Normal endothelial and epithelial cells undergo apoptosis when cell adhesion and spreading are
prevented, implying a requirement for antiapoptotic signals from the extracellular matrix for
cell survival. We investigated some of the molecular changes occurring in focal adhesions during growth factor deprivation-induced apoptosis in confluent monolayers of human umbilical
vein endothelial cells. Among the first morphologic changes after initiation of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the matrix followed by detachment. We observe a specific proteolytic cleavage of focal adhesion kinase
(pp125FAK), an important component of the focal adhesion complex, and identify pp125FAK as a
novel substrate for caspase-3 and caspase-3-like apoptotic caspases. The initial cleavage precedes detachment, and coincides with loss of pp125FAK and paxillin from focal adhesion sites
and their redistribution into the characteristic membrane blebs of apoptotically dying cells.
Cleavage of pp125FAK differentially affects its association with signaling and cytoskeletal components of the focal adhesion complex; binding of paxillin, but not pp130Cas (Cas, Crk-associated
substrate) and vinculin, to the COOH terminally truncated pp125FAK is abolished. Therefore,
caspase-mediated cleavage of pp125FAK may be participating in the disassembly of the focal adhesion complex and actively interrupting survival signals from the extracellular matrix, thus propagating the cell death program.
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Introduction |
Anchorage of cells to the extracellular matrix and the
resulting integrin-mediated signaling events are
thought to play a crucial role in cell survival (1). Endothelial and epithelial cells undergo apoptosis when cell anchorage is prevented in vitro or in vivo (2). However, recent
evidence suggests that the shape of the cell and the rigidity
of the nucleocytoskeletal architecture, both intricately connected to integrin engagement, have a major impact on the
cell's decision between life and death (7). The focal adhesion complex is the multifunctional structure that the cell
uses for integrating integrin-mediated signaling events from the matrix with the dynamic cytoskeletal meshwork that
mechanically couples the cell with the extracellular matrix.
Appropriate orientation of the signaling machinery of the
cell is dependent on the structure of the focal adhesion
complex, which provides an intricate interrelationship between the extracellular matrix, the cytoskeleton, and signaling cascades (8).
A central member of the focal adhesion complex is the
tyrosine kinase pp125FAK (FAK, focal adhesion kinase1; reference 12), which has been implicated in the integration of
signals from integrins, oncogenes, and neuropeptides (13). Although pp125FAK has been shown to be dispensible for
the assembly of focal adhesions, it has a key role in the assembly of various signaling proteins recruited to focal adhesions and the downstream events initiated by these components (14). pp125FAK has also been shown to play an
important role in cell survival; constitutively active forms of
pp125FAK can rescue epithelial cell lines from apoptosis in
suspension (15). Inversely, fibroblasts undergo apoptosis
when the interaction of pp125FAK with the cytoplasmic domain of the beta 1 integrin is inhibited by microinjection of
a peptide identical to the pp125FAK binding site on the integrin, or by microinjection of an antibody interfering with
this binding (16). Proteolytic alteration of pp125FAK during
c-myc-induced apoptosis has been suggested (17), but the
nature of this change remains unclear. Evidence has also
been provided for proteolytic degradation of pp125FAK
during platelet aggregation (18).
In this study, we characterize a distinct pattern of specific
proteolytic cleavage of pp125FAK during apoptosis of human umbilical vein endothelial cells (HUVECs) induced by
growth factor (GF) deprivation. Further, we identify the
enzymes responsible for this cleavage to be members of the family of death proteases (caspases), related to the mammalian interleukin 1
-converting enzyme (ICE), and the
product of the Caenorhabditis elegans ced-3 gene, which are
instrumental in the execution phase of apoptosis (19). Evidence for the influence of this cleavage on potential downstream events in the apoptotic process is also provided.
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Materials and Methods |
Protein Analysis and Immunoprecipitations.
