Cleavage of Focal Adhesion Kinase by Different Proteases during
Src-regulated Transformation and Apoptosis
DISTINCT ROLES FOR CALPAIN AND CASPASES*
Neil O.
Carragher
,
Valerie J.
Fincham,
Deborah
Riley, and
Margaret C.
Frame
From the the Beatson Institute for Cancer Research, Cancer Research
Campaign Beatson Laboratories, Glasgow G61 1BD, United Kingdom
Received for publication, October 2, 2000, and in revised form, November 3, 2000
 |
ABSTRACT |
Integrin-associated focal adhesion complexes
provide the main adhesive links between the cellular actin cytoskeleton
and the surrounding extracellular matrix. In vitro, cells
utilize a complex temporal and spatially regulated mechanism of focal
adhesion assembly and disassembly required for cell migration. Recent
studies indicate that members of both calpain and caspase protease
families can promote limited proteolytic cleavage of several components
of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such
as tumor invasion. v-Src-induced oncogenic transformation is associated
with loss of focal adhesion structures and transition to a less
adherent, more motile phenotype, while inactivating temperature-sensitive v-Src in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that
v-Src-induced disassembly of focal adhesions is accompanied by
calpain-dependent proteolysis of focal adhesion kinase.
Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion
disruption, loss of substrate adhesion, and cell migration. In
contrast, focal adhesion loss during detachment and apoptosis induced
after switching off temperature-sensitive v-Src in serum-deprived
transformed cells is accompanied by caspase-mediated proteolysis of
focal adhesion kinase. Thus, calpain and caspase differentially
regulate focal adhesion turnover during Src-regulated cell
transformation, motility, and apoptosis.
 |
INTRODUCTION |
The transforming viral Src gene (v-src) is
associated with classic characteristics of oncogenic cell
transformation, including deregulated growth control, cell rounding,
and substrate detachment resulting from adhesion loss and disruption of
the actin cytoskeleton (1-6). The stability of the actin cytoskeleton
and adhesive properties of cells are mediated, at least in part, by
focal adhesion complexes (7, 8). Dynamic regulation of these adhesive
links through assembly of focal adhesions at the leading edge of cells,
coordinated with focal adhesion disassembly at the trailing edge, plays
a key role in controlling cell migration (9). The mechanisms regulating
turnover of focal adhesions are not well understood. However, a recent
study suggests that proteolysis of specific components of the focal
adhesion complex by the calpain family of proteolytic enzymes promotes
disassembly of smooth muscle focal adhesions in response to collagen
fragments (10). Furthermore, calpain activity has been implicated in
promoting migration of Chinese hamster ovary cells (11). The calpains
are defined as a well conserved family of intracellular, nonlysosomal
calcium-dependent cysteine proteases consisting of two
ubiquitously expressed calpain isozymes, µ-calpain (calpain-I) and
m-calpain (calpain-II) and several tissue-specific isoforms (12-14).
Colocalization of calpain II with focal adhesion structures (15) and
the identification of several focal adhesion proteins as calpain
substrates (16-21) suggest that calpains are functional at focal
adhesion sites.
Oncogenic transformation of cells by v-Src is associated with an
overall loss in abundance of focal adhesions as well as changes in
focal adhesion architecture to smaller more condensed structures (22).
The precise mechanisms by which v-Src promotes the disassembly of focal
adhesions have so far not been elucidated. The focal adhesion kinase
(FAK)1 is both a substrate of
Src kinase activity and a central component of focal adhesions and may
be a strong candidate for mediating v-Src-induced disassembly of focal
adhesions. Through multiple protein-binding domains, FAK can interact
with and phosphorylate several members of the focal adhesion complex
(23). Studies on cells derived from FAK knock-out embryos demonstrate
that focal adhesions devoid of FAK are larger and that these cells have
a reduced migratory capacity, suggesting that FAK may be required to
regulate the turnover of focal adhesion structures (24).
To specifically examine the role of calpain-mediated proteolysis in
tyrosine kinase-induced focal adhesion disruption, we have used a
temperature-dependent mutant of v-Src (ts LA29
v-Src). Activation of v-Src by shift to the permissive temperature
initiates focal adhesion disassembly (5). Our previous work has
indicated that this and consequent morphological transformation are
dependent on the catalytic activity of v-Src that induces
phosphorylation of focal adhesion components, including FAK, followed
by dissociation of the Src-FAK complex and degradation of FAK,
events that precede focal adhesion loss and cell rounding (5, 6).
