hsp72 Inhibits Focal Adhesion Kinase Degradation in ATP-depleted
Renal Epithelial Cells*
Haiping
Mao
,
Fanghong
Li§,
Kathleen
Ruchalski§,
Dick D.
Mosser¶,
John H.
Schwartz§,
Yihan
Wang
, and
Steven C.
Borkan§**
From the § Renal Section, Department of Medicine, Boston
Medical Center, Boston University, Boston, Massachusetts 02118-2518, the
Department of Nephrology, First Affiliated Hospital,
Zhongshan University, GuangZhou 510080, China, the
Department of Pathology, Tufts University and New England
Medical Center, Boston, Massachusetts 02111, and the
¶ Department of Molecular Biology and Genetics, University of
Guelph, Ontario N1G 2W1, Canada
Received for publication, January 6, 2003, and in revised form, February 21, 2003
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ABSTRACT |
Prior heat stress (HS) or the selective
overexpression of hsp72 prevents apoptosis caused by exposure to
metabolic inhibitors by protecting the mitochondrial membrane and
partially reducing caspase-3 activation. Focal adhesion kinase (FAK), a
tyrosine kinase, exhibits anti-apoptotic properties and is a potential target for degradation by caspase-3. This study tested the hypothesis that hsp72 interacts with FAK, preventing caspase-3-mediated
degradation during ATP depletion. ATP depletion (5 mM
NaCN and 5 mM 2-deoxy-D-glucose in the absence
of medium glucose) caused FAK degradation within 15 min. FAK
degradation was completely prevented by a caspase-3-specific inhibitor.
HS induced the accumulation of hsp72, increased the interaction between
hsp72 and FAK, and significantly inhibited FAK degradation during ATP
depletion. Selective overexpression of wild-type hsp72 (but not
hsp72
EEVD) reproduced the protective effects of HS on FAK cleavage.
Purified hsp72 prevented the degradation of FAK by caspase-3 in
vitro in a dose-dependent manner without affecting
caspase-3 activity. Interaction between hsp72 and FAK is critical
because both exogenous ATP and deletion of the substrate-binding site
decreased protection of FAK by hsp72. These data indicate that FAK is
an early target of injury in cells exposed to metabolic inhibitors and
demonstrate that hsp72 reduces caspase-3-mediated proteolysis of FAK,
an anti-apoptotic protein.
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INTRODUCTION |
Transient ischemia in vivo (1) and ATP depletion
in vitro (2-4) induce apoptosis in renal epithelial cells.
Apoptosis in renal cells (like many eukaryotic cells) is characterized
by mitochondrial injury and the subsequent activation of caspase-3 (5).
In addition to mitochondrial membrane injury, ischemia and ATP
depletion perturb cell-cell contact sites (6, 7) and cause cell
detachment (7, 8). These untoward events predispose cells to apoptosis
and contribute to the acute deterioration of renal function (9-12).
Several studies suggest that hsp72 exerts anti-apoptotic effects in a
variety of cell types subjected to diverse stresses (5, 13-18). In
renal epithelial cells, prior heat stress protects cell contact sites
(6) and inhibits apoptosis caused by ATP depletion (3). Recent studies
in renal epithelia show that heat exposure and the selective
overexpression of hsp72 decrease, but do not abolish, mitochondrial
membrane injury and the subsequent activation of caspase-3 (5, 17).
These observations suggest that hsp72 could provide additional
cytoprotection by inhibiting caspase-3 and/or by preventing caspase-3
from degrading anti-apoptotic proteins.
Focal adhesion kinase (FAK)1
is a multifunctional non-receptor protein-tyrosine kinase that promotes
the formation of cell-substrate contact sites in response to
integrin-mediated contact with the extracellular matrix (19, 20).
Integrin-associated focal adhesion complexes localize to these contact
sites and provide the primary "adhesive link" between the actin
cytoskeleton and extracellular matrix proteins (21). FAK regulates the
turnover of the focal adhesion complex (22) that is required for
migration (23), differentiation (24), and cell growth (21, 25). FAK
itself is regulated by its state of tyrosine phosphorylation, its
intracellular distribution, and protease-mediated cleavage of its
kinase and focal adhesion-targeting domains (26-28).