Cells were lysed in
50 mM Tris/HCl, pH 7.4, 250 mM NaCl, 0.5% NP-40, 10%
glycerol, 5 mM EDTA, 50 mM NaF, 0.5 mM Na3VO4, 10 mM
-glycerophosphate, PMSF, leupeptin, and aprotinin. Lysates were separated on 10% SDS-PAGE, proteins transferred to Immobilon membrane (Millipore, Bedford, MA), and immunoblotted with specific antibodies. All immunoblots were visualized by
enhanced chemiluminescence (ECL; Amersham Corp., Arlington
Heights, IL). The following antibodies were used: polyclonal antibodies to pp125FAK (Santa Cruz Biotechnology, Santa Cruz,
CA), monoclonal antibodies to pp125FAK, paxillin, p130Cas (Cas,
Crk-associated substrate) (Transduction Labs., Lexington, KY),
vinculin (Sigma Chemical Co., St. Louis, MO), and poly(ADP-ribose) polymerase (PARP) (Enzyme Systems Products, Dublin,
CA). Proteins associated with pp125FAK were detected by immunoprecipitation with the NH2-terminal pp125FAK antibody, followed by immunoblotting with specific antibodies.
Immunocytochemistry, Cell Fractionation Analysis, and Inhibition of
Apoptosis by Benzyloxycarbonyl-Val-Ala-Asp Fluoromethyl Ketone.
HUVECs were plated on gelatin-coated chamber slides, grown
for 48 h at 37°C, and starved for 4 h. Cells were fixed in 4%
paraformaldehyde for 20 min at room temperature, washed two
times with PBS, and permeabilized in 0.5% NP-40 in PBS for 10 min. After another two washes, quenching was performed by 3 × 5 min incubations with 50 mM NH4 acetate. Fixed cells were incubated with monoclonal antibodies to pp125FAK (1:50) or paxillin (1:200) in 0.1% BSA/PBS for 1 h at room temperature followed by incubation with a fluorescein isothiocyanate-labeled anti-mouse IgG antibody (Cappel, Durham, NC). Actin was
stained using phalloidin (1:1,000, Sigma Chemical Co.). Cell
fractionation was performed as described for cyclin A (20) using
0.1% digitonin to gently solubilize the plasma membranes. In experiments using the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (ZVAD-fmk; Alexis Biochemicals,
San Diego, CA), cells were preincubated with the inhibitor for
1 h and then exposed to GF deprivation in the presence of the inhibitor.
In Vitro Cleavage of pp125FAK.
When endogenous pp125FAK
from cell lysates was used as a substrate, control cells were lysed
on ice in 10 mM Hepes/KOH, pH 7.4, 2 mM EDTA, 5 mM
dithiothreitol, 1% NP-40, and protease inhibitors leupeptin and
aprotinin as previously described (21). Lysates were cleared by
centrifugation at 27,000 g for 5 min, and 50 µg cell lysate was incubated with 500 ng (10 pmol) of the individual caspases at 37°C
for 2 h in a total volume of 10 µl in reaction buffer (50 mM
Hepes/KOH, pH 7.4, 0.1 M NaCl, 0.1% 3-[3-cholamidopropyl]dimethyl-ammonio-1-propanesulfonate (CHAPS), and
10% sucrose). In vitro transcription and translation of pp125FAK
were performed using the TNT® coupled reticulocyte lysate system (Promega, Madison, WI) and [35S]methionine (1,000 Ci/
mmol; Amersham Corp.) according to the manufacturer's instructions. The expression plasmid for pp125FAK was a gift from
Dr. J.T. Parsons (University of Virginia, Charlottesville, VA). 1/
500 of the reaction was used as a substrate and incubated with 250 ng (5 pmol) of the individual caspases, or with 10 µg apoptotic or
control cell lysates, respectively, and prepared as described above,
in the presence or absence of 10 nM N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO; Bachem Bioscience, King of Prussia, PA) in a total volume of 9 µl in reaction buffer at 37°C for
1.5 h. The reactions were stopped in all cases by the addition of
4× sample buffer. Purified caspase-3, -7, and -6 were a gift from
Drs. K. Orth and V.M. Dixit (University of Michigan, Ann Arbor, MI).
 |
Results and Discussion |
Time-dependent Cleavage of pp125FAK in Apoptotic Endothelial Cells.