Despite this characterization of the sequence of events during the
initial stages of v-Src-induced cell transformation, we still lack any understanding of the nature of the process of FAK proteolytic cleavage
induced by v-Src, and specifically, the identities of the proteases
involved have not been elucidated. As mentioned above, calpains
represent good candidates; however, FAK has also been demonstrated to
be cleaved by caspases as focal adhesions are lost during apoptosis
(25, 26). In this study, we have addressed whether FAK cleavage that is
triggered prior to focal adhesion disassembly during cell
transformation is mediated by members of either the calpain or caspase
family of proteolytic enzymes.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
Primary chicken embryo
fibroblasts (CEF) were subcultured as described previously (5). Low
density cultures were transfected with replication-competent avian
retroviral constructs, RCAN-v-src, encoding the
temperature-sensitive ts LA29 v-Src mutant. Transfected CEF
were cultured at the permissive temperature of 35 °C until cells
were uniformly infected and expressing v-Src protein. For analysis of
v-Src-induced transformation, cells were cultured at the restrictive
temperature (41 °C) and then examined following shift to the
permissive temperature (35 °C). Rat 1 fibroblasts expressing
ts LA29 v-Src were maintained in DMEM supplemented with 5%
newborn calf serum, 1 mM sodium pyruvate, and 2 mM L-glutamine at the permissive temperature
(35 °C). Rat 1 cells were serum-starved for 24 h (Dulbecco's
modified Eagle's medium supplemented with 0.2% newborn calf serum)
prior to a shift to the restrictive temperature (39.5 °C).
Antibodies and Reagents--
Calpain inhibitor studies were
performed using calpain inhibitor 1 (ALLN; Calbiochem), calpain
inhibitor 2 (ALLM), and the cell-permeable sense and scrambled
calpastatin peptides (Calbiochem). Caspase inhibitor studies were
carried out with caspase inhibitor 1 (ZVAD-FMK; Calbiochem). CEF were
preincubated with ALLN, ALLM, or ZVAD-FMK (50-100
µM) for 1 h prior to shift to 35 °C and then subsequently incubated at 35 °C in the presence of ALLN, ALLM, or
ZVAD-FMK (50-100 µM) or sense and scrambled
calpastatin peptides (50 µM) for the indicated time
periods. Serum-starved Rat 1 fibroblasts were preincubated with
ZVAD-FMK or ALLN (100 µM) for 1 h prior to
shift to restrictive temperature 39.5 °C and then subsequently incubated at this temperature in the presence of the inhibitors. Antibodies for Western blot detection and immunocytochemistry included
2-18N pp125FAK and 903-1058C
pp125FAK (Santa Cruz Biotechnology, Inc.),
354-534N pp125FAK (Transduction Laboratories),
paxillin (Transduction Laboratories), and calpain-II (Research
Diagnostics, Inc.). Anti-mouse and -rabbit peroxidase-conjugated
secondary antibodies were purchased from New England Biolabs, Inc.
Immunocytochemistry--
Nontransformed and transformed ts
LA29 CEF were cultured on permanox plastic chamber slides (Nalge
Nunc International). Cells were fixed in 3.7% formaldehyde for 10 min
at room temperature, permeabilized in 0.5% Nonidet P-40 in PBS for 10 min at room temperature, and washed serially in PBS, 0.15 M
glycine/PBS plus 0.02% NaN3, and PBS. Cells were blocked
in 10% fetal calf serum/PBS prior to 1 h incubation at room
temperature with primary antibodies, including affinity-purified
monoclonal anti-paxillin (Transduction Laboratories) and polyclonal
rabbit antiserum generated against the large subunit of m-calpain
(calpain II) purified from human placenta (Research Diagnostics Inc.).
Primary antibody incubation was followed by several washes in PBS and
subsequent incubation with FITC- or Texas Red-conjugated secondary
antibodies (Jackson Immunoresearch Laboratories). Cells were also
incubated with fluorescein isothiocyanate-labeled phalloidin (Sigma).
Immunostaining of cells was analyzed by confocal microscopy.
Immunoblotting--
Cells were washed twice with PBS and lysed
in low detergent lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM NaF, 10 mM 
glycerophosphate, 10 mM Na4P2O7, 100 µM NaVO4 with protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and
10 µg/ml aprotinin). Lysates were clarified by high speed
centrifugation at 4 °C, supplemented with SDS-sample buffer,
separated by 10% SDS-polyacrylamide gel electrophoresis, and
immunoblotted with specific antibodies.
Adhesion Analysis--
ts LA29
v-Src-expressing CEF were cultured on tissue culture plastic substrates
at 41 or 35 °C for 18 h in the absence or presence of calpain
inhibitor 1 and 2 (ALLN, ALLM; 100 µM), or herbimycin A
(5 mM) cells were washed three times with PBS, and those
cells remaining attached to the culture substrate were quantified by
counting the number of cells per high power field (magnification × 200). Data representing the mean of four high power fields were expressed as the number of cells per high power field remaining attached to the culture substrate.
Wound-healing Migration Assay--
2 × 105 CEF
expressing ts LA29 v-Src were cultured at 41 °C in 60-mm
dishes until 70-80% confluent. The cell monolayers were wounded by
scoring with a sterile micropipette tip, and cultures were incubated at
35 °C for a further 18 h in the absence or presence of ALLN (50 µM), ALLM (50 µM), and ZVAD-FMK (100 µM).
 |
RESULTS |
To analyze the role of the calpain proteolytic enzymes in focal
adhesion loss associated with v-Src induced morphological transformation, we examined protein levels and localization of the
ubiquitously expressed calpain II in nontransformed (41 °C) and
v-Src-transformed CEF (35 °C). Immunoblotting cell lysates prepared
at sequential time points following shift to 35 °C demonstrated a
consistent rapid increase in levels of calpain II following activation
of v-Src, which subsequently declined at 24 h (Fig. 1A). Calpain I protein was
detected at extremely low levels in CEF, and levels of calpain I did
not vary during the early stages of transformation (results not shown).