Recent evidence suggests that FAK suppresses apoptosis (26-33). The
selective overexpression of FAK protects against apoptosis induced by a
variety of noxious insults (29, 32, 34). Conversely, FAK proteolysis
(26, 28, 35) or the overexpression of mutant FAK proteins (33) promotes
apoptosis. During cell stress, cysteinyl aspartate-specific proteases
(caspases) cause the sequential cleavage of intact FAK into several
smaller protein fragments (26, 27). Of the intracellular proteases,
caspase-3 has been shown to exert a major role in the degradation of
FAK into pro-apoptotic fragments (20, 27, 36).
In this study, we hypothesized that hsp72, an inducible
cytoprotective protein, prevents FAK cleavage by caspase-3
during ATP depletion. Our study shows that transient exposure to
metabolic inhibitors results in the rapid proteolysis of FAK. Heat
stress with recovery induced hsp72 and significantly inhibited FAK
proteolysis during ATP depletion. Acute heat stress without recovery
did not increase hsp72 content and failed to prevent FAK degradation in ATP-depleted cells. In intact cells, caspase-3 inhibition or the selective overexpression of hsp72 reproduced the protective effect of
heat stress on FAK proteolysis. Wild-type hsp72 co-immunoprecipitated with FAK. Prior heat stress and ATP depletion markedly increased the
interaction between hsp72 and FAK. In an in vitro assay,
purified human caspase-3 produced virtually complete fragmentation of
intact human FAK. Purified human hsp72 inhibited FAK cleavage by
caspase-3 in a dose-dependent manner. Preservation of FAK
was more striking in the absence of ATP and was not observed when the
hsp72
EEVD deletion mutant was overexpressed, suggesting that
interaction with hsp72 mediates protection of FAK. This study supports
the hypothesis that hsp72 inhibits apoptosis, at least in part, by preventing caspase-3-mediated proteolysis of FAK.
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EXPERIMENTAL PROCEDURES |
Materials--
All reagents were obtained from Sigma unless
otherwise indicated.
Cell Culture--
Renal epithelial cells (CRL-1840) derived from
opossum kidney (OK) were purchased from American Type Culture
Collection. Previously characterized human kidney cells (37)
were generously provided by Dr. Richard Zager (University of
Washington, Seattle, Washington). Cells were grown in Dulbecco's
modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf
serum. Human kidney cells required the addition of 50% Dulbecco's
modified Eagle's medium and 50% Ham's F-12 medium in the presence of
10% fetal calf serum. Cells were used within 72 h of achieving confluence.
ATP Depletion--
To induce ATP depletion, cells were incubated
for 15-60 min at 37 °C in glucose-free medium (Dulbecco's modified
Eagle's medium) containing sodium cyanide and
2-deoxy-D-glucose (5 mM each) as previously
described (38). In control cells, parallel medium changes were
performed using 10 mM glucose-containing Dulbecco's modified Eagle's medium.
Induction of Wild-type hsp72 and hsp72
EEVD--
Wild-type
hsp72 content was increased either by transient heat stress (42.5 ± 0.5 °C for 45 min) in a temperature-regulated incubator followed
by incubation at 37 °C for 16-18 h (38) or by co-infection of OK
cells with adenoviruses containing human wild-type hsp72 and green
fluorescent protein (AdTR5/hsp70-GFP) expressed on separate cistrons
and a tetracycline-regulated promoter (AdCMV/tTA) as previously
described (18). Control cells were co-infected with AdTR5/GFP and
AdCMV/tTA. To increase hsp72 content, cells were infected for 24 h
at 37 °C with 3 × 107 plaque-forming
units/35-mm2 Petri dish. hsp72
EEVD, a deletion mutant of
hsp72 lacking the C-terminal EEVD sequence that is essential for
peptide binding, was also overexpressed using adenovirus as previously
described (18). After removing the virus, cells were incubated for an additional 24 h. Infection efficiency was >95% as estimated by direct visualization of GFP. Preliminary experiments were performed to
determine the amount of adenovirus and the incubation conditions required to approximate hsp72 content in heat-stressed cells as measured by immunoblot analysis (described below).
