 μl of the lysates were set aside for analysis by
47
immunoblotting. In a separate tube, the remaining 650 μl of the cell lysates were added to
35 μl of Protein-G sepharose beads (Pierce, Thermo Fisher Scientific Inc.) and 35 μl of
anti-HA antibody. For GST pull-downs, 650 μl of cell lysates were added to 35 μl of
glutathione-sepharose beads (Pierce, Thermo Fisher Scientific Inc.). Protein samples
were rotated overnight at 4°C. The beads were then pelleted by centrifugation at 13,000
rpm for 2 min and the supernatant was discarded. The beads were then washed once with
high salt wash buffer (20 mM Tris pH 7.4, 2 mM EGTA, 10 mM MgCl2, 0.1 (v/v) % βmercaptoethanol, 0.1 % (v/v) Triton X-100, 100 μM PMSF, 1 μM aprotinin, 2 μM
leupeptin, 2 μM pepstatin and 250 mM LiCl) and twice with wash buffer (same as high
salt wash buffer but lacks the LiCl). The beads were then suspended with 35 μl of 6X
SDS sample buffer and boiled for 5 min before analysis SDS-PAGE.
2.7 MLK4 kinase assay
Lysates were prepared from HCT116 cells overexpressing human MLK4 (HAMLK4 or FLAG-MLK4), the negative control (pCMV5-FLAG) and the positive controls
(HA-ASK1 or FLAG-MLK3). The lysis buffer consisted of 20 mM Hepes, pH 7.4, 2 mM
EGTA, 50 mM β-glycerophosphate, 1 mM DTT, 1 mM Na3VO4 (prepared from a 20 mM
stock that is heat-activated), 1% (v/v) Triton X-100, 10% (v/v) glycerol, 2 μM leupeptin,
1:1000 aprotinin and 400 μM PMSF. The lysates were then centrifuged at 13,000 rpm for
10 min. Three hundred and fifty μl of the lysates were kept for immunoblotting and the
remaining 650 μl were added to 35 μl of Protein-G beads sepharose beads with either 35
μl of anti-HA or 0.5 μl of anti-FLAG antibody. Immunoprecipitation was performed by
rotating the samples at 4°C for an hour and a half. The beads were then pelleted by
48
centrifugation for 2 min at 13,000 rpm. The supernatant was then discarded and the beads
were washed once with lysis buffer, once with high salt wash buffer (500 mM LiCl, 100
mM Tris-Cl, pH 7.6, 0.1 % (v/v) Triton X-100, 1mM DTT) and three times in assay
buffer (20 mM Mops, pH 7.2, 2 mM EGTA, 10 mM MgCl2, 1 mM DTT, 0.1% (v/v)
Triton X-100). After the last wash, beads were resuspended in 40 μl of assay buffer. The
substrate (GST-SEK1-KR or the human H2A histone (New England, Biolabs, Ipswich,
MA, USA)) in 18 μl of assay buffer and 15 μl of Mg-ATP mix (50 mM MgCl2 and 0.5
mM ATP) were then added to each tube. For the radioactive kinase assay, 57 μCi of γ32p
[ATP] (PerkinElmer, Waltham, MA, USA) were added to the Mg-ATP mix before
addition to each tube. The samples were then incubated for 30 min at 30 °C with shaking
at 450 rpm. Twenty μl of 6X SDS buffer was then added to the samples and they were
boiled for 5 min and then analyzed by 15% SDS-PAGE. The MLK4 kinase activity was
detected by probing with phospho-SEK1 antibody and autoradiography.
2.8 Cell proliferation assay
HCT116 were either transfected with nonspecific or MLK4 siRNA oligo. 18-20
hours post-transfection, 10,000 cells/well, from each sample, were seeded in 24 well
plates. The effect of MLK4 on cell proliferation was then studied by using the MTT
(Thiazolyl blue tetrazolium bromide) assay (Sigma-Aldrich, Saint Louis, MO, USA) at
days 0, 3 and 4 as described by the manufacturer. Briefly, on the first day after plating, 25
μl of MTT solution (50 mg of MTT reagent dissolved in 10 ml of 1x PBS pH 7.4) was
added in each well. The plates were then incubated at 37 °C for 2 hours. 500 μl of MTT
buffer (isopropanol with 10% (v/v) Triton X-100 and 0.1 N HCl) was then added to each
49
well and the contents of the wells were mixed thoroughly by repeated pipetting. The
plates were incubated at room temperature on the shaker for half an hour after which
absorbance of MTT formazan, that is generated by live cells, was measured at a
wavelength of 570 nm on an ELISA plate reader. The graphs represent the fold change in
proliferation over time. All experiments were run in triplicate and repeated twice.
Statistical analysis of data was performed by using the student t-test.
2.9 Luciferase assay
The luciferase assay was used to determine the effect of MLK4β on NF-κB
activity in HEK293 cells. In this assay, the reporter plasmid 3XkappaB-Luc (a gift from
Dr. Brian Ashburner, University of Toledo) was used. This reporter contains three copies
of the DNA sequence that encodes for the NF-κB binding site, in the major
histocomptability complex class I gene, upstream of the luciferase reporter gene. Briefly,
cells were seeded in 12-well plates and 24 hours later were transfected with either the
empty vector, 100 ng of FLAG-MLK4β or 200 ng FLAG-MLK4β together with 100 ng
3XkappaB-Luc reporter plasmid and 100 ng β-Gal plasmid using Polyjet as described
earlier. 18 h later, the cells were either left untreated or treated with 10 ng/ml of TNF-α
for 6 h. The luciferase assays were then performed (Promega Biosciences, CA, USA).
Luciferase activity was normalized to β-Gal activity. The assays were all run in triplicate.
2.10 Invasion assays
Cell invasion assays were performed with 100 μl of 1 mg/ml BD matrigel matrix
(BD Bioscience, San Jose, CA, USA), mixed with serum free media, in 24-well
50
Transwells with 8.0 μm pore size and 24 mm diameter polycarbonate membrane
(Corning, Acton, MA, USA). Cells were washed in DMEM containing 0.5% (v/v) FBS.
10,000 cells were seeded onto the upper chamber. Matrigel and cells remaining in the
upper chamber were removed after 36 h incubation. The cells on the underside surface of
the membrane were fixed using the Diff Quick Stain Kit (Fisher Scientific, Pittsburgh,
PA, USA) and the number of cells per field of view was counted. The average of number
of cells from four different fields of view was calculated. All experiments were run in
triplicate and repeated three times.
2.11 Gelatin zymography
MMP-2 and MMP-9 enzymatic activities in the cell culture medium were
determined by SDS-PAGE gelatin zymography. Cells were seeded at 5x105 cells per 6
cm dish. Conditioned cell culture medium from each dish was normalized to an equal
amount of protein (100μg), denatured in the sample buffer in the absence of reducing
agents, and subjected to 10% SDS-PAGE containing 0.1% (w/v) gelatin (Acros Organics,
Morris Plains, New Jersey, USA). The gel was incubated in the presence of 2.5% (v/v)
Triton X-100 at room temperature for 2 h and then at 37 °C for 40 h in digestion buffer
containing 1% (v/v) Triton X-100, 10 mM CaCl2, 0.15M NaCl, and 50 mM Tris (pH 7.5).
Gels were stained with Coomassie Blue R-250 and destained with a buffer containing
10% (v/v) methanol and 5% (v/v) acetic acid. Proteolysis was detected as a white band
against a blue background (Schmalfeldt, et al., 2001).
51
Chapter 3
Results
3.1 The role of MLK4β in p38 signaling
p38 is activated in response to different stresses and pro-inflammatory cytokines
(Raingeaud, et al., 1995). Once activated, p38 regulates a variety of cellular responses
including invasion, inflammation and transformation (Hanahan and Weinberg, 2011; Lee,
et al., 1994; Shin, et al., 2005). Multiple MAP3Ks, including MLK3, mediate stimulusinduced p38 activation (Cuenda and Rousseau, 2007; Kim, et al., 2004; Tibbles, et al.,
1996). However, whether MLK4β affects p38 signaling is yet unknown.