Messenger RNA levels of calpain II and the endogenous calpain inhibitor
calpastatin are not altered in response to v-Src transformation
(results not shown), suggesting that elevation of calpain II protein
levels and proteolytic activity following v-Src activation are
regulated at a post-transcriptional level.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
Modulation of calpain II protein levels
during v-Src-induced cell transformation of chick embryo
fibroblasts. A, total cell lysates were prepared from
ts LA29 v-Src CEF cultured at the restrictive temperature
(41 °C) or at sequential time points following shift to the
permissive temperature (35 °C). Cell lysates were separated by 10%
SDS-polyacrylamide gel electrophoresis and immunoblotted with an
antibody specific for calpain II. ts LA29 v-Src CEF were
cultured at 41 °C for 24 h and then for a further 6 h
either at 41 °C (B) or 35 °C (C).
Immunocytochemistry was performed with an antibody specific for calpain
II or paxillin. Colocalized staining of calpain II with paxillin at
focal adhesion attachment sites is indicated by arrows.
Bar, 25 µm.
|
|
Immunostaining of nontransformed CEF (41 °C) with an antibody
against calpain II was generally weak and diffuse throughout the
nucleus and cytoplasm; however, some calpain II immunoreactivity was
evident at peripheral focal adhesion sites colocalizing with the focal
adhesion protein paxillin (indicated by arrows) (Fig. 1B). Six hours following shift to the permissive temperature
(35 °C), immunostaining of ts v-Src-expressing CEF with
anti-calpain II was of greater intensity than that of nontransformed
cells (Fig. 1C), consistent with the elevated protein levels
observed by immunoblotting (Fig. 1A). In v-Src-transformed
CEF, calpain II staining often localized at peripheral podosome-like
structures (indicated by arrows) that may represent
substrate attachment sites consisting of residual focal adhesions (Fig.
1C). Immunostaining of anti-calpain II at these peripheral
sites again colocalized with focal adhesion proteins such as paxillin
(Fig. 1C).
To determine whether calpain activity was involved in the process of
oncogenic transformation, CEF expressing ts v-Src were studied following treatment with pharmacological inhibitors of calpain
proteolytic activity as well as a calpastatin peptide highly specific
for calpain inhibition. ts v-Src-expressing CEF were shifted
from the restrictive temperature (41 °C) to the permissive temperature (35 °C) to induce cell transformation. Characteristic of
v-Src transformation, untreated CEF lost focal adhesion structures, exhibited a disorganized actin cytoskeleton, and developed a rounded morphology with many cells having detached from the culture substrate 18 h following shift to 35 °C (Fig.
2B). Treatment of cells
cultured at 35 °C with a well characterized pharmacological peptide
aldehyde inhibitor of calpain activity, ALLN (100 µM)
(27-29) suppressed the loss of focal adhesions, cell rounding, and
remodeling of the actin cytoskeleton (Fig. 2C). Calpain
inhibitor I-ALLN has also been reported to inhibit the lysosomal
proteases cathepsin B, cathepsin L, and the
ubiquitin-dependent proteasome complex (27, 30, 31).
Treatment of ts v-Src-expressing CEF undergoing transformation with ammonium chloride, a reputed inhibitor of lysosomal
cathepsins (32, 33), or the specific proteasome inhibitor, lactacystin
(34), had no effect on the characteristics of v-Src transformation
(results not shown), indicating that the effects of ALLN were most
likely due to calpain inhibition. In addition, treatment of
ts v-Src-expressing CEF with ALLM (100 µM),
which is inhibitory against calpains, but not the proteasome, can
suppress v-Src-induced loss of focal adhesions and remodeling of the
actin cytoskeleton and cell morphology (results not shown). A more
specific approach to inhibiting intracellular calpain activity can be
achieved by exploiting the characteristics of the naturally occurring
endogenous inhibitor of calpain, known as calpastatin. Calpastatin is
highly specific for calpain I and calpain II and has not been
demonstrated to inhibit other proteases (35, 36). Studies demonstrate
that a 27-amino acid synthetic calpastatin peptide corresponding to the
minimal inhibitory segment of domain I of calpastatin specifically
inhibits activity of calpain both in vitro (37) and in a
cell-permeable fashion in vivo (38). ts
v-Src-expressing CEF treated with the sense (Fig. 2D), but not the scrambled (Fig. 2E), cell-permeable calpastatin
peptide demonstrate a reduction in the loss of focal adhesions,
disorganization of the actin cytoskeleton, and cell rounding normally
associated with v-Src transformation at 35 °C. In contrast,
treatment of cells with the broad spectrum caspase inhibitor
ZVAD-FMK has no effect on transformation (Fig. 2F).