Immunoblot Analysis and Co-immunoprecipitation--
Harvested
cells were resuspended in cell lysis buffer (150 mM NaCl,
10 mM Tris-HCl, 5 mM EDTA, 1 mM
EGTA, and 1% Triton-X-100) and a mixture of protease inhibitors (5 µM 4-(2-aminoethyl)benzenesulfonyl fluoride HCl, 10 nM leupeptin, 1.5 nM aprotinin, 10 nM E-64, and 5 µM EDTA, pH 7.40;
Calbiochem-Novabiochem). The cells were sonicated and then centrifuged
at 10,000 × g for 10 min at 4 °C. hsp72 (SPA-810, Stressgen Biotech Corp., Victoria, British Columbia, Canada), hsp70
EEVD (SPA-811, Stressgen Biotech Corp.), and FAK (BD
Biosciences) were detected in the supernatant by immunoblotting using
commercially available monoclonal antibodies. Specific protein bands
were detected with an anti-IgG antibody coupled to a horseradish
peroxidase-based enzyme-linked chemiluminescence system (Lumigolow,
Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). After
digitizing the image of each immunoblot (Desk Scan II, Hewlett-Packard
Co.), band densities were quantified using NIH ImageQuant software. Immunoprecipitation of FAK using a specific monoclonal antibody (2 µg/mg of protein/ml of immunoprecipitation buffer; Santa Cruz Biotechnology, Santa Cruz, CA) was performed as recently described by
us for cytochrome c (5). Apyrase (10 units/ml), a compound that causes ATP hydrolysis, was used to prevent the ATP-mediated release of bound ligands from hsp72 (3). Co-immunoprecipitation was
assessed by probing the membranes with an antibody directed against hsp72.
Caspase-3 Activity--
Caspase-3 enzyme activity was measured
as previously described by us (5) using a fluorometric assay (ApoAlert
caspase-3 fluorescence assay, Clontech) according
to the manufacturer's protocol. To assess caspase-3-specific activity,
assays were also performed either without substrate or in the presence
of a caspase-3-specific inhibitor (1 µl of DEVD-aldehyde; Clontech).
FAK Proteolysis in Vivo--
In intact cells, FAK proteolysis
was assessed by comparing the quantity of cleaved FAK by immunoblot
analysis of homogenates obtained from control and ATP-depleted OK cells
in the presence and absence of caspase-3 inhibition. Immunoblot
analysis was performed with an antibody that detects both intact FAK
(125 kDa) and FAK cleavage products between 70 and 80 kDa (BD
Biosciences). To inhibit caspase-3, cells were treated with
DEVD-aldehyde (100 µM) for 10 min prior to ATP depletion.
Both attached and detached cells were collected and resuspended in cell
lysis buffer as described above and then subjected to immunoblot
analysis using an anti-FAK antibody.
FAK Proteolysis in Vitro--
FAK was isolated from human kidney
cells by immunoprecipitation (as described above for
immunoprecipitation of proteins from OK cells) or obtained directly
from whole cell lysates. FAK cleavage was assessed in this cell-free
system by immunoblot analysis as described above. FAK was preincubated
in the presence or absence of purified hsp72 (0.01-3 µg/60 µl;
SPP-755, Stressgen Biotech Corp.) in reaction buffer (50 mM
Hepes, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA,
10% glycerol, and 10 mM dithiothreitol, pH 7.40) at 37 °C for 20 min. Purified human caspase-3 (100-200 ng; Upstate Biotechnology, Inc., Lake Placid, NY) was then added, and the reaction mixture was incubated at 37 °C for 1 h. The final
volume of the reaction mixture was 50-100 µl. Under these
conditions, virtually complete (
93%) FAK degradation was observed.
Reactions were terminated by the addition of 4× SDS sample buffer. All
samples were separated by 10-12% SDS-PAGE and transferred and
immunoblotted onto nitrocellulose membranes.
Protein Assay--
Protein concentrations were determined with a
colorimetric dye binding assay (BCA assay, Pierce). Results are
expressed in mg of protein/ml.
Statistical Analysis--
Data are expressed as means ± S.E. Comparison of two groups was performed using one- or two-tailed
Student's t test. Bonferroni's adjustment was applied to
correct the p value when multiple tests were performed using
a single control group. A result was considered significant if
p < 0.05.
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RESULTS |
Exposure to metabolic inhibitors caused the de novo
appearance of several FAK cleavage products with apparent molecular
sizes of 70-90 kDa (Fig. 1A).
Although virtually no FAK fragments were detected in control cells
maintained at 37 °C (first lane), FAK proteolysis was
evident after only 15 min of exposure to metabolic inhibitors
(second lane). The magnitude of FAK degradation was greatest
after 30 min. More prolonged ATP depletion (60-90 min) was associated
with less immunoreactive FAK cleavage products (fourth and
fifth lanes). Prior heat stress inhibited FAK degradation at
all time points of ATP depletion (sixth through
tenth lanes). Prior heat stress significantly decreased FAK
degradation after 15, 30, and 60 min of ATP depletion
(p < 0.05 versus control) (Fig.
