Colocalization of Calcium-Dependent Protease II and One of Its

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Colocalization of Calcium-Dependent Protease II and One of Its
Cell, Vol. 51, 569-577,
November
20, 1967, Copyright
0 1967 by Cell Press
Colocalization of Calcium-Dependent
Protease II
and One of Its Substrates at Sites of
Cell Adhesion
Mary C. Beckerle:
Keith Burridge,t
George N. DeMartino,*
and Dorothy E. CroalP
* Department of Biology
University of Utah
Salt Lake City, Utah 84112
tDepartment
of Anatomy
University of North Carolina
Chapel Hill, North Carolina 27514
t Department of Physiology
University of Texas Health Science Center
Dallas, Texas 752359040
Adhesion plaques, specialized
regions of the plasma
membrane where a cell contacts its substratum,
are
dynamic structures.
However, little is known about
how the protein-protein
interactions
that occur at
adhesion plaques are controlled.
One mechanism by
which a cell might modulate its associations
with the
substratum is by selective, regulated proteolysis of an
adhesion plaque component.
Here we show that the
catalytic subunit of the calcium-dependent
protease
type II (CDP-II) is localized in adhesion plaques of several cell types (BS-C-1, EBTr, and MDBK). We have
compared the susceptibility
of the adhesion plaque
constituents
vinculin, talin, and a-actinin to calciumdependent
proteolysis in vitro and have found talin to
be the preferred substrate for CDP-II. The colocalization of a calcium-requiring
proteolytic
enzyme and talin in adhesion
plaques raises the possibility
that
calcium-dependent
proteolytic
activity
provides
a
mechanism
for regulating
some aspect of adhesion
plaque physiology and function via cleavage of talin.
Introduction
A transmembrane
linkage between the extracellular matrix and the cytoskeleton occurs at specialized regions of
the plasma membrane where a cell is in very close contact with the substratum. These areas, called adhesion
plaques or focal contacts, are the primary structures mediating a cell’s attachment to the extracellular environment.
Adhesion plaques are also the sites at which actin filament bundles (stress fibers) are found in apparent end-on
association with the plasma membrane. Because of the
importance of controlled adhesion for cellular activities
such as directed migration and normal growth, much attention has been focused on determining the molecular
mechanisms by which these associations between a cell
and its substratum are established, mediated, and modified. A number of proteins have been identified as constituents of these specialized regions of the plasma membrane. A transmembrane
glycoprotein complex that has
been shown to interact with extracellular matrix components such as fibronectin (Akiyama et al., 1986; Horwitz
et al., 1985) and laminin (Horwitz et al., 1985) is localized
in adhesion plaques (Damsky et al., 1985; Chen et al.,
1985b). This fibronectin receptor complex interacts with
talin (Horwitz et al., 1986), a 225 kd cytoplasmic protein
that is also found at focal contacts (Burridge and Connell,
1983). Talin, in turn, associateswith a 130 kd protein called
vinculin (Otto, 1983; Burridge and Mangeat, 1984). By the
elucidation of such tandem protein-protein
interactions,
the organization of the functional linkage between the extracellular matrix and the cytoskeleton is becoming more
clear.
That there is indeed some “communication”
between
the extracellular matrix and the cytoskeleton via the focal
contact has been apparent for some time. Thin section
electron microscopy has revealed a structural continuity
between fibronectin and actin that appears to be mediated
by the electron-dense
adhesion plaques (Singer, 1979).
Moreover, treatment of well-spread, adhesive cells with
the actin-disrupting
agent cytochalasin B induces both
loss of ordered actin filament bundles and reduced adhesion (Ali and Hynes, 1977). Similarly, virally transformed
cells, which have a disrupted actin cytoskeleton and exhibit decreased adhesiveness, undergo phenotypic reversion to the normal morphology
upon exposure to exogenously supplied fibronectin (Yamada et al., 1976; Ali et
al., 1977). Thus bidirectional
integration of information
across the cell membrane appears to be occurring at
adhesion plaques.
Adhesion plaques are dynamic structures. In cultured
cells they are disassembled
and then reestablished at
specific times during the cell cycle. Viral transformation
and growth factors such as PDGF affect the structural integrity of the focal contacts (David-Pfeuty and Singer,
1980; Herman and Pledger, 1985) but the mechanism
by which adjustments in the stability of these actinmembrane-substratum
interactions occur has not been
elucidated. It has been suggested that phosphorylation
of
adhesion plaque components could contribute to the dynamic nature of the focal contact (Sefton et al., 1981). A
number of adhesion plaque components are phosphoproteins. One of these, the fibronectin receptor, is phosphorylated on tyrosine when cells are infected with Rous sarcoma virus strains that induce disruption
of cellular
adhesion and actin organization (Hirst et al., 1986). This
provocative observation raises the possibility that phosphorylation of the receptor complex could produce altered
interactions among the adhesion plaque proteins and
contribute to the virus-induced
loss of organization
at
these sites.
Another mechanism with the potential to modulate focal
contact organization is limited proteolysis. Proteolysis of
extracellular matrix has been implicated for some time in
the changes in cellular adhesion associated with transformation (Unkeless et al., 1973; Chen et al., 1985a; Chen
and Chen, 1987). Proteolysis of an intracellular protein at
the focal contact could also theoretically affect the structural integrity of actin-membrane-substratum
interactions.