The results obtained with the above panel of inhibitors indicate that
calpains are the proteases involved in promoting disassembly of focal
adhesions and the actin cytoskeleton and transition to a less adherent
and more rounded morphology during v-Src transformation of CEF.

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 2.
v-Src-induced focal adhesion disassembly,
disorganization of the actin cytoskeleton, and cell rounding are
prevented by inhibitors of calpain activity. ts LA29
v-Src-expressing CEF were cultured at 41 °C (A) or
35 °C (B) for 18 h in the absence or presence of the
following inhibitors: ALLN (100 µM) (C),
calpastatin peptide (calpa.P.; 50 µM)
(D), scrambled calpastatin peptide (calpa.S.; 50 µM) (E), and ZVAD-FMK (100 µM) (F). Cell morphology was evaluated by
phase-contrast microscopy (magnification × 100). Focal adhesion
structures and the actin cytoskeleton were analyzed by
immunocytochemistry utilizing an anti-paxillin antibody and fluorescein
isothiocyanate-labeled phalloidin, respectively. Bar, 25 µm.
|
|
We have previously observed proteolytic degradation of the recognized
calpain substrate FAK during v-Src transformation and focal adhesion
loss, although the antibody used in these studies did not detect
cleavage products (5). To characterize FAK cleavage in more detail, we
have utilized three epitope-specific antibodies raised against defined
regions of the FAK protein. Using a low detergent lysis buffer to
enrich for proteolytic fragments of FAK present in the cytoplasm (see
"Experimental Procedures"), total cell lysates were prepared from
ts v-Src-expressing CEF cultured at 41 °C or at
sequential time points following v-Src activation by shift to 35 °C.
Lysates were subjected to immunoblotting using three antibodies
recognizing the amino terminus (N-pp125FAK), residues
354-534 downstream of the amino terminus
(354-534N-pp125FAK), and the carboxyl terminus
(C-pp125FAK) of FAK as probes (Fig.
3). Immunoblotting with all three
antibodies indicated that cleavage of native FAK occurred between 6 and
8 h following activation of v-Src. The loss of native FAK was
paralleled by the generation of approximately sized 95- and 40-kDa
amino-terminal fragments and a 30-kDa C-terminal cleavage product (Fig.
3, A and C). Additionally, 55-, 42-, and 30-kDa
fragments of FAK derived from the internal region of the protein can be
identified by the 354-534N pp125FAK antibody
(Fig. 3B). The initial cleavage of FAK to approximately 95-kDa amino-terminal and 30-kDa C-terminal fragments is consistent with the pattern of calpain-mediated cleavage described previously for
other stimuli (16, 39, 10). Pretreatment and subsequent incubation of
ts v-Src-expressing CEF at 35 °C in the presence of ALLN
at 100 µM significantly inhibited cleavage of FAK and generation of all cleavage products associated with v-Src
transformation (Fig. 3, A-C), whereas treatment with a
caspase inhibitor (ZVAD-FMK; 100 µM) had no effect
(results not shown).

View larger version (105K):
[in this window]
[in a new window]
|
Fig. 3.
Calpain-mediated FAK degradation during v-Src
transformation of CEF. Total cell lysates were prepared from
v-Src-expressing CEF cultured at 41 °C or at sequential time points
following shift to 35 °C with and without ALLN (100 µM). Cell lysates were separated by 10%
SDS-polyacrylamide gel electrophoresis and immunoblotted with
antibodies recognizing the amino terminus of FAK (N-FAK)
(A), an epitope 354 residues downstream from the amino
terminus (N354-534 FAK) (B), and the carboxyl
terminus of FAK (C-FAK) (C). The location and approximate
molecular masses of native (125-kDa) FAK and putative cleavage
fragments are indicated by arrows. The alternatively spliced
c-terminal isoform, FRNK, is also indicated.
|
|
To examine the influence calpain inhibition had on the adhesive
strength of v-Src-transformed CEF for their culture substrate, ts v-Src-expressing CEF were cultured for 18 h at
41 °C or at 35 °C in the absence or presence of the calpain
inhibitors ALLN and ALLM. The number of cells remaining attached to the
substrate after extensive washing was quantified and expressed as the
number of cells per high power field (Fig.