HUVECs undergo apoptosis when deprived of
GFs. Among the first morphologic changes after initiation
of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the substratum followed by detachment. After detachment, the apoptotic
cells appear in the culture medium as "floaters", displaying
characteristic morphologic (membrane blebbing, nuclear condensation, and fragmentation; reference 22) and biochemical (DNA laddering, caspase activation, cleavage of
PARP) features of apoptosis (data not shown). Apoptotic
floaters are observed as early as 4 h after GF withdrawal and
after 12-16 h account for 40-45% of the total cell population, whereas the remaining adherent cells are viable ("viable
cells") and would survive and proliferate if supplemented
with GFs in the culture medium (data not shown).
To determine whether alterations in a central member of
the focal adhesion complex, the tyrosine kinase pp125FAK,
coincide with apoptosis and the detachment of apoptotic
cells from the matrix, we probed lysates from HUVECs exposed to GF deprivation for increasing periods of time with
different antibodies to pp125FAK (Fig. 1 A). pp125FAK undergoes distinct proteolytic changes with time, and completely disappears in the pure apoptotic cell population (the
floaters). Three different NH2-terminal proteolytic fragments of ~100, 90, and 48 kD are recognized with a polyclonal NH2-terminal antibody. The 100-kD fragment appears as early as 2 h after GF deprivation, decreases in
intensity over the next 10 h, and is almost totally absent in
the floaters. The 90-kD fragment appears first at 2 h, but
increases in intensity with time, and constitutes the principal high molecular weight proteolytic pp125FAK fragment
present in the floaters. The 48-kD fragment first appears at
4 h, and its levels also increase with time. A monoclonal
antibody to amino acids 354-534 of pp125FAK also recognizes both the 90- and 100-kD fragments, but fails to detect the 48-kD fragment. This monoclonal antibody recognizes an additional 32-kD fragment, which appears first at 4 h,
suggesting generation of an internal proteolytic pp125FAK
fragment devoid of both a COOH and an NH2 terminus.
A polyclonal antibody to the COOH terminus of pp125FAK
does not recognize the 100-, 90-, 48-, or 32-kD fragments,
but detects two different fragments: a principal fragment of
36 kD, and a smaller one of 29 kD, with the main COOH-terminal fragment of 36 kD appearing as soon as 2 h after
GF deprivation, which then remains constant with time.
Substantial amounts of the 100-kD pp125FAK cleavage fragment are present after just 2 h of GF deprivation, and appearance of the 100-kD fragment of pp125FAK precedes
cleavage of PARP. Significant amounts of cleaved PARP product are detectable 4-8 h after GF deprivation (Fig. 1 B).

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Fig. 1.
Appearance of proteolytic
fragments of pp125FAK during GF deprivation-induced apoptosis. (A) HUVECs were deprived of GF for the indicated times and cell lysates of pooled
cell populations (0-12 h) or only apoptotic floaters at 12 h (A) were immunoblotted for pp125FAK with three different antibodies: a polyclonal antibody to residues 2-18 at the NH2 terminus (N-pp125FAK), a monoclonal antibody to residues 354-534 (354-534N-pp125FAK), and a polyclonal antibody to residues 903-1,052 at the COOH terminus (C-pp125FAK). Arrowheads, approximate molecular weights of
the fragments (kD). (B) Cleavage of PARP during endothelial apoptosis
was analyzed as described in A and detected using a monoclonal antibody
to PARP.
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Caspase-mediated Cleavage of pp125FAK.