Cell
570
A
B
1986; DeMartino et al., 1986); however, the in vivo srgnificance of autoproteolytic processing remains to be determined. Although much is known about the regulation
of these proteolytic enzymes in vitro, their regulation and
function(s) in vivo are unclear. Platelets provide perhaps
the best example of a dynamic system in which calciumdependent proteolysis appears to play a functionally significant role in vivo. When platelets are activated by a
physiological stimulus, such as thrombin, they assemble
actin filaments, reorganize their cytoskeletons, and aggregate (for reviews, see Nachmias, 1983; Fox and Phillips,
1983). Calcium-dependent
proteolysis of two abundant,
high molecular weight platelet proteins-actin-binding
protein and P235-follows
thrombin stimulation (Fox et
al., 1983, 1985). We have recently demonstrated
that
platelet P235, one of the physiological
substrates of
calcium-dependent
proteases, is homologous
to talin
(O’Halloran et al., 1985; Beckerle et al., 1986). Here we
show that CDP-II is colocalized with talin in adhesion
plaques of cultured cells, and we discuss the implications
of this observation for the regulation of actin-membranesubstratum interactions.
Results
Figure
1. Antibody
Characterization
(A) A Coomassie
blue-stained
gel of standard proteins (lane l), total
BS-C-1 protein (lane 2) and purified CDP-II from bovine heart (lane 3).
Antibody
was affinity-purified
against
the high molecular
weight
subunit (JO kd) of the protease and analyzed by Western blot with the
result shown in the autoradiograph
in (B). The affinity-purified
antibody
recognizes
the 80 kd subunit of the protease (lane 3’) as well as a protein of comparable
molecular mass in the BS-C-1 cells (lane 2’). A similar result was obtained with proteins from EBTr and MDBK cells (not
shown).
Many of the stimuli that cause disruption of adhesion
plaques and of the associated actin cytoskeleton induce
a rise in intracellular calcium levels. Thus it is an attractive
hypothesis that stimulation of an intracellular calciumdependent protease could be responsible, in part, for
mediating dramatic changes in cell adhesion and morphology.
Calcium-dependent
proteases (also known as calpains
or calcium-activated
neutral proteases) are widely distributed in animal cells. There are two unique but related
enzymes: calcium-dependent
protease I (CDP-I), which
requires l-10 uM calcium for half-maximal activity; and
calcium-dependent
protease II (CDP-II), which requires
200-300 FM calcium for half-maximal activity. Each enzyme consists of a catalytic subunit (84 kd and 80 kd,
respectively) and a regulatory subunit (26 kd) (Pontremoli
and Melloni, 1986; DeMartino and Croall, 1987; Suzuki et
al., 1984; Murachi, 1983). Limited autoproteolysis of CDPII occurs in vitro and lowers the enzyme’s calcium requirement 20- to 50-fold (Suzuki et al., 1981; Coolican et al.,
Localization
of CDP-II
A polyclonal antibody was raised against CDP-II, purified
from bovine heart. Affinity-purified
antibody directed
against the 80 kd catalytic subunit of the protease was prepared as described in Experimental
Procedures. The
affinity-purified
antibody (anti-CDP-II-80kd) recognizes a
single 80 kd protein in BS-C-1 cells, as determined by immunoblot analysis (Figure 1, lane 2’). This 80 kd protein
comigrates with the authentic 80 kd subunit of CDP-II (Figure 1, lane 3’). Prior to affinity purification, the antibody
also recognized the 26 kd subunit of CDP-II and CDP-I (not
shown) and, less prominently, some other proteins in the
cell lysate. To ensure maximum specificity of the antibody,
affinity-purified
antibody was used exclusively in the
studies reported here.
In order to determine the location of CDP-II within cells,
the affinity-purified
antibody was used to stain cells by
indirect immunofluorescence.
The result obtained with
BS-C-1 cells, epithelial-like
cells derived from African
green monkey kidney, is shown in Figure 2. The distribution of actin filament bundles (stress fibers) was visualized
by staining with rhodamine-phalloidin
and is shown in Figure 28. In the same cells, anti-CDP-II-80kd
stains the
adhesion plaques where the actin filament bundles terminate (Figure 2C). This distribution of the 80 kd subunit of
CDP-II is the same as that described for talin. Staining of
adhesion plaques in BS-C-1 cells was obtained with two
independently produced polyclonal antisera against CDPII. We did not detect any specific staining of adhesion
plaques in these cells with antisera raised against CDP-I.
Characterization
of the Calcium-Dependent
Proteolytic
System of BS-C-1 Cells
Although all mammalian cells contain the calcium-dependent proteases I and II and their endogenous inhibitor pro-
Protease
571
and Substrate
at Sites of Cell Adhesion
hgzoo0
0
40
50
60
70
Frocficn
Figure
3. Fractionation
of the CDP
Number
System
from
BS-C-l
Cells
A soluble extract from BS-C-1 cells was fractionated
by gel filtration on
Sephacryl
S-300 (A). Five milliliter fractions were collected and 90 PI
samples were assayed for proteolytic
activity in the presence
of calcium (7 mM) by incubation with [Wjcasein
at 25OC for 1 hr (0). Purified CDP-I (0.1 ng per assay) was assayed (20 min at 25%) for caseinolytic activity in the presence
of 80 pl of each fraction to identify the
endogenous
inhibitor activity (0).
Fractions
61-69 (A) were pooled and chromatographed
on DEAEcellulose. Fractions (~5 ml) were collected and assayed for caseinolytic activity as described
above in the presence
of calcium
(0).