4). These results demonstrated that after
18 h at 35 °C there was a significant loss in the number of
ts v-Src-expressing cells remaining strongly adherent to the substrate. Transformation of cells at 35 °C in the presence of the
calpain inhibitor ALLN or ALLM (100 µM) resulted in a
greater than 2-fold increase in the number of cells remaining attached to the culture substrate. Incubation of CEF at 35 °C in the presence of the general inhibitor of kinase function herbimycin A, which inhibits Src kinase activity, also partially rescued substrate anchorage (Fig. 4). These results indicate that
calpain-dependent disassembly of focal adhesions that
accompanies v-Src transformation results in a general decrease in the
adhesion of transformed CEF to their culture substrate. Thus, treatment
of v-Src-transformed CEF with calpain inhibitors stabilized focal
adhesion structures and maintained substrate anchorage.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Inhibitors of calpain activity limit the loss
of cell anchorage that accompanies v-Src transformation. ts
LA29 v-Src-expressing CEF were cultured at 41 or 35 °C for
18 h in the absence or presence of the calpain inhibitors ALLN,
ALLM (100 µM), and the general kinase inhibitor
herbimycin A (5 µM). Cells were then washed three times
with PBS, and the number of cells remaining attached to the culture
dish were counted and expressed as the number of cells per high power
field (magnification × 200).
|
|
Since we have shown that the proteolysis of FAK during transformation
by v-Src is most likely calpain-dependent and since we have
shown previously that Src-induced focal adhesion disassembly is
required for cell migration (6), we addressed whether inhibitors of
calpain could suppress cell migration into a wounded monolayer of
v-Src-transformed cells. After wounding, ts v-Src-expressing CEF cultured at 35 °C for 18 h demonstrated extensive migration into the denuded area (Fig. 5).
Incubation of ts v-Src-expressing CEF at 35 °C in the
presence of the calpain and proteasome inhibitor (ALLN; 50 µM) inhibited any motility of transformed CEF into the wound (Fig. 5). Treatment of CEF with the inhibitor ALLM (50 µM), which inhibits calpain and not the proteasome
complex, also substantially prevented migration. Since ALLN and ALLM
have previously been reported to exhibit cathepsin-inhibitory activity
(27, 30), we also treated ts v-Src-expressing CEF with the
reported inhibitor of lysosomal cathepsins, ammonium chloride, as a
negative control and found this had no significant influence on
migration (results not shown). Treatment of cells with the caspase
inhibitor ZVAD-FMK also has no effect on migration of
v-Src-transformed CEF (Fig. 5). These results demonstrate that
v-Src-transformed CEF are dependent on calpain activity for optimal
migration, most likely as a result of its proposed role in FAK cleavage
and focal adhesion turnover.

View larger version (122K):
[in this window]
[in a new window]
|
Fig. 5.
Migration of v-Src-transformed CEF is
repressed by inhibitors of calpain activity. ts LA29
v-Src CEF were initially cultured at 41 °C. A wound was generated,
and wound size was recorded at time 0 (T0, 41 °C).
Cells were subsequently incubated at 35 °C for a further 18 h
in the absence or presence of the following inhibitors; ALLN (50 µM), ALLM (50 µM), and ZVAD-FMK (100 µM).
|
|
Our previous studies demonstrate that Rat-1 fibroblasts expressing the
ts LA29 v-Src protein undergo apoptosis when cultured in low
serum following attenuation of v-Src activity by culture at the
restrictive temperature (39.5 °C). Following shift of serum-starved ts LA29 Rat-1 cells from permissive (35 °C) to
restrictive temperature (39.5 °C), the cells rapidly round up and
become less adherent (40). Proteolytic cleavage of FAK to 85- and
77-kDa N-terminal fragments accompanies these morphological changes
(Fig. 6); however, in contrast to v-Src
transformation, FAK cleavage in serum-deprived cells switched to the
restrictive temperature was not inhibited by ALLN but instead was
inhibited by ZVAD-FMK (Fig. 6), indicating that
caspases and not calpains are responsible for FAK cleavage that
accompanies adhesion loss under these circumstances.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 6.
FAK cleavage mediated by caspases during
apoptosis of Rat-1 fibroblasts. Total cell lysates were prepared
from serum-starved ts v-Src-expressing Rat-1 fibroblasts
cultured at the permissive (35 °C) and restrictive temperature
(39.5 °C) for 2, 4, and 6 h in the absence or presence of
calpain (ALLN; 100 µM) or caspase (ZVAD-FMK; 100 µM) inhibitors. Protein samples were separated by 10%
SDS-polyacrylamide gel electrophoresis and immunoblotted with the N-FAK
antibody.
|
|
 |
DISCUSSION |
Cell adhesion to extracellular matrix substrates regulates many
aspects of normal cell physiology, including proliferation, migration,
and cell survival (9, 41, 42). An integral role for calpain activity in
regulating critical aspects of cell behavior has been suggested by
previous studies demonstrating that calpain activity can degrade focal
adhesion components (39, 10), mediate substrate detachment at the
trailing edge of cells (9), and regulate filipodia and
lamellipodia formation and forward protrusion at the leading edge of
cells (43). In addition calpain may also contribute to caspase-mediated
apoptotic cell death pathways (44-46). However, evidence demonstrating
either a physiological or pathological role for
calpain-dependent proteolysis of focal adhesion components has not been reported.