3.1.1 The effect of ectopic expression of MLK4β on p38 activation
MLK4β shares high sequence homology with MLK3 (Kashuba et al. 2011).
Therefore, we postulated that MLK4β, similar to MLK3 and other MAP3Ks, is an
upstream activator of the p38 pathway. To test this possibility, MLK4β was ectopically
expressed in HCT116 cells, human colon cancer cells that constitutively express active
Ras, and the levels of active p38 were analyzed using an activation state phospho-specific
p38 antibody (Thr180/Tyr182; phosphorylated-p38 (p-p38)). Surprisingly, cells
overexpressing MLK4β had substantially reduced levels of p-p38 in comparison to that
52
observed in cells transfected with an empty vector, while total p38 levels were unaffected
(Figure 10A, left panel). External osmolarity conditions are critical for cell integrity and
any changes, whether hypo- or hyperosmotic, induce osmotic stress that affects different
cellular processes, including cell survival (Lezama, et al., 2005). The p38 pathway is
activated in response to the hyperosmotic stress inducer, sorbitol (Chan, et al., 2008). To
determine whether MLK4β overexpression would affect sorbitol-induced p38 activation,
HCT116 cells were treated with sorbitol. Sorbitol treatment increased the levels of p-p38
in cells transfected with the empty vector (Figure 11A, right panel). MLK4β
overexpression, however, completely blocked p38 activation by sorbitol (Figure 10A,
right panel). These results show a negative regulatory effect for MLK4β on p38 activity.
p38 signaling is also activated by cytokines (Kyriakis and Avruch, 2001;
Raingeaud, et al., 1995). To test whether MLK4β also affects the activation of p38 by
cytokines, HCT116 cells were treated with the pro-inflammatory cytokine, TNFα, and the
effect of MLK4β overexpression on p38 activity was analyzed. Similar to the results
obtained with sorbitol, TNF-α treatment elevated the levels of active p38 in cells
expressing the empty vector alone (Figure 10B). Overexpressing MLK4β reduced the
levels of TNFα-induced p-p38, while the total levels of p38 remained unchanged (Figure
10B). These results suggest that MLK4β negatively regulates sorbitol- and TNFα –
induced p38 activition.
53
A.
B.
Figure 10: MLK4β overexpression inhibits basal and stimulus-induced p38
activation. (A) HCT116 cells were transiently transfected with pCMV5-FLAG or
FLAG-MLK4β and left untreated, or treated with 0.5 M sorbitol for 30 min. Cell lysates
were immunoblotted with FLAG, p38, p-p38 and β-Actin antibodies. (B) HCT116 cells
were transfected as described in (A) and left untreated or treated with 10 μM TNFα for 20
min. Cell lysated were immunoblotted as described in (A).
3.1.2 The effect of endogenous MLK4 on the activation of p38
The data in Figure 10 were obtained upon ectopic expression of MLK4β.
Therefore, in order to confirm that endogenous MLK4 also inhibits p38 activity, small
interfering RNA (siRNA)-mediated knockdown of MLK4 in HCT116 cells was
performed and the levels of active p38 were analyzed. MLK4-knockdown cells had
elevated levels of p-p38 as compared with cells treated with a non-specific siRNA oligo,
while the total p38 levels remained unchanged (Figure 11, left panel). We also tested
whether MLK4 knockdown would affect sorbitol-induced p38 activation. Sorbitol
54
treatment increased the basal level of p-p38 (Figure 11, right panel). A further increase in
the level of p-p38 was observed in the MLK4-knockdown cells as compared with cells
treated with the nonspecific siRNA oligo (Figure 12, right panel). These results, together
with the results in Figure 10, indicate that MLK4β negatively regulates basal, sorbitoland TNFα-induced activation of p38.
Figure 11.
Elevated active p38 in MLK4 knockdown cells. HCT116 cells were
transfected with nonspecific or MLK4 siRNA. The cells were left untreated (left panel) or
treated (right panel) with 0.5 M sorbitol for 30 min. Cell Lysates were then
immunoblotted with MLK4β, p-p38, p38 and β-Actin antibodies.
55
3.2 The effect of MLK4 on MEK3/MEK6 activation
MEK3 and MEK6 are MAP2Ks that specifically phosphorylate and activate p38
(Cuenda, et al., 1997; Raingeaud, et al., 1996). To explore whether MLK4 has any effect
on MEK3 and MEK6 activities, MLK4 was knocked down in HCT116 cells and the
levels of active MEK3/MEK6 phosphorylated at serines 187 and 207 were analyzed
using an activation state specific antibody that detects these phosphorylation sites (pMEK3/MEK6). The levels of p-MEK3/MEK6 were increased in cells with MLK4
knockdown as compared with cells treated with the nonspecific siRNA (Figure 12A).
This was consistent with the increase in p-p38 levels that was observed in the MLK4knockdown cells (Figure 12A). As expected, the levels of total MEK3/MEK6 and p38
did not change (Figure 12A). Similar results were obtained in ovarian epithelial
carcinoma HEY1B cells (Figure 12B), suggesting that the effect observed on p38 and
MEK3/MEK6 activation by MLK4 is not cell-type specific. These data show that MLK4
inhibits not only active p38 but also the activation of its upstream activator,
MEK3/MEK6.
56
Figure 12. MLK4 negatively regulates MEK3/MEK6 activation. (A) HCT116 cells
and (B) HEY1B cells were transfected either with a nonspecific oligo or with MLK4
siRNA. Cell lysates were subsequently prepared and immunoblotted with MLK4β, p-p38,
p38, p-MEK3/MEK6, MEK3/MEK6 and β-Actin antibodies.
3.3 The role of MLK4β in NF-κB signaling
In addition to activating p38 and JNK, TNFα also activates the NF-κB pathway
(Karin, et al., 2002). As shown earlier, MLK4β reduced TNFα-induced activation of p38.
This prompted us to investigate if MLK4β also affects TNFα-induced NF-κB activation.
NF-κB is sequestered in the cytoplasm in an inactive state by IκBα (Karin, et al., 2002).
In response to TNFα, IκBα is phosphorylated and targeted for degradation, thus leading
57
to NF-κB activation (Karin, et al., 2002). Degradation of IκBα is considered to be an
indicator of NF-κB activity. To test whether MLK4β plays a role in the NF-κB signaling
pathway, MLK4β was ectopically expressed in HCT116 cells and TNFα-induced IκBα
degradation was detected by analyzing the protein levels of IκBα. TNFα treatment
activated the NF-κB pathway as evidenced by the complete abolishment of IκBα (Figure
13A). Untreated cells overexpressing MLK4β had elevated levels of IκBα as compared to
cells expressing the empty vector. However, MLK4β had no effect on TNFα-induced
degradation of IκBα in cells transfected with the empty vector (Figure 13A).
Furthermore, MLK4 knockdown in HCT116 cells reduced the levels of IκBα as
compared with cells treated with a nonspecific siRNA oligo (Figure 13B). IκBα levels
however, were not affected in MLK4-knockdown cells treated with TNFα.
The effect of MLK4β on the NF-κB pathway was further investigated by using an
NF- κB reporter plasmid that was co-transfected into HEK293 cells with the MLK4β
expression vector or the empty vector. TNFα significantly induced expression of the
reporter gene and this induction was not affected by MLK4β expression (Figure 13C).