Column fractions (A and 8) were also assayed for proteolytic activity
in the absence of calcium (data not shown). All protease peaks observed were totally dependent
on calcium for activity.
tein (Mellgren and Carr, 1983; DeMartino and Croak,
1984) the relative and absolute amounts of each of these
proteins vary widely from cell type to cell type (Murachi,
1983; Suzuki et al., 1984). In order to determine the relative amounts of each protease and their inhibitor in BS-C-1
cells, cellular extracts were prepared and fractionated by
standard methods. As expected, CDP-I, CDP-II, and their
inhibitor protein were present in these cells and fractionated similarly to CDPs from all other sources that we have
examined (Figure 3). From measurements of CDP activity
in fractionated, partially purified samples, we estimate the
CDP-II concentration
of BS-C-l cells to be 3- to 5fold
greater than that of CDP-I (data not shown). As in many
other tissues and cells, the endogenous
CDP inhibitor
activity is present in excess of total CDP activity in BS-C-1
cells.
Figure
Cells
2. Localization
of the Calcium-Dependent
Protease
in BS-C-1
(A) Phase contrast.
(B) Actin filament distribution
as revealed
by
rhodamine-phalloidin.
(C) Distribution
of CDP-II. The 80 kd subunit of
CDP-II is localized in adhesion plaques where bundles of actin filaments terminate at the plasma membrane.
Bar = 20 urn.
Localization
of CDP-II in Other Cell Types and in
Other Adherens Junctions
The localization of CDP-II in adhesion plaques is not peculiar to BS-C-1 cells. A fibroblastic cell line derived from embryonic bovine trachea (EBTr) also exhibits adhesion
plaque staining with the anti-CDP-II-8Okd antibody (Figure
4). Comparison of the fluorescence pattern (Figure 48)
with the interference reflection image (Figure 4A) reveals
that the 80 kd subunit of CDP-II is indeed localized at sites
of close cell-substratum
contact. We have also examined
the distribution of CDP-II in MDBK cells, bovine kidney epithelial cells that have well-developed
zonulae adherens
as well as adhesion plaques. The calcium-dependent
protease is present in the adhesion plaques but is not detected in the cell-cell junctional complexes (Figures 4C,
4D). In contrast, vinculin is localized in both zonula adherens and adhesion plaques in MDBK cells (data not
shown).
Cell
572
Figure
4. Dlstrlbution
of CDP-lL8Okd
I” Other
Cells
(A) and (B) EBTr fibroblast
(A) An mterference
reflectlon microscopic
view of an EBTr cell. This optical techmque enables visualization
of regions
where a cell IS In close contact with the substratum.
Adhesion plaques appear black by this approach. (6) lndlrect tmmunofluorescence
with affinitypurified anti-CDP-II-8Okd
antibody demonstrates
that the CDP-lL80kd
antigen is indeed localized at sites of cell-substratum
adhesion as defined
by interference
reflection microscopy.
(C-F) MDEK cells. (C) and (E) Phase contrast. (D) CDP-lL80kd
IS detected in adhesion plaques but not In zonula adherens of MDBK cells. (F) Prelmmune serum yields no staining of adhesion plaques, though faint nuclear staining is observed.
Bar = 20 pm.
A number
of control
experiments
were performed
in order to determine
the specificity
of the anti-CDP-II-80kd
antibody
staining.
Preimmune
IgG does not stain adhesion
plaques,
but does
show
some
nuclear
staining
(see
Figures
4E, 4F for example);
consequently,
we believe
that the faint nuclear
staining
observed
with the anti-CDPII-80kd
represents
nonspecific
background
staining.
Affinity-purified
anti-CDP-II-8Okd
adsorbed
against
native
CDP-II
lost the ability to recognize
a component
in adhesion plaques,
but continued
to give some nuclear
staining,
again suggesting
that the nuclear
staining
may be nonspecific.
Antibody
affinity-purified
against
a control
strip of
nitrocellulose
CDP did not
Substrate
Adhesion
that did not contain
the 80 kd subunit
stain adhesion
plaques.
of the
Specificity of CDP-II for
Plaque Proteins
We have
compared
the susceptibility
of the adhesion
plaque
proteins
vinculin,
talin, and a-actinin
to cleavage
by CDP-II.
Talin, vinculin,
and a-actinin
were purified
and
incubated
individually
with CDP-II
at a ratio of 5O:l (w:w).
The time course
of the digestion
is shown
in Figure
5. As
can be seen in Figure
5A, talin is cleaved
by the protease
to generate
two major
fragments
of approximately
190-
Protease
573
and Substrate
at Sites of Cell Adhesion
v
T
Figure 5. Susceptibility
of Some Adhesion
Plaque Proteins to Calcium-Dependent
Proteolysis
a-A
,”2’ cp
TIME
(Min.):
0
1
1
5
10
234
5670
A
30
f
30
4
30
0
1
30
2
60
3
0
30
60
Talin, vinculin, and a-actinin were incubated at
23% with purified CDP-II at a ratio of 5O:l by
weight. Samples of the reaction mixture were
taken at the times indicated and subjected
to
SDS-PAGE.
Talin (A) is rapidly cleaved by
CDP-II, whereas vinculin (6) and a-actinin (C)
are resistant
to proteolysis
under the conditions of this experiment.
Proteolysis
of talin is
completely dependent on the addition of exogenous protease (A, lane 7) and calcium (A, lane
8) to the reaction mixture.
4
B
200 kd and 46 kd. In the absence of enzyme or calcium,
no proteolysis occurs (Figure 5A, lanes 7 and 8). Even after 80 min of incubation with this concentration of enzyme,
neither vinculin (Figure 58) nor a-actinin (Figure 5C) has
been cleaved by CDP-II. Talin is effectively cleaved by
CDP-II even at lo-fold lower enzyme concentrations (data
not shown). Vinculin and a-actinin may be cleaved in the
presence of a higher concentration of CDP-II than used
here (Croall and DeMartino, 1984; Gache et al., 1984).