In this study, we demonstrate that levels of calpain II protein are
regulated at the post-transcriptional level following v-Src
transformation of CEF. Furthermore, in response to activation of v-Src,
calpain activity promotes proteolytic cleavage of FAK, focal adhesion
disassembly, and morphological transformation. Using epitope-specific
antibodies, we have identified putative cleavage fragments generated by
calpain-mediated proteolysis of FAK taking place during v-Src
transformation. FAK cleavage data indicate that a 30-kDa C-terminal
fragment containing the focal adhesion targeting sequence and a
second proline-rich domain dissociates from a 95-kDa amino-terminal
fragment containing the kinase domain. Since the C-terminal domains of
FAK are important in targeting FAK to focal adhesions (47), this
cleavage event is likely to influence the localization of the kinase
domain to focal adhesion sites and may lead to reduced FAK activity at
adhesions, something that is known to negatively regulate FAK function
and cell motility (48). In addition, the C-terminal cleavage fragment
is relatively stable following its generation, so it could potentially
act in a dominant negative role, competing with functionally intact
full-length FAK for focal adhesion substrates.
Calpain-dependent cleavage of FAK would also be expected to
impair the ability of FAK to act as an adapter protein, thereby
compromising the integrity of the focal adhesion complex. Thus,
modulation of FAK function through calpain-dependent
cleavage is likely to play a significant role in the process of
oncogenic transformation.
The induction of calpain-dependent proteolytic cleavage
following v-Src activation appears to be selective for FAK, since other
known calpain substrates within focal adhesions, such as paxillin,
talin, or Src itself do not undergo calpain-dependent processing during v-Src transformation (results not shown). Although the detailed regulation of calpain-mediated FAK cleavage induced by
v-Src remains to be elucidated, we have found that calpain II protein
levels are modulated during transformation. In addition, calpain-induced cleavage of FAK may be linked to specific tyrosine phosphorylation of FAK that leads to complex dissociation, perhaps releasing FAK and permitting its cleavage by calpain. Previous studies
demonstrate that Src-mediated tyrosine phosphorylation of cortactin and
the NR2 subunits of NMDA receptors influences the ability of
these proteins to be cleaved by calpain (49, 50). It is therefore
tempting to speculate that v-Src-dependent phosphorylation
of FAK may influence the suitability of FAK to act as a substrate for
calpain. We are currently investigating the mechanism by which Src
activity controls calpain II protein levels and the influence
phosphorylation of specific FAK residues has on its ability to be
cleaved by calpain.
FAK cleavage mediated by the caspase family of cysteine proteases has
previously been demonstrated in T lymphocytes and endothelial cells
undergoing apoptosis (25, 26). Treatment of v-Src-expressing CEF with
inhibitors against a broad spectrum of caspase family members
(ZVAD-FMK) had no effect on FAK cleavage or morphological transformation. In contrast, FAK cleavage in serum-starved Rat-1 fibroblasts induced to detach and undergo apoptosis after switching off
v-Src activity is suppressed by caspase inhibitors, whereas calpain
inhibitors had no effect. These data lead us to conclude that
proteolysis of FAK is a general phenomenon associated with focal
adhesion disassembly but can be mediated by distinct proteases under
different biological Src-regulated processes: caspases during apoptosis
and calpain (most likely calpain II) during tyrosine kinase-induced
cell transformation.
From the data presented in this report, we propose that calpain- and
caspase-mediated proteolysis of FAK are important mechanisms for
regulating focal adhesion integrity in both viable and apoptotic cells,
respectively. During v-Src transformation of CEF,
calpain-dependent cleavage of FAK precedes disassembly of
the focal adhesion complex and disorganization of the actin
cytoskeleton, leading to a loss of substrate anchorage and increased
motility. This implicates calpain as a major effector in the process of
v-Src-induced oncogenic transformation and Src-dependent
cell migration. A recent study examining the expression of calpain I in
human renal cell carcinomas demonstrated significantly higher levels of
calpain I expression in tumors that presented evidence of metastases to
peripheral lymph nodes relative to low expression levels in tumors that
apparently had not metastasized (51). This study together with our own data suggests that calpain family proteases could mediate disassembly of focal adhesions and loss of substrate anchorage that may contribute to tumor cell motility and invasion. Targeting the inhibition of
calpain activity in transformed cells may provide a useful therapeutic
strategy for the prevention of tumor invasion and metastases.
 |
ACKNOWLEDGEMENT |
We are grateful to John Wyke for critical
review of the manuscript.
 |
FOOTNOTES |
*
This work was supported by the Cancer Research Campaign
(UK).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-141-330-3956;
Fax: 44-141-942-6521; E-mail: n.carragher@beatson.gla.ac.uk.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008972200
 |
ABBREVIATIONS |
The abbreviations used are:
FAK, focal adhesion
kinase;
ALLM, N-acetyl-leucyl-leucyl-methional (calpain
inhibitor I), ALLN, N-acetyl-leucyl-leucyl-norleucinal
(calpain inhibitor II), CEF, chicken embryo fibroblasts;
ZVAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (caspase inhibitor I);
ts (temperature sensitive), PBS, phosphate-buffered
saline.
 |
REFERENCES |
1.
|
Wyke, A. W.,
Frame, M. C.,
Gillespie, D. A.,
Chudleigh, A.,
and Wyke, J. A.