Consistent with the results in Figure 13A, overexpression of MLK4β reduced the basal
expression levels of the NF-κB reporter gene observed in the sample expressing the
empty vector alone (Figure 13D). This effect however, was not dose-dependent as both
concentrations of the MLK4β expression vector used, induced comparable levels of
reduction in the expression of the reporter gene (Figure 13D). Collectively, these results
show that MLK4β negatively regulates the basal activation of NF-κB but has no effect on
TNFα-induced activation of NF-κB.
58
A.
B.
C.
D.
E.
Figure 13. MLK4β negatively regulates basal NF-κB activation but has no effect on
TNFα-induced NF-κB signaling. (A) HCT116 cells were transiently transfected with
pCMV5-FLAG or FLAG-MLK4β and left untreated or treated with 10 ng/ml TNFα for
10 min. Cell lysates were blotted and immunoblotted with FLAG, IκBα and β-Actin
59
antibodies. (B) HCT116 cells were transfected with nonspecific or MLK4 siRNA. Cell
lysates were then immunoblotted with MLK4β, IκBα and β-Actin antibodies. (C)
HEK293 cells were transfected with the NF-κB reporter plasmid and the β-gal control
reporter plasmid together with the empty vector, 100 ng or 200 ng of FLAG-MLK4β.
The cells were then left untreated or treated with 10 ng/ml of TNFα for 6 h and the
luciferase assay was performed. (D) Untreated samples from (C) are presented in a
separate graph with a different scale. Statistically significant differences from the control
are indicated (*P < 0.01). (E) A portion of the cells from (C) was lysed and
immunoblotted with FLAG and β-Actin antibodies.
3.4 Comparison of the effects of MLK4β and MLK3 on p38 activation
MLK3 activates MEK3 and MEK6, which in turn phosphorylate and activate p38
(Alonso, et al., 2004; Gallo and Johnson, 2002). However, our data thus far indicate that
MLK4β inhibits, rather than activates p38. To confirm that MLK4β, in contrast to MLK3
and other MAP3Ks, is a negative regulator of p38, we compared the effects of MLK3 and
MLK4β expression on p38 signaling. MLK3 or MLK4β were expressed in HCT116 cells
and the levels of p-p38 were analyzed by immunoblotting (Figure 14A). Overexpression
of MLK3 elevated the basal level of p-p38, whereas MLK4β overexpression reduced pp38 levels, as compared with cells expressing the empty vector alone (Figure 14A). The
total p38 levels were unchanged (Figure 14A). Consistent with these results, while
knocking down MLK3 in HCT116 cells had no effect on the basal levels of p-p38,
60
MLK4-knockdown cells displayed higher levels of active p38 as compared with cells
transfected with nonspecific RNA oligo, while the total p38 levels were not affected
(Figure 14B). These data suggest that unlike MLK3, MLK4β is a potent inhibitor of p38
signaling.
Figure 14. MLKβ, unlike MLK3, inhibits activation of p38 (A) HCT116 were
transiently expressed with pCMV5-FLAG, FLAG-MLK3 or FLAG-MLK4β. Cell lysates
were prepared and immunoblotted with FLAG, p-p38 and p38 antibodies. (B) HCT116
cells were transfected with nonspecific, MLK3 and MLK4 siRNA oligos. Cell lysates
were immunoblotted with MLK4β, MLK3, p-p38, p38 and β-Actin antibodies.
61
3.5 The effects of MLK3 and MLK4 on ERK and JNK activation
MLK3 can mediate multiple cellular processes such as cell proliferation, invasion
and apoptosis, not only by activating p38, but also through the activation of JNK or ERK
(Chadee and Kyriakis, 2004; Chen, et al., 2010). The effect of MLK3 on JNK and ERK
activity was also assessed in this study. MLK3 was ectopically expressed in SKOV3 and
HEY1B and the levels of active ERK and JNK were analyzed using activation state
phospho-specific ERK (Thr202/Tyr204; p-ERK) and JNK (Thr283/Tyr185; p-JNK)
antibodies respectively. As shown in Figure 15, while the total levels of ERK and JNK
remained unchanged upon MLK3 overexpression, the levels of both p-ERK and p-JNK
were elevated in both cells lines as compared with cells transfected with the empty vector
(Figure 15). These results support the previous findings of a role for MLK3 as an
upstream activator of ERK and JNK.
Figure 15. MLK3 promotes the activation of ERK and JNK in SKOV3 and HEY1B
cells. SKOV3 and HEY1B cells were transfected with pCMV5-FLAG or FLAG-MLK3.
Cell extracts were prepared and immunoblotted with FLAG, p-ERK, p-JNK, ERK and
JNK antibodies.
62
Our data so far showed that MLK4β, unlike MLK3, inhibits p38. However, it was
not known whether this inhibitory effect was exclusive for p38 or if it extended to other
MAPKs. Therefore, we set out to test the effect of MLK4 on ERK and JNK. MLK3- or
MLK4-knockdown cells were treated with sorbitol and the levels of the p-JNK and pERK were analyzed. MLK3 knockdown did not affect the sorbitol-induced activity of
any of the three MAPKs tested, whereas MLK4 knockdown increased the levels of not
only p-p38 but also p-JNK and p-ERK, as compared with cells treated with the
nonspecific siRNA (Figure 16A). This suggests that MLK4 also acts as a negative
regulator of ERK and JNK activation.
Knockdown experiments with siRNA can often yield nonspecific effects. To verify
that our data were a result of MLK4 deficiency and not a nonspecific effect of the siRNA
oligo, we repeated the experiments with another siRNA (siRNA2) that targets a sequence
300 bp upstream of the complementary sequence for MLK4 siRNA (1). The second
siRNA oligo was used to knock down MLK4 in HCT116 cells, and the levels of p-p38, pERK and pJNK were analyzed. MLK4-knockdown cells had higher levels of the active
forms of all p38, JNK and ERK as compared with cells transfected with the nonspecific
oligo (Figure 16B). The total levels of p38, ERK and JNK were not changed with the
siRNA treatment (Figure 16B). These results further support a role for MLK4 as a
negative regulator of MAPK signaling.
63
A.
B.
Figure 16. MLK4 negatively regulates ERK and JNK. (A) HCT116 cells were
transfected with nonspecific, MLK3 or MLK4 siRNA, and then treated with 0.5 M
sorbitol for 30 min. Cell lysates were then immunoblotted with MLK4β, MLK3, p-p38,
p38, p-ERK, ERK, p-JNK, JNK and β-Actin antibodies. (B) HCT116 cells were either
transfected with nonspecific siRNA or MLK4 siRNA2. Cell lysates were then prepared
and immunoblotted as described in (A).
64
3.6 MLK4β is not an upstream activator of MEK4
MAP3Ks, when activated by overexpression or other stimuli, trigger different
cellular responses by directly phosphorylating and activating MAP2Ks (Johnson and
Lapadat, 2002). Therefore, one way by which the kinase activity of MAP3Ks can be
studied is by testing their ability to activate MAP2Ks. MLK4β shares more than 70%
homology with MLK3 in the kinase domain and, similar to other MAP3Ks, retains the
essential conserved sequences of serine/threonine kinases (Kashuba, et al. 2011).
However, our data thus far revealed a negative regulatory role for MLK4β on MAPKs
and MEK3/MEK6 and prompted us to investigate whether MLK4β harbors any kinase
activity towards MAP2Ks. MEK4, also known as SEK1, is a MAP2K that is directly
phosphorylated and activated by multiple MAP3Ks, including ASK1 and MLK3 (Ichijo,
et al., 1997; Tibbles, et al., 1996). Therefore, to study the kinase activity of MLK4β, an in
vitro kinase assay was carried out using purified GST-SEK1-K129R, or GST-SEK1-KR,
as a substrate, and ASK1 or MLK3 as a positive control. Either HA-ASK1 or HAMLK4β were ectopically expressed in HCT116 cells, and a kinase assay was performed
(Figure 17A). Cells expressing the empty vector alone were used as a negative control.