Nevertheless, it is clear that talin is significantly more susceptible to CDP-II-mediated
proteolysis than are these
other adhesion plaque constituents.
Discussion
In this paper we have shown that the catalytic subunit of
the calcium-dependent
protease II is present in focal contacts of cells from several established lines. Talin, a physiological substrate for calcium-dependent
proteolysis in
platelets, is also localized in focal contacts in these cultured cells. These observations raise the possibility that
specific, calcium-regulated
proteolysis of talin in adhesion
plaques could provide a mechanism by which protein-
protein interactions that occur at focal contacts are modified to effect a change in the structural integrity of the
adhesion plaque.
Distribution
of Talin and the Calcium-Dependent
Protease: Specific Localization
in Adhesion Plaques
The distribution of talin in a variety of cells and tissues has
been extensively examined. For example, talin has been
identified in adhesion plaques (Burridge and Connell,
1983) in the myotendinous junction (Tidball et al., 1986)
in the postsynaptic neuromuscular
junction (Sealock et
al., 1986) and in membrane-associated
densities (probablydense plaques) of smooth muscle (Geiger et al., 1985).
In general, talin appears to be localized at regions of the
plasma membrane specialized for both actin filament attachment and adhesion. The calcium-dependent
protease has previously been described as having a diffuse
cytoplasmic distribution in brain (Hamakubo et al., 1986),
pancreas (Kitahara et al., 1985) and kidney (Yoshimura et
al., 1984) as well as in some cultured cells (Kitahara et al.,
1986). However, in prefusion L6 myoblasts the calciumdependent protease has been shown to exhibit a punctate, peripheral distribution, suggesting that it is asso-
Cell
574
ciated with specialized regions of the plasma membrane
(Schollmeyer, 1986); talin is localized in similar membrane-associated
densities in smooth muscle. We have
demonstrated here that CDP-II is colocalized with talin in
adhesion plaques of several ceil lines. In the future it will
be interesting to compare the distributions of the protease
and talin in various tissues to determine if they are always
colocalized.
The majority of the proteins found at adhesion plaques
are also localized in another adherens junction, the zonula adherens of epithelial cells (Geiger et al., 1985). Both
cell-substratum
and cell-cell adherens junctions have associated actin filaments and contain vinculin, a-actinin,
and the 82 kd protein (Geiger et al., 1985; Beckerle, 1986).
Interestingly, talin and CDP-II are colocalized in cellsubstratum
adherens junctions (focal contacts), and
neither protein is detected in cell-cell adherens junctions
(zonula adherens). Because of their similar molecular
weights, we have frequently been asked whether the 82
kd component of adhesion plaques identified in chicken
embryo fibroblasts (Beckerle, 1986) is related to CDP-II.
Although this question has not been resolved definitively,
the different subcellular distributions of the 82 kd polypeptide and of CDP-II suggest that these proteins are not identical.
Attempts to Compare the Cellular Localization
of CDP-I and CDP-II
In addition to examining the distribution of CDP-II in cultured cells, we examined the distribution of CDP-I with two
antibodies raised against this enzyme. We were unable to
detect any localized distribution of CDP-I with these polyclonal antibodies. Only a diffuse cytoplasmic staining pattern was observed with antibodies raised against CDP-I.
In BS-C-1 cells, the concentration of CDP-I is several-fold
less than that of CDP-II, and this may have contributed to
our inability to detect any localized cytoplasmic distribution for CDP-I. Alternatively, CDP-I and CDP-II may have
distinct functions and, therefore, distinct cellular localizations. It is also possible that the anti-CDP-I antibodies may
recognize determinants that are masked when the enzyme is associated with other proteins in a complex structure like an adhesion plaque.
Significance
of Calcium-Dependent
Proteolysis
In Vivo
Despite recent detailed biochemical characterization
of
the CDPs, there is little evidence as to their physiological
function(s). However, they appear to be necessary for
mammalian
cells since the enzymes are ubiquitous
(Murachi, 1983) and their sequences are highly conserved across species (Ohno et al., 1984; Emori et al.,
1986). A number of functions have been proposed for
these proteases (Pontremoli and Melloni, 1986); however,
in most cases there is little direct experimental evidence
to support these ideas.
Calcium-dependent
proteolysis is perhaps best documented in vivo in the blood platelet. In this case, several
proteins are specifically cleaved in response to physiological platelet activators such as thrombin (White, 1980; Fox
et al., 1983). Cleavage of actin-binding
protein (filamin)
has been proposed to be important for the remodeling of
cortical actin filaments that occurs when platelets are
stimulated (Fox, 1986). Proteolysis of platelet talin (P235)
occurs at a time when platelets are adhering to each other
and to fibrin strands, and are participating in clot retraction (Fox and Phillips, 1983). Calcium-dependent
proteases have been isolated from platelets (Sakon et al.,
1981; Yoshida et al., 1983), and it has been demonstrated
in vitro that both talin- and actin-binding
protein are substrates for the endogenous
platelet CDP (Collier and
Wang, 1982; Fox et al., 1983; Beckerle et al., 1986) as well
as for CDPs purified from heterologous sources (Fox et al.,
1985; O’Halloran et al., 1985). Cleavage products of talinand actin-binding protein that are generated with purified
CDP in vitro are not detectably different from those seen
in vivo upon platelet stimulation. Therefore, it appears
likely that specific CDP-mediated proteolysis occurs in activated platelets. It remains to be determined how directly
the events of platelet activation can be related to adhesion
plaque physiology and function. However, the colocalization of talin and CDP in focal contacts raises the possibility
that physiologically
significant proteolysis of talin occurs
at these sites as well. Although limited proteolysis would
produce an irreversible change in an individual protein,
resultant changes in adhesion plaque integrity would be
reversible in any cell capable of replacing the protein by
new synthesis.