1B). To confirm that in situ FAK proteolysis was
mediated by caspase-3, a cell-permeable caspase-3-specific inhibitor
was added prior to exposure to metabolic inhibitors. DEVD-CHO
completely prevented the degradation of FAK after 30 min of ATP
depletion (Fig. 1C). In contrast to FAK, ATP depletion
minimally increased the proteolysis of paxillin, another regulatory
focal adhesion protein that is susceptible to degradation by caspase-3
(39). Unlike FAK, prior heat stress did not inhibit ATP
depletion-mediated degradation of paxillin (Fig.
2).

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Fig. 1.
A and B,
effect of ATP depletion on FAK fragmentation. A,
FAK proteolysis was examined in control and previously heated OK cells
immediately after 15-90 min of ATP depletion using an antibody that
detects both intact FAK (125 kDa) and its cleavage products (70-85
kDa). B, densitometric analysis was used to assess the
protective effect of prior heat stress on FAK degradation after 15, 30, and 60 min of ATP depletion in control cells (white bars)
and previously heat-stressed cells (black bars). All FAK
cleavage products were included in the analysis. *, p < 0.05 (n = 5). C, effect of a caspase-3
inhibitor on FAK cleavage. OK cells were subjected to 30 min of ATP
depletion in the presence and absence of a cell-permeable caspase-3
inhibitor (100 µM DEVD-CHO). Data are
representative of three separate experiments. Each lane contains 20 µg of total protein. Both intact and degraded FAKs are indicated by
arrows.
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Fig. 2.
Effect of ATP depletion on paxillin
fragmentation. Paxillin fragmentation was examined in control and
previously heated OK cells immediately after 15-90 min of ATP
depletion using an antibody that detects both intact paxillin (68 kDa)
and its cleavage product (42 kDa). Each lane contains 20 µg of total
protein. This immunoblot is representative of three separate
experiments.
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To determine whether hsp72 per se inhibits FAK cleavage in
heat-stressed cells subjected to ATP depletion, hsp72 content was selectively increased by transiently infecting renal epithelial cells
with adenovirus encoding human hsp72. Exposure to an hsp72-expressing adenovirus increased hsp72 to a level similar to that observed after
heat stress (Fig. 3A). Both
heat stress and adenoviral infection resulted in a significant 5-fold
increase in steady-state hsp72 content (Fig. 3B).
Approximately 95-99% of the adenovirus-infected cells were positive
for GFP (data not shown). Compared with control cells (infected with
GFP- and tTA-encoding adenoviruses), increased expression of hsp72
inhibited FAK proteolysis after 15, 30, and 60 min of ATP depletion
(Fig. 3C). The decrease in FAK degradation associated with
selective hsp72 overexpression was significant at all three periods of
ATP depletion (Fig. 3D). Of note, differences in the
conditions of gel electrophoresis (10 versus 12%) accounted for the apparent alteration in the sizes of FAK fragments observed in
Fig. 3C compared with Fig. 1A. The observation
that hsp72 per se inhibited FAK proteolysis during ATP
depletion suggests that hsp72 and FAK might interact with one
another.

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Fig. 3.
A and B, effect of
selective hsp72 overexpression on FAK cleavage during ATP
depletion. A, hsp72 content was examined in control
(C), previously heat-stressed (HS), and
adenovirus-infected (AdV) OK cells. Each lane contains 10 µg of total protein. The effect of selective overexpression of hsp72
on ATP depletion-mediated FAK cleavage was examined in cells infected
with adenovirus containing human hsp72. B, densitometric
analysis was used to assess the hsp72 content after heat stress or
adenoviral infection. Data are expressed as means ± S.E. *,
p < 0.05 (n = 3). C, cells
were subjected to transient ATP depletion for 15-60 min. Cells were
infected with AdCMV/tTA and either AdTR5/GFP (( ) HSP72) or
AdTR5/hsp70-GFP ((+) HSp72). Each lane contains 20 µg of
total protein. D, densitometric analysis of FAK degradation
after 15-60 min of ATP depletion in control cells (white
bars) or in cells infected with adenovirus (black
bars). All FAK cleavage products were included in the analysis.