Since phosphorylation of Thr261 is required for SEK1 activity, kinase activity was
analyzed by immunoblotting using an activation state phospho-specific SEK1 antibody
(Thr261; p-SEK1). Phosphorylated GST-SEK1-KR was only detected in cells expressing
ASK1 but not in cells expressing MLK4β or the negative control. GST-SEK1-KR total
protein levels were similar in all samples (Figure 17A). To further investigate the kinase
activity of MLK4β, another kinase assay was performed with lysates prepared from
HCT116 cells overexpressing either FLAG-MLK3 or FLAG-MLK4β (Figure 17B). As
65
expected, only with MLK3, phosphorylation of GST-SEK1-KR was observed, and no
phosphorylation of SEK1 was detected in samples with MLK4β or the empty vector
(Figure 17B). These results demonstrate that, unlike ASK1 and MLK3, MLK4β fails to
phosphorylate SEK1 on Thr261, suggesting that MLK4β does not activate SEK1 through
direct phosphorylation of these activation sites.
A.
B.
Figure 17. MLK4β does not phosphorylate Thr261 of GST-SEK1-KR. (A) Lysates
from HCT116 cells transiently expressing pCMV5-FLAG, HA-ASK1 or HA-MLK4β
were prepared and immunoprecipitations of HA-ASK1 and HA-MLK4β were performed
with the HA antibody. A kinase assay was then performed using purified GST-SEK1-KR
as a substrate. The protein samples were then separated by SDS-PAGE and
immunoblotted with p-SEK1 (Thr261) and GST antibodies. (B) HCT116 cells were
transiently transfected with pCMV5-FLAG, FLAG-MLK3 and FLAG-MLK4β and the
cell
lysates
were
prepared
and
FLAG-MLK3
and
FLAG-MLK4β
were
immunoprecipitated with anti-FLAG. The kinase assay and the detection of activity were
performed as described in (A). Overexpression of MLK4β and MLK3 was analyzed by
immunoblotting the cell lysates with HA antibody in (A) and FLAG antibody in (B).
66
3.7 MLK4β kinase activity
As observed in Figure 17, MLK4β was not able to phosphorylate GST-SEK1-KR
on Thr261, which is required for SEK1 activity. Although these results show that MLK4β
does not activate SEK1, they do not give a clear indication about the kinase activity of
MLK4β. Thus MLK4β kinase activity was further investigated by using a radioactive
kinase assay. FLAG-MLK4β and/or FLAG-MLK3 were ectopically expressed in
HEK293 cells and the lysates were subjected to immunoprecipitation. Lysates prepared
from cells expressing the empty vector were used as a negative control. The kinase assay
was then performed using γ32P [ATP] and GST-SEK1-KR or histone H2A as substrates.
MLK4β and MLK3 autophosphorylation, and phosphorylation of the GST-SEK1-KR
substrate were then analyzed. Consistent with the results obtained in Figure 17, 32P-GSTSEK1-KR was only detected in the sample expressing FLAG-MLK3 (Figure 18). These
results indicate that MLK4β fails to phosphorylate GST-SEK1-KR (Figure 18).
Interestingly,
32
P-MLK4β was detected in samples overexpressing MLK4β which may
indicate autophosphorylation of MLK4β, but could also be due to another phosphoprotein that was present in the immunoprecipitation (Figure 18). Autophosphorylation of
MLK3 was also observed (Figure 18). Furthermore, immune complexes prepared with the
FLAG antibody from cells overexpressing FLAG-MLK4β were also able to
phosphorylate H2A (Figure 18). Although this observation could indicate that MLK4β
has kinase activity towards H2A, it is possible other kinases that may have precipitated
with MLK4β could have also phosphorylated H2A in these samples. Expression of both
MLK3 and MLK4β in these samples was analyzed by immunoblotting with anti-FLAG
antibody (Figure 18). The sample where MLK3 and MLK4β were co-expressed showed
67
reduced expression of both kinases as compared to the other samples (Figure 18). Hence,
a conclusion regarding the effect of MLK4β on MLK3 kinase activity cannot be drawn
from this experiment. These findings demonstrate that MLK4β could undergo
autophosphorylation. In addition, the data reveal that MLK4β, unlike other MAP3Ks,
does not phosphorylate and/or activate SEK1, but may harbor kinase activity towards
other substrates.
Figure 18. MLK4β: autophosphorylation and substrate specificity. HEK293 cells
were ectopically expressed with the empty vector, FLAG-MLK3 and/or FLAG-MLK4β.
A kinase assay was performed with γ32P [ATP], with GST-SEK1-KR and histone H2A
being added as substrates in the first four lanes and the last lane, respectively.
Autophosphorylation and substrate phosphorylation were detected using autoradiography.
Samples were then immunoblotted with anti-FLAG and anti-GST antibodies to check for
overexpression and the presence of the GST-SEK1-KR substrate.
68
3.8 The effect of MLK4β on MLK3 activation
MLK3 is an activator of p38, JNK and ERK signaling pathways (Brancho, et al.,
2005; Chadee and Kyriakis, 2004; Gallo and Johnson, 2002; Rana, et al., 1996). Our
results showed that MLK4β negatively regulates MAPK signaling. Thus, we postulated
that MLK4β might negatively regulate MAPK signaling by inhibiting MLK3 activation.
To test this possibility, MLK4β and MLK3 were ectopically expressed in HCT116 cells
either each alone or together and the level of active, phosphorylated MLK3 (p-MLK3)
was analyzed. Interestingly, ectopically expressed MLK3 was active and MLK4β
overexpression with MLK3 completely abolished the levels of p-MLK3, suggesting that
MLK4β inhibits MLK3 activation (Figure 19).
Figure 19. MLK4β inhibits induced MLK3 activation. pCMV5-FLAG, FLAG-MLK3
or FLAG-MLK4β were ectopically expressed in HCT116 cells. Cell lysates were
prepared and immunoblotted with p-MLK3 and FLAG antibodies.
69
The effect of MLK4β on MLK3 activation was also analyzed in another cell line,
SKOV3 cells. SKOV3 cells have a detectable level of active MLK3 (Figure 20A).
Ectopic expression of MLK4β, however, substantially reduced the levels of p-MLK3 that
were observed in cells expressing the empty vector alone, without affecting total MLK3
levels (Figure 20A). This negative effect of MLK4β on MLK3 activation was also
confirmed in SKOV3 MLK4-knockdown cells (Figure 20B). Silencing MLK4 elevated
the levels of p-MLK3 as compared with cells treated with the nonspecific siRNA,
whereas the levels of total MLK3 remained unchanged (Figure 20B). Moreover,
consistent with the results obtained for HCT116 cells, MLK4β expression also reduced pp38 levels in SKOV3 cells (Figure 20A), whereas MLK4 knockdown elevated the basal
levels of active p38 (Figure 20B). The effect of MLK4 on MLK3 and p38 activations was
also studied using MLK4 siRNA2. Knocking down MLK4 in HCT116 cells using the
second oligo also led to elevated levels of both p-MLK3 and p-p38 as compared with
cells treated with the nonspecific oligo without affecting the total levels of either protein
(Figure 20C). The data from both the HCT116 and SKOV3 cell lines reveal an unknown
inhibitory role for MLK4β in MLK3 activation.
70
Figure 20. MLK4 inhibits the basal activation of MLK3 in SKOV3 cells. (A) SKOV3
cells transiently transfected with pCMV5-FLAG or FLAG-MLK4β. Cell lysates were
then prepared and immunoblotted with MLK4β, p-MLK3, MLK3, p-p38 and p38
antibodies. (B) SKOV3 cells were transfected with nonspecific or MLK4 siRNA and cell
lysates were immunoblotted with MLK4β, p-MLK3, MLK3, p-p38, p38 and β-Actin
antibodies. (C) SKOV3 cells were transfected with nonspecific oligo or with MLK4
siRNA2. Cell lysates were immunoblotted as described in (B).