One potentially significant difference between talin and
its 190-200 kd proteolytic product has been observed.
Comparison of native talin and its 190-200 kd fragment by
gel filtration and rotary shadowing has revealed that intact
talin undergoes an ionic strength-dependent
conformational change from an e!ongated rod to a more compact
globular shape, whereas the fragment consistently exhibits the more elongated morphology (O’Halloran and
Burridge, 1986; Molony and O’Halloran,
unpublished
data). It will be very interesting to determine whether these
physical differences translate into functional differences
at the focal contact or within the platelet.
As discussed above, CDP-II, the enzyme localized in focal contacts, has an in vitro calcium requirement
of
200-300 PM for half-maximal activity. The calcium requirement of CDPs decreases after autoproteolytic
processing (Suzuki et al., 1981; Coolican et al., 1986; DeMartino et al., 1986) and may also be affected by lipids
(Coolican et al., 1986). This latter observation suggests
that proximity to biological membranes (Pontremoli and
Melloni, 1986; Croall and DeMartino, 1984) could affect
the level of calcium necessary for proteolytic activity.
Conclusions
The demonstration that CDP-II is colocalized with talin in
adhesion plaques of several cell types raises the possibility that cleavage of talin by this protease could have
regulatory significance in these cells. It is clear from other
work that there is transmembrane
“communication”
between the extracellular matrix and the cytoskeleton at the
adhesion plaque, and that perturbation of elements inside
the plasma membrane has dramatic effects on the interac-
Protease
575
and Substrate
at Sites of Cell Adhesion
tions that occur on the extracellular face of the plasma
membrane, and vice versa. Already there is some evidence that a proteolytic fragment of talin exists in cells
@‘Halloran and Burridge, 1986). We can now investigate
whether activation of CDP-II and proteolysis of talin are
correlated with situations in which adhesion plaque integrity is modulated by living cells.
Experimental
Procedures
Preparation
of Antibodies
against Calcium-Dependent
Ploteases
from Bovine Heart
Calcium-dependent
proteases
I and II were purified from bovine
cardiac muscle as described
elsewhere (Croak and DeMartino,
1984).
Each native enzyme was dialyzed against phosphate-buffered
saline
and mixed 1 :l with Freund complete (first injection) or incomplete adjuvant. New Zealand White rabbits were injected subcutaneously
at several sites with a total of 1 mg of CDP for the first injection, and were
reinjected at 2 week intervals for 8 weeks with 0.5 mg of enzyme protein. Animals were bled 7-10 days after the final injection and once every 2 weeks thereafter
for 8 weeks. Immune rabbits were boosted every 6 months with 0.2-0.3 mg of CDP and bled as described
above.
Heat-inactivated
sera (56%, 30 min), untreated sera, or partially purified IgG have all been used with similar results on Western immunoblots to demonstrate
antibody specificity.
Affinity-purified
antibody was prepared according
to a previously
published method (Cox et al., 1983). Briefly, purified CDP-II from bovine heart (80 pg) was electrophoresed
under denaturing
conditions
and transferred
to nitrocellulose
by electroblotting
(20 hr, 80 mA). Protein was visualized by staining with Ponceau S (1% in 5% acetic acid).
The 80 kd subunit was excised and the nitrocellulose
strip was destained in water. Control strips of nitrocellulose
containing
no visible
CDP were also processed
in this manner. After incubation for 1 hr in
3% gelatin in Ris-buffered
saline (TBS: 50 mM Tris, pH 7.5, at 25%;
0.5 M NaCI), the strips were incubated with 1.5 ml of antisera (25% sera
in TBS) for 4 hr at 20%. The nitrocellulose
strips were subsequently
washed twice with TBS containing
0.05% Tween-20 and twice with
TBS. Bound antibody was eluted in 1.0 ml of 0.1 M glycine (pH 2.9) for
2 min in the presence of 100 pg of BSA. The eluted affinity-purified
antibody was immediately
neutralized
with Tris base and concentrated
(Amicon-Centricon
10,000) to two-thirds
the original sera volume.
Affinity-purified
antibodies were stored in TBS containing
0.5% NaNs
at 4°C until used.
Affinity-purified
antibody was characterized
by a modification
(Beckerle, 1986) of the immunoblot method of Towbin and co-workers
(1979).
Characterization
of CDPs from BS-C-1 Cells
BS-C-1 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine sera and 50 units/ml of penicillin, 50 units/ml
of streptomycin.
To examine CDPs, cells were grown to confluence
on
100 mm dishes (15-25 plates per preparation).