Data are expressed as means ± S.E. *, p < 0.05 (n = 3).
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Acute heat stress per se (42.5 °C for either 40 or 60 min) did not cause FAK degradation (Fig.
4, first lanes versus
seventh and tenth lanes). In contrast to heat stress
with recovery, acute heat exposure without recovery did not prevent FAK
degradation after 15 or 30 min of ATP depletion (Fig. 4A).
In addition, acute heat stress without recovery did not increase
steady-state hsp72 content (Fig. 4B).

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Fig. 4.
Effect of acute heat stress on FAK
degradation during ATP depletion. A, FAK degradation
assessed at the base line (0 min) or after 15 and 30 min of ATP
depletion in cells as follows: control (CTL;
first through third lanes), after heat stress + recovery (HS; fourth through sixth
lanes), and immediately after either 40 (AHS40;
seventh through ninth lanes) or 60 (AHS60; tenth through twelfth
lanes) min of exposure to 42.5 °C. B, hsp72 content
determined by immunoblot analysis in each of the samples in
A. Each lane in A and B contains 20 µg of total protein. These immunoblots are representative of
three separate experiments.
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In control cells, interaction between hsp72 and FAK could not be
detected by co-immunoprecipitation prior to ATP depletion (Fig.
5A, upper panel),
and only minimal interaction was observed after 15-60 min of ATP
depletion (second through fourth lanes). In
contrast, prior heat stress sufficient to increase hsp72 content increased the interaction between these two proteins at all time points
(fifth through eighth lanes). Maximal interaction
between hsp72 and FAK occurred after 30 min of ATP depletion
(seventh lane). Increased interaction between hsp72 and FAK
could not be attributed to differences in the amount of
immunoprecipitable protein given that FAK content was virtually
identical in each sample (Fig. 5A, lower panel).
Because the content of intact FAK differed in control and heat-stressed
cells subjected to ATP depletion, the amount of antibody used to
immunoprecipitate FAK was calibrated to permit an equivalent yield of
protein from both experimental groups. hsp72 content was similarly
increased in all lysates obtained from cells subjected to heat stress
(Fig. 5B).

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Fig. 5.
Interaction between FAK and hsp72.
A, immunoblotting of hsp72 in immunoprecipitates
(IP) obtained from whole cell lysates of control and
previously heated cells with an antibody directed against FAK
(upper panel). Samples were obtained prior to ATP depletion
(first and fifth lanes) and after ATP depletion
for 15 min (second and sixth lanes), 30 min
(third and seventh lanes), and 60 min
(fourth and eighth lanes). To assess
the amount of FAK in each sample, immunoprecipitates were probed with
an antibody directed against FAK (lower panel).
Immunoprecipitation was performed on 1 mg of whole cell lysate, and the
resulting immunoprecipitates were separated by SDS-PAGE. B,
hsp72 content in whole cell lysates used for the
immunoprecipitation in A.
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To examine the role of substrate binding in preventing FAK proteolysis,
hsp72
EEVD, a mutant hsp72 protein that exhibits defective substrate
binding (13, 18), was selectively overexpressed in OK cells (Fig.
6). In contrast to wild-type hsp72
(ninth through twelfth lanes), hsp72
EEVD
(fifth through eighth lanes) failed to prevent
FAK proteolysis in cells subjected to ATP depletion (Fig.
6A) despite a marked increase in mutant hsp72 content (Fig. 6B). Overexpression of hsp72
EEVD did not affect the
steady-state content of wild-type hsp72 (Fig. 6C).

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Fig. 6.
Effect of selective overexpression of
hsp72 EEVD on FAK degradation during ATP
depletion. A, FAK proteolysis examined after 30 min of ATP
depletion in whole cell lysates of OK cells after co-infection with
adenoviruses encoding GFP and AdCMV/tTA ( EEVD;
first through fourth), hsp72 EEVD/GFP and
AdCMV/tTA (+ EEVD; fifth through
eighth lanes), and wild-type hsp72 (wtHsp72;
ninth through twelfth lanes). B,
hsp72 EEVD content in each of the samples shown in A. The
antibody used to detect hsp72 EEVD cross-reacts with wild-type hsp72.
C, wild-type hsp72 content in each sample. The antibody used
detects only wild-type hsp72. These immunoblots are representative of
three separate experiments. Each lane in the immunoblots contains
10-20 µg of protein.