71
3.9 The correlation between MLK4β expression and active MLK3 in different cell lines
MLK3 expression and activity are elevated in breast and ovarian cancer cells
(Chen, et al., 2010; Zhan, et al., 2012). MLK4β, as our data suggest, inhibits MLK3
activity. Thus, we were interested in testing if there is a correlation between the
expression level of MLK4β and MLK3 activity. We analyzed the expression levels of
MLK4β and MLK3, as well as the levels of p-MLK3 in the immortalized epithelial
ovarian cell line, T80; the colon cancer cell lines, HCT116 and HT-29; the ovarian cancer
cell lines, HEY1B and SKOV3; and the glioblastoma cell line, T98G. HCT116, HT-29
and SKOV3 cells displayed higher expression levels of MLK4β as compared to HEY1B
cells which had very low level of expression (Figure 21). T80 and T98G cells expressed
barely detectable levels of MLK4β (Figure 21). No correlation between the expression
levels of MLK4β and MLK3 was observed (Figure 21). Interestingly, low MLK4β
expression was associated with an increased level of p-MLK3 in T80, HEY1B and T98G
cells; and high MLK4β expression was associated with low MLK3 activity in HCT116,
SKOV3 and HT-29 cells (Figure 21). The levels of p-ERK were also analyzed in these
cell lines. Higher levels of p-ERK were detected in the cell lines expressing a high level
of p-MLK3 and a low level of MLK4β (Figure 21). However, high p-ERK was observed
in HEY1B cells as compared to T98G cells, although the latter express higher levels of
active MLK3 (Figure 21). This could be attributed to other upstream activators of ERK
that could be expressed more in HEY1B. Among the cancer cell lines, T98G cells that
expressed the lowest levels of MLK4β harbored the highest levels of active MLK3
(Figure 21). Overall these results suggest that there may be an inverse correlation
between the levels of active MLK3 and MLK4β expression in the cell lines tested.
72
Relative protein level
of ERK
1
0.8
0.6
0.4
0.2
0
Relative protein level of
p-MLK3
Relative protein level
of MLK4β
L.E
0.5
0.4
0.3
0.2
0.1
0
Relative protein level of
MLK3
Relative protein
level of p-ERK
S.E
73
0.2
0.15
0.1
0.05
0
1.5
1
0.5
0
0.8
0.6
0.4
0.2
0
Figure 21. Correlation between MLK4β expression and active MLK3. Protein
extracts were prepared from T80, HCT116, HT-29, HEY1B, SKOV3 and T98G cells.
Cell lysates were then immunoblotted with MLK4β, p-MLK3, MLK3, p-ERK, ERK and
β-Actin antibodies (upper, left panel). Quantification of MLK4β (upper, right panel), pMLK3, MLK3, p-ERK and ERK levels (lower panels) was determined using Image J
software (NIH, Bethesda, Maryland, USA). MLK4β, p-MLK3, MLK3, p-ERK or ERK
levels were normalized to the levels of β-Actin in each sample and the values presented
represent the relative active levels (p-ERK and p-MLK3) or the relative protein
expression levels (MLK4β, MLK3 and ERK) between the different cell lines tested. S.E
stands for short exposure and L.E for long exposure. Quantification of p-MLK3 levels
was done using the L.E p-MLK3 blot.
74
3.10 MLK4β associates with MLK3
MLK4β and MLK3 share > 65% sequence homology in all of the structural
domains, including the leucine zipper domain, while the C-terminal regions are more
divergent (Kashuba et al. 2011). Thus, we postulated that these two proteins may be
associated in cells. To test if MLK4β interacts with MLK3, (GST)-tagged MLK3 and
HA-tagged MLK4β were expressed in HCT116 cells and co-immunoprecipitation
experiments were performed. In cells expressing both GST-MLK3 and HA-MLK4β, coimmunoprecipitation of GST-MLK3 and HA-MLK4β was observed (Figure 22A).
Moreover, MLK3 was not detected in lysates from untransfected cells, cells expressing
the empty vector alone, or cells expressing only GST-MLK3 or HA-MLK4β (Figure
22A). The interaction between MLK3 and MLK4β was also observed in reciprocal
experiments where GST-MLK3 was pulled down and immunoblotting was performed to
detect HA-MLK4β (Figure 22B). Again, MLK4β was not detected in untransfected or
empty vector controls or cells expressing GST-MLK3 or HA-MLK4β (Figure 22B).
These results suggest that GST-MLK3 and HA-MLK4β are associated in cells.
The association observed between overexpressed MLK3 and overexpressed
MLK4β does not necessarily mean that the endogenous proteins interact. To address this
possibility, endogenous MLK4β was immunoprecipitated from HCT116 cells and the
immunoprecipitates were analyzed by immunoblotting (Figure 22C). Indeed, coimmunoprecipitation of endogenous MLK3 and MLK4β was detected (Figure 22C).
Collectively, these results demonstrate that endogenous MLK4β and MLK3 are
associated in cells.
75
A.
B.
C.
Figure 22. MLK4β associates with MLK3. (A) HCT116 cells were transfected with
pCMV5 vector, GST-MLK3, HA-MLK4β or both GST-MLK3 and HA-MLK4β. HAMLK4β was immunoprecipitated from cell lysates with HA antibody. Coimmunoprecipitation of GST-MLK3 was assessed by immunoblotting with MLK3
antibody. HA-MLK4β was immunoprecipitated and immunoblotted with HA antibody to
verify MLK4β expression. (B) GST pull-downs were performed with lysates from the
cells as described in A. The presence of HA-MLK4β and GST-MLK3 in the GST pulldowns was determined by immunoblotting with HA and GST antibodies, respectively.
(C) Endogenous MLK4β was immunoprecipitated from HCT116 cell lysates and
associated endogenous MLK3 was detected by immunoblotting with MLK3 antibody.
The level of endogenous MLK4β in the immunoprecipitation was assessed by
immunoblotting with MLK4 antibody. Control immunoprecipitations were performed
with no antibody or with rabbit IgG.
76
3.11 The effect of MLK4 on cell proliferation
MAP3Ks, including MLK3, through activation of MAPKs, have been reported to
play a role in proliferation (Brancho, et al., 2005; Chadee and Kyriakis, 2004; Dhillon, et
al., 2007). Based on the inhibitory role of MLK4β on MLK3 activity and MAPK
signaling, we postulated that MLK4β would have a negative effect on proliferation. As
shown in Figure 21, HCT116 cells expressed the highest levels of MLK4β among the cell
lines tested. Therefore, to test if MLK4 has any effect on cell proliferation, an MTT assay
was performed with MLK4 knockdown HCT116 cells. MLK4 knockdown HCT116 cells,
similar to cells transfected with the nonspecific oligo, had approximately a 7 fold increase
in cell number 3 days post-seeding. After 4 days, the MLK4 knockdown cells exhibited
approximately a 14 fold increase, which was comparable to that of cells treated with the
nonspecific oligo (Figure 23, upper panel). The extent of MLK4 knockdown was detected
by immunoblotting (Figure 23, lower panel). These results show that MLK4 does not
affect HCT116 cell proliferation. Stable expression of MLK4β in cells, that normally
have low MLKβ and a high level of active MLK3, such as T98G cells, would be a better
approach to elucidate any role for MLK4β in cell proliferation.
77
Fold increase in cell number
18
16
14
12
10
Nonspecific oligo
8
MLK4 siRNA (2)
6
4
2
0
Day 0
Day 3
Day 4
Figure 23. MLK4 has no effect on HCT116 cell proliferation. HCT116 cells were
transfected with either the MLK4 siRNA or the nonspecific oligo. Transfected cells were
then seeded in 24-well plates and cell proliferation was assessed using the MTT assay at
days 0, 3 and 4 (upper panel). MLK4 knockdown was detected by immunoblotting using
MLK4β and β-Actin antibodies (lower panel). Statistical analysis was performed using
the Student t-test.