Cell monolayers
were
washed three times with phosphate-buffered
saline prior to scraping
into 5 mM potassium
phosphate (pH 7.5) 5 mM 2-mercaptoethanol,
2
mM EDTA, 2 mM EGTA, at 4°C (BufferA). Cells were homogenized
with
20 strokes, B pestle, in a Dounce-type
homogenizer,
and were subsequently centrifuged
at 15,000 x g for 30 min. The supernatant
was incubated with 3 ml of packed volume DE52 ion exchange resin (Whatman) that had been equilibrated
with Buffer A. Bound protein was
eluted with 1.25 M KCI in Buffer A. After extensive dialyses against 50
mM Tris (pH 7.5) at 4OC, 0.5 mM EGTA, 5 mM P-mercaptoethanol,
the
sample was centrifuged
to remove insoluble protein and was applied
to a Sephacryl
S-300 gel filtration column (2.5 x 100 cm). Protease activity was assayed by hydrolysis
of methyl-14C-a casein as previously
described
(Croall and DeMartino,
1983; DeMartino
et al., 1986). Assays for the inhibition of CDPs were carried out with purified bovine
heart CDP-I (0.1 ttg per assay) (DeMartino
and Croall, 1984). Assays
of caseinolytic
activity in fractions containing both CDPs and the endogenous inhibitor protein reflect the CDP activity that is in excess of
the inhibitor activity. CDP-I and CDP-II were separated by ion exchange
chromatography
at 4% on DE52 (Whatman)
(6 x 2.5 cm) by using a
300 ml gradient of O-O.4 M KCI in 50 mM X-is (pH 7.5). 0.5 mM EGTA,
5 mM P-mercaptoethanol.
Partial purification
of CDP-I was accom-
plished by chromatography
on phenyl-Sepharose,
and CDP-II was purified on reactive red agarose (Croall and DeMartino, 1983, 1984). Each
enzyme was concentrated
by binding to and elution from DEAESephacel followed by Centricon (Amicon) concentration.
The specific
activity of purified CDP-I or CDP-ii from bovine heart, expressed
as
cpm of TCA-soluble
j4C released per minute of incubation per pg of
protease, was used to estimate the amounts of CDP-I and CDP-Ii recovered from BS-C-1 cells.
Ceil Culture and Indirect
lmmunofluorescence
The established cell lines EBTr, MDBK. and BS-C-1 were obtained from
the American Type Culture Collection.
The cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum
and supplemented
with penicillin-streptomycin.
Cells that had been
plated on 12 mm diameter glass coverslips
were used for the localization studies.
Indirect immunofluorescence
was performed
as described previously
(Beckerle,
1986).
Pmteolytic
Digests
Vinculin, taiin, and a-actinin were purified by established
procedures
(Burridge and Conneli, 1983; Feramisco
and Burridge, 1980; Collier
and Wang, 1982). These purified proteins were incubated with CDP-II
at a mass ratio of 5O:l in 38.5 mM Tris-HCI (pH 7.5) 1 mM dithiothreitol,
in the presence or absence of CaCls. Aliquots of the reaction mixture
were withdrawn at the indicated time intervals and the digestion was
stopped by boiling of the sample with an equal volume of gel sample
buffer, The resultant
proteolytic
peptides
were analyzed
on 10%
SDS-polyacrylamide
gels (Laemmii, 1970) containing 0.13% bisacrylamide. Human platelet talin and chicken smooth muscle talin gave the
same results.
Acknowledgments
We are particularly
grateful to Maurine Vaughan for patience and skill
in typing this manuscript.
This research was supported
by grants from
the National Institutes of Health (NIH) to K. Burridge (GM 29860) and
from the Texas Affiliate of the American
Heart Association
and the
American Cancer Society (IN-142) to D. E. Croall. Support is also acknowledged
from the National Science Foundation (DCB 8602131) and
American Heart Association
to M. C. Beckerle, and from the NIH (AM
29829) to G. N. DeMartino.
The costs of publication of this article were defrayed in part by the
payment
of page charges.
This article must therefore
be hereby
marked “advertisement”
in accordance
with 18 U.S.C. Section 1734
solely to indicate this fact.
Received
July 6, 1987; revised
September
4, 1987.
References
Akiyama, S. K., Yamada, S. S., and Yamada,
ization of a 150-kD avian cell surface antigen
molecule. J. Cell Biol. 102, 442-448.
K. M. (1986). Characteras a fibronectin-binding
Ali. I. U., and Hynes. R. 0. (1977). Effects of cytochalasin
Band colchitine on attachment
of a major surface protein of fibroblasts.
Biochim.
Biophys. Acta 477, 16-24.
Ali, I. U., Mautner, V., Lanza, Ft., and Hynes, R. 0. (1977). Restoration
of normal morphology:
adhesion and cytoskeleton
in transformed
cells
by addition of a transformation-sensitive
surface
protein. Cell 17,
115-126.
Beckerle, M. C. (1986). Identification
of a new protein localized
of cell-substrate
adhesion. J. Cell Biol. 103, 1679-1687.
at sites
Beckerle, M. C., O’Halloran, T., and Burridge, K. (1986). Demonstration
of a relationship
between talin and P235, a major substrate of the calcium-dependent
protease in platelets. J. Cell Biochem. 30, 259-270.
Burridge, K., and Connell, L. (1983). A new protein of adhesion
and ruffling membranes.
J. Cell Biol. 97, 359-367.
plaques
Burridge, K., and Mangeat. l? (1984).
and talin. Nature 308, 744-745.
vinculin
An interaction
between
Chen. J.- M., and Chen, W.-T. (1987). Fibronectm-degrading
proteases
from the membranes
of transformed
cells. Ceil 48, 193-203.