Analysis of the
amino acid sequence of pp125FAK reveals several putative
cleavage sites for caspases (23, 24): seven sites for caspase-3-like caspases, which contain the conserved motif DXXD, and eight sites with the sequence (IVL)XXD,
which are cleaved preferentially by caspase-6-like caspases
(Fig. 2). To investigate whether cleavage by caspases is involved in the generation of the proteolytic pp125FAK fragments in endothelial cell apoptosis, we incubated in vitro translated pp125FAK with purified caspase-3, -6, and -7, a
caspase recognizing the same substrate motif as caspase-3
(Fig. 3 A). We further tested whether cell lysates from apoptotic cells contain endogenously activated caspases that
could cleave in vitro translated pp125FAK (Fig. 3 B). Both
recombinant caspase-3 and -7 cleave pp125FAK into two
main fragments, which are ~100 and 36 kD. The apoptotic cell lysates, used as a source of active endogenous
caspases, generate a cleavage pattern identical to the one
observed with recombinant caspase-3 and -7. This
pp125FAK cleaving activity in apoptotic cell lysates was entirely inhibited in the presence of 10 nM Ac-DEVD-CHO, a specific inhibitor of caspase-3-like caspases (25),
suggesting active endogenous caspases to be responsible for
the cleavage. Cell lysates from control cells do not cleave
pp125FAK, with or without the caspase inhibitor. Caspase-6
is much less efficient in cleaving the native in vitro translated pp125FAK molecule. The cleavage fragments generated by caspase-6 also differ from those resulting from
caspase-3-mediated cleavage, and the predominant fragment is ~90 kD. A fragment of the same size is also seen as
a result of pp125FAK cleavage by apoptotic cell lysates (Fig.
3 B), and appears to correspond to the final, ~90-kD fragment in apoptotic cells (Fig. 1).

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Fig. 2.
Schematic diagram
of pp125FAK and FRNK. The
domains of pp125FAK and FRNK
responsible for interaction with
different binding partners are indicated, and the putative cleavage sites for caspase-3 (black arrowheads) and caspase-6 (white
arrowheads) are shown. The putative fragments resulting from
caspase-mediated cleavage observed during GF deprivation-
induced endothelial cell apoptosis and the epitopes of the antibodies used for analysis are illustrated diagrammatically in relation
to pp125FAK.
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Fig. 3.
pp125FAK is a substrate for caspases. (A) In vitro-translated
and [35S]methionine-labeled pp125FAK was incubated with reaction
buffer alone (C), caspase-3, -7, -6, and 10 µg apoptotic (apo) or control
(control) cell lysates in the presence (+) or absence ( ) of the caspase inhibitor Ac-DEVD-CHO (10 nM) at 37°C for 1.5 h. The samples were
subjected to SDS-PAGE and pp125FAK cleavage fragments were detected
by autoradiography. (B) Cell lysates from control cells were used as a
source of endogenous pp125FAK and incubated with caspase-3, -7, -6, and
reaction buffer alone (control) for the indicated times. Caspase-generated
pp125FAK cleavage fragments were compared to those present in cells exposed to GF deprivation for 12 h (C, control, V, viable, and A, apoptotic floaters) by immunoblotting using the monoclonal antibody to an internal
(amino acids 354-534) pp125FAK epitope.
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We next compared, side by side, the in vitro-generated
pp125FAK cleavage fragments with those observed during
HUVEC apoptosis by incubating recombinant caspases
with cell lysates from control cells as a source of endogenous pp125FAK, which also provides the molecular environment in which pp125FAK cleavage occurs (Fig. 3 B).
Both caspase-3 and -7 cleave endogenous pp125FAK completely and generate the upper pp125FAK cleavage fragment
of 100 kD. Surprisingly, under these experimental conditions, caspase-6 is as effective as caspase-3 in completely cleaving endogenous pp125FAK and generating the initial
100-kD fragment, and then proceeds further to efficiently
generate the lower 90-kD fragment. The 90-kD fragment
appears to be derived from the 100-kD fragment, since the
100-kD fragment disappears completely after 90 min, whereas the 90-kD fragment increases in intensity. Since
caspase-6 can activate caspase-3 (26), it is possible that exogenously added caspase-6 activates endogenous caspase-3,
which then initiates the first cleavage of pp125FAK and acts
together with caspase-6 to further degrade pp125FAK to the
final 90-kD fragment in a manner similar to the indirect cleavage of U1-70 kD and PARP mediated by caspase-6
through its activation of caspase-3 (26). Using both the antibody to the internal epitope (354-534N-pp125FAK; Fig. 3 B)
and the NH2-terminal pp125FAK antibody (data not
shown), we compared the pp125FAK cleavage fragments in
the in vitro cleavage reactions with those in straight cell lysates from control, viable, and apoptotic cells, and observed
that the purified caspases generate an identical pattern to
that observed in the HUVEC apoptosis time course (Fig. 3
B). From these observations we conclude that caspase-3
and/or caspase-3-like caspases are responsible for the initial
cleavage of pp125FAK to a 100-kD fragment, and that they,
together with caspase-6 and/or caspase-6-like caspases,
participate in the further degradation of the molecule (see
Fig. 2).