78
3.12 MLK3 is required for cell invasion in ovarian cancer cells
MLK3, in addition to cell proliferation, is involved in numerous other biological
functions in which a role for MLK4β could be implicated. Cell invasion represents one of
these functions. For instance, MLK3 was shown to be required for cell invasion in
multiple cancer systems, such as breast, gastric and lung cancer cell lines (Chen, et al.,
2010; Mishra, et al., 2010; Swenson-Fields, et al., 2008). The invasive ovarian cancer cell
lines, SKOV3 and HEY1B, express basal levels of active MLK3 which suggests that
MLK3 might play a crucial role in promoting invasion in these cells. To test this
possibility, MLK3 was knocked down in both SKOV3 (Figure 24A) and HEY1B (Figure
24B) cells, and the capacity to invade through the Matrigel in vitro was analyzed. In
MLK3-knockdown SKOV3 cells, invasion was reduced by approximately 66% with
siRNA oligo 1 and 82% with siRNA oligo 2 as compared with cells transfected with the
nonspecific oligo (Figure 24A). Similarly, HEY1B cells with MLK3 knockdown
exhibited approximately 70% (siRNA oligo 1) and 80% (siRNA oligo 2) reduction in cell
invasion as compared with cells treated with the nonspecific oligo (Figure 24B). These
results clearly demonstrate a specific requirement for MLK3 in the invasion of the
ovarian cancer cell lines, SKOV3 and HEY1B.
79
Figure 24. MLK3 is essential for SKOV3 and HEY1B cell invasion. SKOV3 (A) and
HEY1B (B) cells were transfected with nonspecific oligo or MLK3 siRNA oligo 1. Cell
invasion was analyzed using Transwell chambers containing Matrigel. Cells that crossed
the membrane were stained and counted (left panel). The experiment was repeated three
times. Each bar represents the mean of data obtained from three independent experiments
and the error bars represent deviation from the mean. Statistically significant differences
from the control are indicated (*P < 0.01). A portion of the cells were lysed and
immunoblotted with MLK3 and β-Actin antibodies (right panel). (The invasion assay for
SKOV3 cells using the siRNA oligo 1 in panel A was performed by Nidhi Modi).
80
3.13 MLK4β inhibits SKOV3 cell invasion
Given the role of MLK3 in cell invasion and our results that MLK4β suppresses
MLK3 activity, we were interested in testing the effect of MLK4β on cell invasion. For
this purpose, the invasive SKOV3 cells were transiently transfected with MLK4β and the
effect on cell invasion was tested in vitro by analyzing the capacity of cells to invade
through the Matrigel (Figure 24). Cells overexpressing MLK4β exhibited a significant
reduction in cell invasion as compared with cells expressing the empty vector alone
(Figure 25, left panel). MLK4β overexpression was detected by immunoblotting (Figure
25, right panel). These results show that MLK4β reduces SKOV3 cell invasion.
81
Figure 25. MLK4β reduces the invasion of SKOV3 cells. SKOV3 cells were
transfected with pCMV5-FLAG or FLAG-MLK4β. Cell invasion was analyzed using
Transwell chambers containing Matrigel. Cells that crossed the membrane were stained
and counted (left panel). The experiment was repeated three times. Each bar represents
the mean of data obtained from three independent experiments and the error bars
represent standard deviation from the mean. Statistically significant differences from the
control are indicated (*P < 0.01). A portion of the cells was lysed and immunoblotted
with FLAG and β-Actin antibodies (right panel).
3.14 MLK3 regulates MMP-2 and MMP-9 enzyme activity
MMPs are proteolytic enzymes that promote tumor cell invasion by breaking
down components of the extracellular matrix (Nagase and Woessner, 1999; Schmalfeldt,
et al., 2001). Such processes are associated with an increase in the expression and activity
of MMP-2 and MMP-9 (Schmalfeldt, et al., 2001). To test whether MLK3 regulates
MMP activity which may in turn impact cell invasion, MLK3 was knocked down in
SKOV3 and HEY1B cells and the effect on MMP-2 and MMP-9 enzyme activities was
analyzed using gelatin zymography (Figure 26A). Culture media collected from SKOV3
and HEY1B siRNA (1 or 2) knockdown cells exhibited a substantial reduction in the
enzyme activities of both MMP-2 and MMP-9 as compared to the MMP activities in the
culture media of cells transfected with the nonspecific siRNA oligo (Figure 26A). These
results indicate that MLK3 is required for MMP-2 and -9 enzyme activities in SKOV3
82
and HEY1B ovarian cancer cells. Possibly, the regulation of MMP-2 and MMP-9
activities is a potential mechanism by which MLK3 promotes invasion in these ovarian
cell lines.
As shown in Figure 15, MLK3 activates JNK and ERK in SKOV3 and HEY1B
cells. Moreover, both ERK and JNK have previously been shown to regulate the
activation of MMP-2 and MMP-9 (Kim, et al., 2010). Thus, we proposed that ERK and
JNK could mediate MLK3-dependent regulation of MMPs. To test this, SKOV3 and
HEY1B cells were ectopically transfected with FLAG-MLK3 and the cells then treated
with pharmacological inhibitors of ERK (U0126) or JNK (SP600125), and MMP-2 and
MMP-9 activation was assessed using the gelatinase assay. ERK and JNK activities were
inhibited by U0126 and SP600125, respectively (Figure 26B). MLK3 overexpression
resulted in an increase in MMP-2 and MMP-9 gelatinase activities in SKOV3 and
HEY1B cells (Figure 26C). Inhibition of either ERK or JNK blocked MLK3-induced
activation of MMP-2 and MMP-9 (Figure 26C). These results suggest a role for both
ERK and JNK in mediating MLK3-dependent activation of MMP-2 and MMP-9.
83
A.
B.
C.
Figure 26. MLK3 mediates MMP-2 and MMP-9 activation in SKOV3 and HEY1B
cells by a mechanism that involves ERK and JNK. (A) SKOV3 and HEY1B cells were
transfected with nonspecific (NS) siRNA or MLK3 siRNA oligo 1 or 2. Cell culture
media was collected and MMP-2 and -9 enzyme activities were studied by subjecting the
collected media to gelatin zymography analysis (upper panel). Cell extracts were
immunoblotted with MLK3 and β-Actin antibodies (lower panel) to check for MLK3
knockdown. (The gelatinase assay for SKOV3 cells using MLK3 siRNA (1) was
performed by Yu Zhan). (B) SKOV3 cells were either treated with U0126 or SP600125
for 4 hours and cell extracts were immunoblotted with p-ERK, ERK, p-JNK, JNK
antibodies. (C) SKOV3 and HEY1B cells were transfected with pCMV5 vector or
FLAG-MLK3. Cells expressing FLAG-MLK3 were treated with U0126 or SP600125.
Cell culture medium was subjected to gelatin zymography analysis (upper two panels).
Cell extracts were immunoblotted with anti-FLAG antibody (lower panel).
84
3.15 MLK4β reduces MMP-9 activity in SKOV3 cells
Our results indicate that MLK3 is required for MMP-2 and MMP-9 activities in
ovarian cancer cells. Since we also observed that MLK4β inhibits MLK3 activity, we
postulated that MLK4β might negatively regulate the enzyme activities of MMP-2 and
MMP-9. Therefore, to investigate if MLK4β affects MMP-2 and MMP-9 enzyme
activities, the levels of active MMP-2 and MMP-9 were analyzed in SKOV3 cells
ectopically expressing MLK4β by gel zymography (Figure 27). Although overexpression
of MLK4β had little effect on MMP-2 gelatinase activity, it decreased the gelatinase
activity of MMP-9 (Figure 27, upper panel). These results suggest that MLK4β may
regulate MMP-9 activity.