Chen,
W.- T., Chen,
J.- M., Parsons,
S. J., and Parsons,
J. T. (1985a).
Cell
576
Local degradation
ing gene product
of fibronectin at sites of expression
ppGO*“. Nature 376, 156-158.
of the transform-
phorylation
of the fibronectin receptor complex in cells transformed
by
oncogenes
that encode tyrosine kinases. Proc. Natl. Acad. Sci. USA
83, 6470-6474
Chen, W.-T., Greve, J. M., Gottlieb, D. I., and Singer, S. J. (1985b). Immunocytochemical
localization
of 140 kD adhesion molecules
in cultured chicken fibroblasts
and in chicken smooth muscle and intestinal
eprthelial tissues. J. Histochem.
Cytochem.
33, 576-586.
Horwitz, A., Duggan, K.. Greggs, R., Decker, C., and Buck, C. (1985).
The cell-substrate
attachment
(CSAT) antigen has properties
of a
receptor for laminin and fibronectin.
J. Cell Biol. 701, 2134-2144.
Collier, N.. and Wang, K. (1982). Purification and properties
of human
platelet P235. A high molecular weight protein substrate
of endogenous calcium-activated
protease(
J. Biol. Chem. 257, 6937-6943.
Horwitz, A., Duggan. K., Buck, C., Beckerle. M. C.. and Burridge. K.
(1986). Interaction of the plasma membrane
fibronectin
receptor with
talin: a transmembrane
linkage. Nature 320, 531-533.
Coolican. S. A., Haiech, J., and Hathaway,
D. R. (1986). The role of
subunit autolysis in activation of smooth muscle calcium-dependent
proteases. J. Biol. Chem. 267, 4170-4176.
Kitahara, A., Ohtsuki, H., Kirahata. Y., Yamagata, Y., Takano, E., Kannagi, R., and Murachi, T. (1985). Selective localization of calpain I (the
low Gas’ requiring form of Gas+-dependent
cysteine proteinase)
in B
cells of human pancreatic
islets. FEBS Lett. 184, 120-124.
Cox, J. V., Schenk, E. A., and Olmsted, J. 8. (1983). Human
mere antibodies: distribution,
characterization
of antigens,
on microtubule
organization.
Cell 35, 331-339.
Croall. D. E., and DeMartmo,
ization of calcium dependent
258, 5660-5665.
G. N. (1983). Purification
protease from rat heart.
anticentroand effect
Kitahara, A., Takano. E., Ohtsuki, H., Kirahata, Y,, Yamagata, Y., Kannagi, R., and Murachi, T. (1986). Reversed distribution of calpains and
calpastatin
in human pituitary gland and selective localization of calpastatin in adreno coriticotropin
producing cells as demonstrated
by
immunohistochemistry.
J. Clin Endo. Metab. 63, 343-348.
and characterJ. Biol Chem.
Croak, D. E., and DeMartino, G. N. (1984). Comparison
of two calciumdependent proteinases from bovine heart. Biochim. Biophys. Acta 788,
348-355.
Damsky, C. H., Knudsen, K. A., Bradley, D., Buck, C. A., and Horwitz,
A. F. (1985). Distribution of cell-substratum
attachment (CSAT) antigen
on myogemc
and fibroblastic
cells in culture. J. Cell Biol. 100,
1528-1539.
David-Pfeuty,
cytoskeletal
transformed
6687-6691.
T., and Singer, S. J. (1980). Altered distributions
of the
proteins vinculin and a-actinin
in cultured
fibroblasts
by Rous sarcoma virus, Proc. Natl. Acad. Sci. USA 77,
DeMartino, G. N.. and Croall, D. E. (1984). Purification and characterization of a protein inhibitor of calcium-dependent
proteases from rat
liver. Arch. Biochem. Biophys. 232, 713-720.
DeMartino,
G. N., and Croall,
teases: a prevalent
proteolytic
Physiol. Sci. 2, 82-85.
D. E. (1987). Calcium-dependent
system of uncertain
function.
proNews
DeMartino. G. N.. Huff, C. A., and Croall. D. E. (1986). Autoproteolysis
of the small subunit of calcium-dependent
protease II activates and
regulates protease activity. J. Biol. Chem. 267, 12047-12052.
Emori, Y., Kawasaki, H., Sugihara. H., Imajoh, S., Kawashima,
S., and
Suzuki, K. (1986). Isolation and sequence
analysis of cDNA clones for
the large subunits of two isozymes
of rabbit calcium-dependent
protease. J. Biol. Chem. 261, 9465-9471.
Feramisco,
J. R., and Burridge,
K. (1980) A rapid purification
of
a-actinin, filamin, and a 130,000-dalton
protein from smooth muscle.
J. Brol. Chem. 255. 1194-1199.
Fox. J. E. B. (1986). ldentifrcahon of actin-binding
lmking the membrane
skeleton to glycoproteins
membranes.
J. Biol. Chem. 260, 11970-11977.
protein as the protern
on platelet plasma
Fox, J. E. B., and Phillips, D. R. (1983). Polymerization
of actin filaments within platelets. Semin. Hematol.
CalciumJ. Biol.
Fox, J. E. B., Gall, D. E., Reynolds, C. C., and Phillips, D. R. (1985). Identlfication of two protems (actin-binding
protein and P235) that are
hydrolyzed
by endogenous
Ca’*-dependent
protease during platelet
aggregation.
J. Biol. Chem. 260. 1060-1066.
Gache, Y., Landon, F., Touiton, M., and Olomucki.
A. (1984). Susceptlbility of platelet a-actinin to a calcium-activated
neutral protease. Biochim. Biophys. Res. Commun.
724, 877-881.
Gerger, B., yolk, T., and Volberg, T. (1985). Molecular
adherens junctions. J. Cell Biol. 701, 1523-1531.
heterogeneity
of
Hamakubo, T., Kannagi, R.. Murachl. T., and Matus, A. (1986). Distribution of calpams I and II In rat brain. J. Neuroscl.