Cleavage Products of pp125FAK Are Observed in Both the Nuclear and Cytoplasmic Fractions of Apoptotic Cells.
Caspase cleavage often results in altered cellular localization of substrates
after cleavage (21). We, therefore, asked whether the subcellular localization of pp125FAK is altered in cells undergoing apoptosis. We isolated nuclear and cytoplasmic extracts
from control cells and cells exposed to GF deprivation for 5 h,
a time when the majority of the cells are viable and most of
the cells undergoing apoptosis remain attached. Although
full-length pp125FAK is detected mainly in the cytoplasmic
fractions of control and GF-deprived cells, the 100- and
90-kD cleavage fragments of pp125FAK in GF-deprived
cells are detected both in the cytosolic and in the nuclear
fraction, 67 and 34% of the total, respectively (Fig. 4), suggesting entry of pp125FAK fragments into the nucleus after
cleavage. Probing the same lysates for proliferating cell nuclear antigen (PCNA) and vinculin as controls show mainly
nuclear PCNA and cytoplasmic vinculin localization, consistent with minimal cross-contamination between nuclear
and cytoplasmic compartments using a digitonin-based
protocol (20). We have no explanation for this putative
nuclear translocation of pp125FAK fragments, and analysis of
the COOH-terminal fragment of pp125FAK generated by
caspase-mediated cleavage did not reveal any obvious nuclear export signal, although partial homology is observed
in the sequence 884LxxLxxL891.

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Fig. 4.
Intracellular distribution of pp125FAK in GF deprivation-induced apoptosis. The
nuclear (N) and cytoplasmic (C)
fractions of control cells and cells
deprived of GF for 5 h were immunoblotted for pp125FAK,
PCNA, and vinculin. The nuclear and cytoplasmic fractions
were normalized to contain equal
cell equivalents (400,000/lane).
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Cleavage of pp125FAK Alters Its Interaction with Components
of the Focal Adhesion Complex.
Kinetic analysis of pp125FAK
cleavage in GF-deprived HUVECs reveals caspase-mediated proteolysis early in the process of apoptosis. We,
therefore, examined how this cleavage may affect pp125FAK
interaction with other focal adhesion components. The first
proteolytic cleavage leads to loss of ~200 COOH-terminal
amino acids (see Fig. 2), the domain that contains the focal
adhesion targeting sequence (COOH-terminal residues
904-1,040), required for efficient localization to focal adhesions, as well as the paxillin binding site (COOH-terminal 148 residues), which overlaps the focal adhesion targetting sequence, but is functionally separate (27). We
therefore examined the ability of the COOH-terminally
truncated pp125FAK in apoptotic cells to bind paxillin. Immunoprecipitation of pp125FAK from control, viable, and
apoptotic cells shows that the NH2-terminal 90-kD
pp125FAK fragment can be efficiently immunoprecipitated
from apoptotic cells (Fig. 5). Immunoblotting for paxillin
on the same blots shows paxillin to coimmunoprecipitate
with pp125FAK in control and viable cells, but to be virtually absent from the 90-kD pp125FAK cleavage fragment in
apoptotic cells, despite the presence of diminished, but substantial amounts of paxillin in total cell lysates from apoptotic cells (Fig. 5). The COOH terminus of pp125FAK also
contains a proline-rich region, P712PKPSR, which has been
shown to mediate the binding of p130Cas through its Src
homology domain (SH)3 domain (28) (Fig. 2). This region
would still be on the principal 90-kD NH2-terminal pp125FAK fragment in apoptotic cells (predicted to end at
the proposed cleavage site DQTD772S), and should therefore be available for interaction with p130Cas. Indeed, we
see residual p130Cas to be associated both with the native
pp125FAK protein and the 90-kD NH2-terminal pp125FAK
fragment in apoptotic cells, in spite of the fact that no
p130Cas is detected in straight lysates from apoptotic cells
(Fig. 5). Vinculin, another component of the focal adhesion complex, can also be detected in pp125FAK immunoprecipitates, and shows no changes between control, viable, and apoptotic cells, and total protein levels of vinculin in
the same lysates are quite similar (Fig. 5). Immunoblotting
of pp125FAK and paxillin immunoprecipitates with an antiphosphotyrosine antibody shows no tyrosine phosphorylation, and no c-Src is associated with the complex (data
not shown). These data suggest that cleavage of pp125FAK
results in a selective modification of the pattern of molecules it associates with, and thus may affect both its cytoskeletal and signaling functions.