Gelatinase
Assay
FLAG
Immunoblot
Actin
Figure 27. MLK4β reduces MMP-9 enzyme activity. SKOV3 cells were transfected
with pCMV5-FLAG or FLAG-MLK4β. Culture media was then collected and subjected
to gelatin zymography analysis (upper panel). Cell extracts were immunoblotted with
FLAG and β-Actin antibodies (lower panel) (duplicated from Figure 25).
85
Chapter 4
Discussion
MAPKs play important roles in a plethora of cellular responses including
proliferation, transformation, invasion, differentiation and apoptosis (Dhanasekaran and
Johnson, 2007; Dhillon, et al., 2007; Raman, et al., 2007). The specific response(s)
triggered by the MAPKs depends on the stimulus, cell type and degree of MAPK
activation (Dhanasekaran and Johnson, 2007; Dhillon, et al., 2007). Controlled regulation
of MAPK signaling is crucial to maintaining normal cellular function. Since deregulated
MAPK signaling can lead to cancer-related processes and cell death, MAP3Ks, as main
upstream activators of MAPKs, are critical players that modulate MAPK pathways to
generate specific biological responses. Enhanced basal activities of MAP3Ks can
promote constitutive activation of one or multiple MAPKs, which can in turn lead to
different pathologies. Indeed, mutations in B-Raf, which result in constitutive activation
of the ERK pathway, occur at high frequencies in multiple cancers (Beeram, et al., 2005;
Davies, et al., 2002). Thus, elucidating the functions of MAP3Ks is important to better
understand the regulation of MAPK signaling and to present MAP3Ks as potential
therapeutic targets for treating cancer and other pathologies.
MLK3 activates JNK, ERK and p38 and is involved in numerous cellular
responses including apoptosis, transformation, proliferation and invasion (Chadee and
86
Kyriakis, 2004; Chen, et al., 2010; Gallo and Johnson, 2002; Hartkamp, et al., 1999).
However, functional characterization of the MLK subfamily members other than MLK3
has so far been limited. Unraveling the cellular function(s) of MLKs 1, 2 and 4 is
necessary to identify redundancies and uncover unique functions of the MLK enzymes.
Activating mutations in MLK4 have been reported in colon cancer (Bardelli, et al., 2003).
These mutations might implicate a role for MLK4 in cancer. Therefore, in this study, we
investigated the role of MLK4β in MAPK signaling. Given that MLK4β and MLK3 share
75% homology in their catalytic kinase domains, it might be assumed that MLK4β
activates MAPK signaling (Kashuba et al. 2011). However, our findings indicate that
MLK4β is a negative regulator of MAPK signaling, and MLK4β inhibits both basal and
sorbitol- and TNFα-induced, p38 activity (Figures 10 and 11). Consistent with this, a
recent study reported that MLK4β overexpression failed to activate co-expressed p38 and
that MLK4β overexpression reduced the levels of LPS-induced p38 activation 15 min
post treatment (Seit-Nebi, et al., 2012). These observations support our findings that
MLK4β functions to inhibit, rather than to activate, p38 activity.
Our results demonstrate an elevation in the basal and sorbitol-induced levels of
active p38, JNK and ERK when mlk4 is silenced, suggesting that the inhibitory role of
MLK4 is not specific to p38, but extends to JNK and ERK as well (Figure 16). These
results are in accordance with similar findings by Seit-Nebi et al. that showed that MLK4
inhibits LPS-induced activation of the JNK and ERK pathways (Seit-Nebi, et al., 2012).
Collectively, our results demonstrate that MLK4β, unlike MLK3 and other MAP3Ks,
acts as a negative regulator of MAPK signaling (Figure 28).
87
MLK4β shares >70% sequence homology with MLK3 in the catalytic domain
(Kashuba et al. 2011). Moreover, MLK4β retains the conserved serine/threonine
sequences present in MLK3 and other serine/threonine kinases. For instance, the kinase
domain of MLK4β contains the conserved KAAR sequence (Lys 151- Arg 154) that is
required for ATP binding and the conserved TTXXS motif, found within the DFG-APE
sequences, that is phosphorylated in many kinases, at the second threonine and serine
residues (residues Thr300 and Ser303 in MLK4β), and is required for activation of the
kinase. Since MLK3 and other MAP3Ks were shown to directly phosphorylate and
activate MAP2Ks, it might be assumed that MLK4β would also phosphorylate and
activate MAP2Ks. However, our in vitro kinase assay data show that unlike MLK3 and
ASK1, MLK4β fails to phosphorylate GST-SEK1-KR at Thr261, a phosphorylation
event that is required for SEK1 activation (Figure 17). MLK4β also failed to
phosphorylate other residues of GST-SEK1-KR (Figure 18). These results demonstrate
that MLK4β does not phosphorylate and/or activate SEK1. Furthermore, MLK4β
undergoes autophosphorylation and phosphorylates histone H2A (Figure 18). These
results demonstrate that although MLK4β, similar to MLK3, can autophosphorylate,
MLK4β and MLK3 exhibit different substrate specificities towards SEK1, which may
suggest why these two enzymes have such different biological effects.
MLK3 signaling promotes the activation of multiple MAPK pathways (Chadee
and Kyriakis, 2004; Gallo and Johnson, 2002). Thus, we proposed that MLK4β might
exert its inhibitory effect on MAPK signaling by inhibiting MLK3 activity. Indeed, we
found that MLK4β expression reduces the level of active MLK3 in HCT116 and SKOV3
cells, while silencing mlk4 elevates the level of p-MLK3 (Figures 19 and 20).
88
Interestingly, comparison of tumor cell lines and normal cell lines revealed an inverse
correlation between the level of MLK4β and the level of active MLK3 (Figure 21).
Moreover, low MLK4β protein levels are associated with an elevation in the levels of
active ERK (Figure 21). Our results, however, revealed that the level of active ERK do
not always correlate with the levels of p-MLK3 (Figure 21). For instance, although T98G
cells have a higher level of active MLK3 than HEY1B cells, higher levels of p-ERK are
present in HEY1B cells (Figure 21). MAPKs can be activated by numerous upstream
activators (Raman, et al., 2007). Therefore, it is possible that HEY1B cells express high
levels of other upstream activators of ERK, in addition to MLK3. Collectively, these
results show that MLK4β inhibits MAPK signaling and MLK3 activition (Figure 28).
Co-immunoprecipitation
experiments
revealed
an
association
between
overexpressed MLK4β and MLK3, and endogenous MLK4β and MLK3 in HCT116 cells
(Figure 22), which to our knowledge is the first demonstration of an association between
two MLK subfamily members. However, the nature of the interaction between MLK4β
and MLK3, whether direct or indirect, and the domains involved remains to be
determined. Because the leucine zipper domains of MLK4β and MLK3 do share 66%
identity (Kashuba et al. 2011), heterodimerization between these two proteins remains a
possibility.
MLK3 activity is autoinhibited by the N-terminal SH3 domain and autoinhibition
is relieved upon binding of active Cdc42 to the MLK3 CRIB domain (Du, et al., 2005;
Gallo and Johnson, 2002). An association between MLK4β and Cdc42 was not observed
(Seit-Nebi, et al., 2012). Therefore, it is still possible that the association between
89
MLK4β and MLK3 blocks MLK3 activation by interfering with Cdc42 binding to the
MLK3 CRIB domain.
p38 can promote cell survival by activating the NF-κB pathway (Maulik, et al.,
1998; Zechner, et al., 1998). Given that MLK4β overexpression reduced basal p38
activity (Figure 10), it might be assumed that MLK4β would act as a negative regulator
of the NF-κB pathway. Indeed, MLK4β overexpression elevated the basal level of IκBα,
while MLK4β knockdown reduced the level of IκBα (Figure 13). Moreover, MLK4β
overexpression significantly reduced NF-κB luciferase activity in HEK293 cells. These
results demonstrate a negative regulatory effect for MLK4β on basal NF-κB activity.