6, 3103-3111.
Herman, B., and Pledger, W. J. (1985). Platelet-derived
induced alterations
In vmculin and actin distribution
cells. J Cell Biol. 700, 1031-1040.
Hirst.
R., Horwrtz,
A., Buck,
C., and Rohrschneider,
growth factor
in BALB/c 3T3
L. (1986).
Mellgren, R. L., and Carr, T. C. (1983). The protein inhrbitor of calciumdependent
proteases:
purification
from bovine heart and possible
mechanisms
of regulation. Arch. Biochem. Biophys. 225, 779-786.
Murachl.
167-169.
T (1983). Calpains
and calpastatin.
Trends
Phos-
Biochem.
Sci. 8,
Nachmias,
V. T. (1983). Platelet and megakaryocyte
shape change:
triggered alterations in the cytoskeleton.
Semin. Hematol. 20,261-281.
O’Halloran, T,, and Burridge. K. (1986). Purification of a 190 kDa protein
from smooth muscle: relationship to talin. Biochim, Biophys. Acta 869,
337-349.
O’Halloran, T., Beckerle, M. C., and Burridge. K. (1985). Identification
of talin as a major cytoplasmic
protein implicated in platelet activation.
Nature 377, 449-451.
Ohno, S., Emori, Y., Imajoh, S.. Kawasaki, H., Kisaragr, M., and Suzuki,
K. (1984). Evolutionary
origin of a calcium-dependent
protease by fusion of genes for a thiol-protease
and a calcium-binding
protein? Nature 312. 566-570.
Otto, J. J. (1983). Detection
of vinculin-binding
proteins
‘*%inculin
gel overlay technique.
J. Cell Biol. 97, 1283-1287.
Pontremok, S , and Melloni, E. (1986). Extralysosomal
tion. Ann. Rev. Biochem. 55, 461-481.
Sakon, M., Kambayashi,
forms of Ca++-activated
24, 207-214.
wrth
protein degrada-
J. I., Ohno, H., and Kosakr,
neutral protease in platelets.
G. (1981). Two
Thromb. Res.
Schollmeyer,
J E. (1986). Role of Cae’ and Cast-achvated
myoblast fusion. Exp. Cell Res. 762, 411-422.
Sealock, R.. Paschal, B., Beckerle, M., and Burridge,
is a postsynaptic
component
of the rat neuromuscular
Cell Res. 763, 143-150.
and organization
20, 243-260.
Fox, J. E B., Reynolds,
C. C., and Phillips, D. R. (1983).
dependent
proteolysls
occurs during platelet aggregation.
Chem. 258, 9973-9981.
Laemmli, U. K. (1970). Cleavage of structural
proteins during the assembly of the head of bacteriophage
T4. Nature 227 680-685.
protease
K. (1986).
junction.
In
Talin
Exp.
Sefton, 8. M., Hunter, T., Ball, E. H., and Singer, S. J. (1981). Vinculin:
a cytoskeletal
target of the transforming
protein of Rous sarcoma virus.
Cell 24, 165-174
Singer, I. I (1979). The fibronexus:
a transmembrane
association
fibronectin-containing
fibers and bundles of 5 nm microfilaments
hamster and human fibroblasts.
Cell 16, 675-685.
of
In
Suzuki, K.. Tsuji, S., Ishiura, S., Klmura, Y., Kubota. S., and Imahori,
K. (1981). Autolysis of calcium-activated
neutral protease of chicken
skeletal muscle. J. Blochem. 90. 1787-1793.
Suzuki. K., Kawashima,
in Biological Systems,
Press), pp 213-226.
S., and Imahori, K. (1984). Calcium Regulation
S. Ebashi et al., eds. (New York: Academic
Tidball, J G , O’Halloran, T., and Burndge, K. (1986).
dtnous junctions
J. Cell Biol. 703, 1465-1472.
Talin at myoten-
Towbin. H.. Staehlln, T, and Gordon, J. (1979). Electrophoretrc
transfer
of proteins from polyacrylamide
gels to nitrocellulose
sheets: procedure and some applications. Proc Natl. Acad. Sci. USA 76, 4350-4354.
Unkeless, J. C , Tobra. A., Ossowski.
L., Quigley, J. P, Rifkin, D. B., and
Reich, E. 11973) An enzymatic function associated
with transformanon
Protease
577
and Substrate
of fibroblasts
transformed
at Sites of Cell Adhesion
by oncogenic viruses. I. Chick embryo fibroblast cultures
by avian RNA tumor viruses. J. Exp. Med. 737, 85-111.
White, G. G. II (1980). Calcium-dependent
protease in platelets. Response of calcium-activated
protease in normal and thrombasthenic
platelets to aggregating
agents. Biochim. Siophys. Acta 637, 130-138.
Yamada, K. M.. Ohanian,
tein decreases
microvilli
cells. Cell 9, 241-245.
S. H., and Pastan, I. (1916). Cell surface proand ruffles on transformed
mouse and chick
Yoshida, N., Weksler, 6, and Nachman,
R. (1963). Purification
of human platelet calcium-activated
neutral protease. J. Biol. Chem. 258,
7l68-7l74.
Yoshimura,
N., Hatanaka,
M., Kitahara,
A., Kawaguchi,
N., and
Murachi, T. (1964). Intracellular
localization of two distinct calcium proteases (calpain I and calpain II) as demonstrated
using discriminative
antibodies.
J. Siol. Chem. 259, 9847-9652.