(1995)
Cell Growth Differ.
6,
1225-1234[Abstract]
|
2.
|
Johnson, D.,
Frame, M. C.,
and Wyke, J. A.
(1998)
Oncogene
16,
2017-2028[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Jove, R.,
and Hanafusa, H.
(1987)
Annu. Rev. Cell Biol.
3,
31-56[CrossRef]
|
4.
|
Kellie, S.
(1988)
Bioessays
8,
25-30[Medline]
[Order article via Infotrieve]
|
5.
|
Fincham, V. J.,
Wyke, J. A.,
and Frame, M. C.
(1995)
Oncogene
10,
2247-2252[Medline]
[Order article via Infotrieve]
|
6.
|
Fincham, V. J.,
and Frame, M. C.
(1998)
EMBO J.
17,
81-92[Abstract/Free Full Text]
|
7.
|
Burridge, K.,
Fath, K.,
Kelly, T.,
Nuckolls, G.,
and Turner, C.
(1988)
Annu. Rev. Cell Biol.
4,
487-525[CrossRef]
|
8.
|
Burridge, K.,
and Chrzanowska-Wodnicka, M.
(1996)
Annu. Rev. Cell Dev. Biol.
12,
463-518[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Huttenlocher, A.,
Sandborg, R. R.,
and Horwitz, A. F.
(1995)
Curr. Opin. Cell Biol.
7,
697-706[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Carragher, N. O.,
Levkau, B.,
Ross, R.,
and Raines, E. W.
(1999)
J. Cell Biol.
147,
619-630[Abstract/Free Full Text]
|
11.
|
Huttenlocher, A.,
Palecek, S. P.,
Lu, Q.,
Zhang, W.,
Mellgren, R. L.,
Lauffenburger, D. A.,
Ginsberg, M. H.,
and Horwitz, A. F.
(1997)
J. Biol. Chem.
272,
32719-32722[Abstract/Free Full Text]
|
12.
|
Mellgren, R. L.
(1980)
FEBS Lett.
109,
129-133[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Croall, D. E.,
and DeMartino, G. N.
(1991)
Physiol. Rev.
71,
813-847[Free Full Text]
|
14.
|
Molinari, M.,
and Carafoli, E.
(1997)
J. Membr. Biol.
156,
1-8[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Beckerle, M. C.,
Burridge, K.,
DeMartino, G. N.,
and Croall, D. E.
(1987)
Cell
51,
569-577[Medline]
[Order article via Infotrieve]
|
16.
|
Cooray, P.,
Yuan, Y.,
Schoenwaelder, S. M.,
Mitchell, C. A.,
Salem, H. H.,
and Jackson, S. P.
(1996)
Biochem. J.
318,
41-47[Medline]
[Order article via Infotrieve]
|
17.
|
Oda, A.,
Druker, B. J.,
Ariyoshi, H.,
Smith, M.,
and Salzman, E. W.
(1993)
J. Biol. Chem.
268,
12603-12608[Abstract/Free Full Text]
|
18.
|
Yamaguchi, R.,
Maki, M.,
Hatanaka, M.,
and Sabe, H.
(1994)
FEBS Lett.
356,
114-116[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Hayashi, M.,
Suzuki, H.,
Kawashima, S.,
Saido, T. C.,
and Inomata, M.
(1999)
Arch. Biochem. Biophys.
371,
133-141[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Selliah, N.,
Brooks, W. H.,
and Roszman, T. L.
(1996)
J. Immunol.
156,
3215-3221[Abstract]
|
21.
|
Yoshida, H.,
Murachi, T.,
and Tsukahara, I.
(1984)
FEBS Lett.
170,
259-262[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
David-Pfeuty, T.,
and Singer, S. J.
(1980)
Proc. Natl. Acad. Sci. U. S. A.
77,
6687-6691[Abstract]
|
23.
|
Hanks, S. K.,
and Polte, T. R.
(1997)
Bioessays
19,
137-145[Medline]
[Order article via Infotrieve]
|
24.
|
Ilic, D.,
Furuta, Y.,
Kanazawa, S.,
Takeda, N.,
Sobue, K.,
Nakatsuji, N.,
Nomura, S.,
Fujimoto, J.,
Okada, M.,
and Yamamoto, T.
(1995)
Nature
377,
539-544[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Wen, L. P.,
Fahrni, J. A.,
Troie, S.,
Guan, J. L.,
Orth, K.,
and Rosen, G. D.
(1997)
J. Biol. Chem.
272,
26056-26061[Abstract/Free Full Text]
|
26.
|
Levkau, B.,
Herren, B.,
Koyama, H.,
Ross, R.,
and Raines, E. W.
(1998)
J. Exp. Med.
187,
579-586[Abstract/Free Full Text]
|
27.
|
Squier, M. K.,
Miller, A. C.,
Malkinson, A. M.,
and Cohen, J. J.
(1994)
J. Cell. Physiol.
159,
229-237[Medline]
[Order article via Infotrieve]
|
28.
|
Sasaki, T.,
Kishi, M.,
Saito, M.,
Tanaka, T.,
Higuchi, N.,
Kominami, E.,
Katunuma, N.,
and Murachi, T.