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Fig. 5.
Differential association of apoptotic pp125FAK cleavage fragments with components of the focal adhesion complex. pp125FAK was immunoprecipitated from control (C), viable (V) and apoptotic (A) cells and
associated paxillin, p130Cas, and vinculin detected by immunoblotting
(left). Western blots for the same proteins present in 50 µg of total cell lysates are shown (right).
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Dissociation of Molecular Components of Focal Adhesions and
Membrane Blebbing.
Among the hallmarks of apoptosis in
virtually all known apoptotic systems is the phenomenon of
plasma membrane blebbing. The sudden onset of surface
blebbing, together with cytoplasmic fragmentation, condensation, and exfoliation is one of the earliest morphologic events in apoptosis. We observe membrane blebbing
very early in the process of GF deprivation-induced apoptosis of endothelial cells, always preceding the detachment
of apoptotic cells as floaters. Membrane blebbing has been
recently described to be independent of caspases during apoptosis induced by oncogenes, DNA damage, and expression of the proapoptotic bcl-2 homologue bak in Rat-1 fibroblasts. The broad-spectrum cell permeable caspase
inhibitor ZVAD-fmk could prevent cleavage of nuclear
lamins and PARP, as well as DNA fragmentation, but was
unable to prevent membrane blebbing (29). In contrast, we
observe ZVAD-fmk to protect HUVEC from apoptosis,
and to be equally effective in dose dependently inhibiting
cleavage of known endogenous substrates, including pp125FAK, and apoptotic membrane blebbing (data not
shown). A possible explanation for this divergence could be
the different methods used to induce apoptosis, which may
affect the point in the signaling cascade where apoptosis is
initiated in the two systems. In addition, we are studying
apoptosis in normal diploid cells, which may have distinct
pathways from transformed cell lines.
To test whether the molecular changes in pp125FAK that
alter its interaction with components of the focal adhesion
complex correlate with changes in the cellular architecture
and composition of focal adhesions, we examined the distribution of pp125FAK, paxillin, and actin in cells undergoing apoptosis 4 h after GF deprivation. At this time point,
the majority of the cells are viable and individual cells begin
to exhibit membrane blebbing but remain attached to the
substratum. Using confocal microscopy, we see single GF-deprived cells to lose pp125FAK immunostaining in the focal
adhesions at the bottom of the cell (Fig. 6), and these same
cells exhibit membrane blebbing with immunoreactivity
for pp125FAK observed in the membrane blebs on the apical
surface 4 µm above the basal section (Fig. 6). We also observe enhanced immunoreactivity for pp125FAK in the cell
nuclei of retracting and blebbing cells, in accordance with
the cell fractionation studies. Immunostaining for paxillin (Fig. 7) also shows loss of paxillin from focal adhesions, but a distinct feature of these cells is a dramatic redistribution and enrichment for paxillin in the membrane blebs. No actin filaments are visible in the blebbing cells (Fig. 6), and
the membrane blebs frequently show an actin ring surrounding the bleb.

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Fig. 6.
Loss of pp125FAK from focal adhesions and its appearance in the membrane blebs of retracting cells. Confocal microscopy of immunostaining for pp125FAK on control cells (C) and cells deprived of GF for 4 h (-GF), as seen at the bottom of the cell and 4 µm above the basal level. pp125FAK immunostaining is observed in focal adhesions in control and viable cells (arrows), but is lost from focal adhesions and redistributes into the membrane blebs of
retracting cells during the process of their detachment from the substratum (arrowheads).
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Fig. 7.