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If hsp72 prevented FAK proteolysis by caspase-3, then similar
protection might be observed in vitro. To test this
hypothesis, FAK was obtained from whole cell lysates (Fig.
7A) or by immunoprecipitation using an anti-FAK antibody (Fig. 7B) from normal human
kidney cells previously characterized as proximal tubule in origin
(37). Both procedures yielded a single band at 125 kDa (Fig. 7,
A and B, lanes 1). The addition of
purified human hsp72 to FAK obtained from whole cell lysates did not
alter the migration or content of FAK (Fig. 7A, lane
2). Exposure of FAK to purified human caspase-3 resulted in the
formation of at least one major cleavage product (at ~85 kDa)
(lane 3). In contrast, co-incubation with human hsp72 reduced FAK degradation (lane 4). If hsp72 was preincubated
with caspase-3 prior to the addition of FAK, FAK cleavage was not
inhibited despite equivalent contact time between FAK and caspase-3
(lane 5). Similar results were obtained with FAK obtained by
immunoprecipitation (Fig. 7B, lanes 2-4).
Exogenous ATP (10 mM) resulted in a relative increase in
FAK degradation by caspase-3 (lane 5). In contrast, ATP
hydrolysis with apyrase (10 units/ml) (3) increased the inhibitory
effect of hsp72 on caspase-3-mediated FAK proteolysis (lane
6).

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Fig. 7.
Effect of purified hsp72 on FAK cleavage by
caspase-3 in vitro. A, FAK obtained
from whole cell lysates (lane 1), FAK incubated with 3 µg
of purified hsp72 (lane 2), FAK incubated with 200 ng of
caspase-3 (lane 3), FAK preincubated with 3 µg of hsp72
prior to the addition of 200 ng of caspase-3 (lane 4), and
hsp72 (3 µg) incubated with 200 ng of caspase-3 prior to the addition
of FAK (lane 5). All incubations were performed at 37 °C
for 60 min. The arrows indicate the locations of intact FAK
and a major degradation product. B, intact FAK
immunoprecipitated from human kidney cells (lane 1) and
subjected to degradation by 200 ng of caspase-3 (lane 2) in
the presence of 1 µg (lane 3) or 3 µg (lane
4) of purified hsp72. FAK cleavage was examined in the presence of
exogenous ATP (10 mM; lane 5) or in the absence
of ATP (10 units/ml apyrase; lane 6). The results are
representative of three separate experiments.
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Exposure of FAK to caspase-3 resulted in a 93% decrease in
the content of intact FAK (Fig. 8,
second lane). Wild-type hsp72 inhibited FAK degradation by
caspase-3 in a dose-dependent manner at hsp72
concentrations between 2.3 and 700 nM (third
through sixth lanes). The correlation coefficient for the
relationship between the dose of hsp72 and the degree of protection of
intact FAK was 0.979 as assessed by densitometry of the 125-kDa bands in Fig. 8.

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Fig. 8.
Dose-response effect of purified hsp72 on
caspase-3-mediated FAK fragmentation in vitro.
FAK obtained from whole cell lysates was incubated with human caspase-3
(200 ng) in the presence of purified hsp72 (0.01-3 µg) for 60 min at
37 °C as described under "Experimental Procedures." The
arrows indicate the locations of intact FAK and its
degradation products. Each lane contains 50 µg of total protein. The
results are representative of two separate studies.
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To examine the possibility that hsp72 interfered with caspase-3,
in vitro enzyme activity was compared in the presence and absence of purified hsp72. The addition of either 1 or 3 µg of hsp72
(130 and 400 nM) did not alter the activity of purified caspase-3 (Fig. 9). These results are in
agreement with a previous report showing that purified hsp70 has no
effect on the activity of caspase-3 (40). To determine whether or not
hsp72 might compete with FAK as a substrate for caspase-3, purified
hsp72 was incubated with 100 ng of caspase-3 (Fig.
10). Caspase-3 failed to degrade intact
hsp72 at doses between 1 and 10 ng of the latter protein. These data
suggest that hsp72-FAK interaction, rather than inhibition of caspase-3
activity or competition between hsp72 and FAK for proteolysis by
caspase-3, is required to protect FAK.

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Fig. 9.
Effect of hsp72 on caspase-3 activity
in vitro. Caspase-3 activity in a cell-free
system was determined by a fluorometric assay in the absence
(white bar) or in the presence of 1 µg (hatched
bar) or 3 µg (checkered bar) of purified hsp72. Data
are reported as means ± S.E. of three separate experiments. Some
of the error bars are too small to be seen. NS, not
significantly different.