Knocking down MLK3 was shown to elevate the levels of active IKK and reduce IκBα
protein levels, suggesting an inhibitory function for MLK3 in the NF-κB pathway (Cole,
et al., 2009). Considering the inhibitory effect for MLK4β on MLK3 activition, it would
be expected that MLK4β would either enhance NF-κB activity or not affect it at all.
MAPKs and NF-κB signaling, however can be triggered by different upstream targets
other than MLK3, including TLR4 (Kawai and Akira, 2007; Zbinden-Foncea, et al.,
2012). Interestingly, MLK4 was found to interact with and inhibit TLR4 (Seit-Nebi, et
al., 2012). Therefore, it is possible that MLK4β negatively regulates basal NF-κB
pathway by inhibiting TLR4. MLK4β, however, fails to block TNFα-induced NF-κB
activation as evidenced by its inability to prevent IκBα degradation in response to TNFα
treatment (Figure 13). This finding is consistent with the observation that MLK4 does not
affect LPS-induced IκB degradation (Seit-Nebi, et al., 2012). Hence, MLK4β negatively
regulates basal but not TNFα- or LPS-induced NF-κB activation. Possibly, in response to
90
stimuli, such as TNFα and LPS, multiple upstream activators of the NF-κB pathway are
activated which can then overcome the inhibitory effect of MLK4β.
HCT116 cells express high levels of MLK4β (Figure 21). Knock down of MLK4
in HCT116 cells, did not affect cell proliferation even four days after siRNA transfection
(Figure 23). Moreover, MLK4-knockdown cells were viable and did not exhibit any overt
signs of stress or changes in morphology (data not shown). Thus, MLK4 is not vital for
cell survival or proliferation in HCT116 cells. In a recent study, MCF7 breast cancer cells
stably expressing MLK4β failed to proliferate and a role for MLK4 in apoptosis was
proposed (Kashuba et al. 2011). Nonetheless, in the same study, transgenic mice
expressing human pCMV-MLK4β were generated and the mice were normal, healthy and
showed no phenotypic abnormalities (Kashuba et al. 2011). A more thorough study is
thus needed to clarify the role of MLK4β in cell proliferation.
Ovarian cancer is one of the leading types of cancer among women and is
characterized by a poor prognosis and a high mortality rate primarily due to the lack of
early detection (Dutta, et al., 2010). Therefore, unraveling the early events involved in
ovarian cancer provides insights for new therapeutic targets that can yield novel
treatments to counteract this disease. Cell invasion is an essential process in the transition
of tumors to a malignant state (Hanahan and Weinberg, 2011). Hence, identifying the key
players in ovarian cancer cell invasion is crucial to the development of new therapeutic
options. Recent studies have shown a requirement for MLK3 in the invasion of mammary
epithelial tumor cells and in the migration of gastric and A459 lung tumor cells (Chen, et
al., 2010; Mishra, et al., 2010; Swenson-Fields, et al., 2008). In addition, we have
demonstrated that MLK3-knockdown SKOV3 and HEY1B cells exhibit impaired cell
91
invasion (Figure 24). These results suggest that MLK3 has a critical function in ovarian
cancer cell invasion. Considering the inhibitory effect of MLK4β on MLK3, we
postulated that MLK4β might hinder the invasion of ovarian cancer cells. As expected,
overexpressing MLK4β reduced SKOV3 cell invasion (Figures 25 and 28), which
suggests a novel role for MLK4β in ovarian cancer cell invasion.
MMPs, particularly MMP-2 and MMP-9, play important functions in promoting
cell invasion by breaking down the ECM and mediating the EMT process (Hua, et al.,
2011; Roy, et al., 2009; Tester, et al., 2007). We proposed that MLK3 may mediate
ovarian cancer cell invasion by regulating MMP-2 and MMP-9 activities. Indeed, using
siRNA-mediated silencing of mlk3, we determined a reduction in the levels of active
MMP-2 and MMP-9 in SKOV3 and HEY1B cells lacking MLK3 (Figure 26A). This
suggests a requirement for MLK3 in the activation of these enzymes. p38, ERK and JNK
can mediate cell invasion by mechanisms that depend on MMP-2 and/or MMP-9
(Spallarossa, et al., 2006; Zhou, et al., 2007). Other than activating transcription factors
that induce MMP gene expression, the ERK and JNK pathways have also been shown to
directly regulate MMP-2 and MMP-9 activation (Kim, et al., 2010). For instance, TIMP2
which plays a role in MMP-2 activation, is activated by ERK and JNK, and in turn MMP2 activates MMP-9 (Butler, et al., 1998; Kim, et al., 2010; Toth, et al., 2003). MLK3 is
required for ERK and JNK activation in colon and lung fibroblasts and also in SKOV3
cells (Chadee and Kyriakis, 2004; Zhan, et al., 2012). We postulated that MLK3 may
induce MMP activity and ovarian cancer invasion through activation of ERK and JNK.
This was demonstrated in HEY1B and SKOV3 cells whereby ERK or JNK inhibition
potently reduced MLK3-dependent MMP-2 and MMP-9 gelatinase activities (Figure 26
92
C). Being a negative regulator of MLK3 and MAPK signaling, we predicted that MLK4β
would inhibit MMP activity. Indeed, we found that MLK4β expression reduces MMP-9
proteolytic activity (Figure 27).
In this study we demonstrated unexpected differences in the signaling between
MLK4β and MLK3. The MLK subfamily members are highly diverse in their C-terminal
domains (Gallo and Johnson, 2002). The fact that MLK3 and MLK4β, despite sharing >
65% homology in all domains but the C-terminal region, have opposing functions
suggests that the C-terminus may have a critical role in regulating MLK functions.
Identification of the proteins that interact with the MLK C-termini is an interesting
direction for future investigation. Our results indicated that wild-type MLK4β has no
kinase activity towards SEK1. Consistent with this, wild type MLK4 exhibited low
kinase activity that was comparable to that of the kinase dead variant of MLK4 (Martini
et al. 2013). Interestingly, in a recent study, mutations in MLK4, H261Y, G291E, R470C
and R555STP were found to substantially increase MLK4 kinase activity (Martini et al.
2013). Moreover, these mutants, unlike wild-type MLK4, significantly enhanced the
transformational capacity of cells expressing the G12V Ras mutant, which readily
triggers cellular transformation (Martini et al. 2013). Possibly these transformation
mutations, allow MLK4β to adopt a tertiary structure that enhances the activation of the
enzyme. Considering that MLK4β associates with and inhibits MLK3 and TLR4, MLK4β
may have a scaffold function that mediates inhibition of these proteins. Hence, the
transforming mutations might induce a conformational change of the MLK4 enzyme that
favors a kinase-directed over a scaffold function. Thus, MLK4β harboring one of the
activating mutation might function as a MAP3K that activates MAPK signaling. The
93
colon cancer cell line, LoVo, harbors one of the activating mutations, R470C (Bardelli, et
al., 2003). Hence, for future studies, it will be interesting to study the role of MLK4β in
MAPK signaling in LoVo cells as compared to other cell lines. Moreover, analysis of the
effect of MLK4 activating mutations on MAPK signaling and cell invasion is important
to better understand the biochemical functions of MLK4β.
Figure 28. Schematic diagram illustrating the role of MLK4β in MAPK signaling.
MLK3, upon activation, activates the p38, JNK and ERK pathways leading to different
cellular responses, including invasion. Our results indicate that MLK4β negatively
regulates MLK3 activation and the ERK, JNK and p38 MAPK-signaling pathways.
Therefore, MLK4β-dependent suppression of MAPK signaling may be mediated through
inhibition of MLK3 activation.
94
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