(1990)
J. Enzyme. Inhib.
3,
195-201[Medline]
[Order article via Infotrieve]
|
29.
|
Squier, M. K.,
Sehnert, A. J.,
Sellins, K. S.,
Malkinson, A. M.,
Takano, E.,
and Cohen, J. J.
(1999)
J. Cell. Physiol.
178,
311-319[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Vinitsky, A.,
Michaud, C.,
Powers, J. C.,
and Orlowski, M.
(1992)
Biochemistry
31,
9421-9428[Medline]
[Order article via Infotrieve]
|
31.
|
Milligan, S. A.,
Owens, M. W.,
and Grisham, M. B.
(1996)
Arch. Biochem. Biophys.
335,
388-395[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Seglen, P. O.,
Grinde, B.,
and Solheim, A. E.
(1979)
Eur. J. Biochem.
95,
215-225[Medline]
[Order article via Infotrieve]
|
33.
|
Ziegler, H. K.,
and Unanue, E. R.
(1982)
Proc. Natl. Acad. Sci. U. S. A.
79,
175-178[Abstract]
|
34.
|
Fenteany, G.,
Standaert, R. F.,
Lane, W. S.,
Choi, S.,
Corey, E. J.,
and Schreiber, S. L.
(1995)
Science
268,
726-731[Medline]
[Order article via Infotrieve]
|
35.
|
Suzuki, K.,
Imajoh, S.,
Emori, Y.,
Kawasaki, H.,
Minami, Y.,
and Ohno, S.
(1987)
FEBS Lett.
220,
271-277[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Mohan, P. S.,
and Nixon, R. A.
(1995)
J. Neurochem.
64,
859-866[Medline]
[Order article via Infotrieve]
|
37.
|
Maki, M.,
Bagci, H.,
Hamaguchi, K.,
Ueda, M.,
Murachi, T.,
and Hatanaka, M.
(1989)
J. Biol. Chem.
264,
18866-18869[Abstract/Free Full Text]
|
38.
|
Eto, A.,
Akita, Y.,
Saido, T. C.,
Suzuki, K.,
and Kawashima, S.
(1995)
J. Biol. Chem.
270,
25115-25120[Abstract/Free Full Text]
|
39.
|
Yuan, Y.,
Dopheide, S. M.,
Ivanidis, C.,
Salem, H. H.,
and Jackson, S. P.
(1997)
J. Biol. Chem.
272,
21847-21854[Abstract/Free Full Text]
|
40.
|
Johnson, D.,
Agochiya, M.,
Samejima, K.,
Earnshaw, W.,
Frame, M.,
and Wyke, J.
(2000)
Death Differ.
7,
685-696[CrossRef]
|
41.
|
Fang, F.,
Orend, G.,
Watanabe, N.,
Hunter, T.,
and Ruoslahti, E.
(1996)
Science
271,
499-502[Abstract]
|
42.
|
Ruoslahti, E.,
and Reed, J. C.
(1994)
Cell
77,
477-478[Medline]
[Order article via Infotrieve]
|
43.
|
Potter, D. A.,
Tirnauer, J. S.,
Janssen, R.,
Croall, D. E.,
Hughes, C. N.,
Fiacco, K. A.,
Mier, J. W.,
Maki, M.,
and Herman, I. M.
(1998)
J. Cell Biol.
141,
647-662[Abstract/Free Full Text]
|
44.
|
Kato, M.,
Nonaka, T.,
Maki, M.,
Kikuchi, H.,
and Imajoh-Ohmi, S.
(2000)
J. Biochem. (Tokyo)
127,
297-305[Abstract]
|
45.
|
Wang, K. K.
(2000)
Trends Neurosci.
23,
20-26[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Nakagawa, T.,
and Yuan, J.
(2000)
J. Cell Biol.
150,
887-894[Abstract/Free Full Text]
|
47.
|
Hildebrand, J. D.,
Schaller, M. D.,
and Parsons, J. T.
(1995)
Mol. Biol. Cell
6,
637-647[Abstract]
|
48.
|
Gilmore, A. P.,
and Romer, L. H.
(1996)
Mol. Biol. Cell
7,
1209-1224[Abstract]
|
49.
|
Huang, C.,
Tandon, N. N.,
Greco, N. J.,
Ni, Y.,
Wang, T.,
and Zhan, X.
(1997)
J. Biol. Chem.
272,
19248-19252[Abstract/Free Full Text]
|
50.
|
Bi, R.,
Rong, Y.,
Bernard, A.,
Khrestchatisky, M.,
and Baudry, M.
(2000)
J. Biol. Chem.
275,
26477-26483[Abstract/Free Full Text]
|
51.
|
Braun, C.,
Engel, M.,
Seifert, M.,
Theisinger, B.,
Seitz, G.,
Zang, K. D.,
and Welter, C.
(1999)
Int. J. Cancer
84,
6-9[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.