Loss of paxillin from focal adhesions and its accumulation in
membrane blebs of retracting cells is accompanied by disassembly of the
actin cytoskeleton. Double staining for paxillin and actin was performed
on control cells (C) and on cells deprived of GF for 4 h (-GF), and the
images were evaluated by confocal microscopy. Arrows, paxillin localized
in focal adhesions and actin stress fibers in the control cells; arrowheads,
membrane blebs devoid of actin and highly enriched with paxillin on individual retracting and detaching cells after GF withdrawal.
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Does Dissolution of Adherence Complexes Accelerate Apoptosis?
In conclusion, we have identified pp125FAK as a new
cleavage substrate of the apoptotic caspases and suggest that
the membrane blebbing observed in GF deprivation-induced
apoptosis of HUVECs results, at least in part, from the disassembly of the regular architecture of the focal adhesions
and from disruption of molecular interactions within focal
adhesions. Besides the changes in focal adhesions, we also
observe a dissolution of adherens junctions in apoptotic endothelial cells, including caspase cleavage of
-catenin and
plakoglobin, and modifications of vascular endothelial cadherin (Herren, B., B. Levkau, E.W. Raines, and R. Ross,
manuscript submitted). Both processes may contribute to
the active interruption of extracellular signals required for
cell survival. It is possible that this controlled disengagement of cell-matrix and cell-cell interactions results in profound changes in cell shape providing a cell geometry permissive for subsequent irreversible apoptotic events (7).
Chen et al. (7) used different patterns on microfabricated surfaces to alter the extent of cell spreading while retaining a constant cell-matrix interaction area. They observed that
the more rounded endothelial cells had a higher apoptotic
index. It is also possible that initiation of apoptosis in large
vessel endothelial cells may be more dependent on modulation of cell adherence than other cell types; once endothelial cells are detached and swept into the circulation, a
mechanism to prevent their survival and proliferation at
distal sites is critical.
Activation of caspases, such as caspase-3, are generally
thought to trigger the final degradative phase of apoptosis
(30), but they constitute an amplified protease cascade
whose sequence and end points have not been fully defined. Although a few targets of caspase-mediated proteolysis during apoptosis have been identified, their relationship
to the apoptotic process is also not clear. Our identification
of caspase cleavage of pp125FAK and adherens junction
components early in the process of endothelial apoptosis
suggests the possibility that initial cleavage of the molecular
elements required for maintenance of extracellular-cytoskeletal interactions and cell shape may be critical for the
progression of the cell death program to the final execution
stage, particularly in cell types so dependent on cell-cell
and cell-matrix interactions for survival as endothelial cells.
Of interest is also the question of whether the main COOH-terminal pp125FAK fragment we observe in apoptotic cells,
which is structurally very similar to the pp125FAK-related
nonkinase (FRNK; see Fig. 2), acts in a manner similar to
FRNK. FRNK competitively blocks the formation of focal
adhesions (31), and thus, if the COOH-terminal pp125FAK
cleavage fragment is also a competitive inhibitor, it may
further promote disassembly of the focal adhesion in cells
induced to undergo apoptosis.
Whether cleavage of pp125FAK through the apoptotic
caspases is an initiating event in the disassembly of the focal
adhesion complex or merely participates in its progression
remains unanswered, although the altered pattern of molecular partners that pp125FAK can associate with after
cleavage suggests a role in this process. The correct assembly and interaction of molecules in the focal adhesion complex, occurring mainly via SH2 and SH3 domains, may be
necessary for the mediation of survival signals from the extracellular matrix. This hypothesis is supported by the ability of SH2 domains from a number of signaling molecules
to inhibit the "initiation phase" of apoptosis in a cell-free
apoptotic system using extracts from Xenopus eggs (32).
The identification of pp125FAK as a target of the apoptotic
caspases, together with previous reports on the necessity of
pp125FAK for survival (15, 16), suggest the possibility that,
once initiated, the apoptotic program may be accelerated
by interrupting pp125FAK-mediated survival signals from
the extracellular matrix through the proteolytic destruction
of the mediator.
Received for publication Received for publication 8 October 1997 and in revised form 8 December 1997..
We would like to thank Bonnie Ashleman for technical assistance with the confocal microscopy, Drs. J. Thomas Parsons and Cheryl
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