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Fig. 10.
Effect of caspase-3 on hsp72
degradation. Caspase-3 (100 ng) was incubated with purified hsp72
(1-10 ng) at 37 °C for 1 h in a cell-free system as described
in the legend to Fig. 7. This immunoblot is representative of three
separate experiments.
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DISCUSSION |
This study demonstrates that focal adhesion kinase is a target for
injury caused by exposure to metabolic inhibitors. In renal epithelial
cells, metabolic inhibitors reduce ATP content to <10% of the basal
level within 10 min (38). Concomitant with the reduction in ATP
content, caspase-3 is activated by 2.5-fold (5). Activated caspase-3
participates in the proteolysis of several substrates, including FAK
(26-28, 30). FAK is preferentially cleaved by caspase-3 after the DQTD
sequence that terminates at residue 772 (27). Sequential degradation by
caspase-3 yields multiple fragments ranging in size from 17 to 90 kDa
(27). Some of these fragments are intrinsically unstable (28, 30),
whereas a 45-kDa N-terminal fragment translocates to the nucleus (41). Either observation could explain the apparent disappearance of FAK
fragments during prolonged ATP depletion (60-90 min) (Fig. 2).
Although caspase-6 and various calpains have also been reported to
cleave FAK (20, 27), caspase-3 is the primary enzyme responsible for
FAK degradation (27, 28). In this study, a cell-permeable caspase-3-specific inhibitor completely prevented FAK proteolysis in
intact cells (Fig. 1C). These results were reproduced in an in vitro system (Figs. 7 and 8), further supporting the
notion that caspase-3 is responsible for the proteolysis of FAK in
renal epithelial cells.
This study tested the hypothesis that heat stress and hsp72 exert
anti-apoptotic effects, at least in part, by decreasing the proteolysis
of FAK. Cell stress has been associated with FAK degradation in a
variety of cell lines (26, 30, 31, 35). FAK cleavage not only separates
the kinase from the focal adhesion-targeting domain, but generates
"FAK-related non-kinase," a peptide fragment that interferes with
the kinase function of intact FAK (27). FAK degradation could
precipitate apoptosis by acting at one or more sites in the cell death
cascade (26-32, 35, 36). FAK regulates the Akt pathway (29) and
stimulates protein inhibitors of apoptosis (32), and intact FAK
prevents anoikis, a form of apoptosis that occurs as a
consequence of the loss of cell adherence (28).
Stewart et al. (41) recently demonstrated that an N-terminal
fragment of FAK derived from the focal adhesion-targeting domain migrates into the nuclei of epithelial cells in a manner that is
independent of its tyrosine phosphorylation or the presence of the C
terminus. Within the nucleus, FAK may directly regulate survival (42,
43). The mechanism by which the N-terminal fragment mediates cell
survival is not presently known (41). It is conceivable that FAK
degradation represents a redundant signaling pathway that ensures death
by apoptosis following caspase-3 activation. Similar redundant
pro-apoptotic functions have been attributed to apoptosis-inducing
factor, a cause of caspase-independent cell death (44).
Several lines of evidence support the contention that hsp72 prevents
the degradation of FAK. First, the selective overexpression of hsp72
(Fig. 3) reproduced the inhibitory effects of prior heat stress (Fig.
1) on the proteolysis of FAK during ATP depletion. In contrast, acute
heat stress did not increase hsp72 content and failed to prevent FAK
degradation during ATP depletion (Fig. 4). Second, hsp72 interacted
with FAK (Fig. 5). This interaction was markedly increased in cells
subjected to prior heat stress and was greatest during the time period
when FAK degradation occurred. The increased interaction was due in
part to the up-regulation of hsp72 content as well as to alterations in
their mutual affinity. Third, hsp72 inhibited FAK cleavage by caspase-3
in an in vitro system (Figs. 7 and 8). The ability of hsp72
to prevent FAK degradation is dose-dependent across a range
of hsp72 concentrations reported to have physiologic effects (15, 26).
These observations may explain, in part, the fact that the selective
overexpression of hsp72 inhibits apoptosis in ATP-depleted renal
epithelial cells (17).
Several mechanisms could explain the ability of hsp72 to inhibit FAK
degradation by caspase-3. For instance, hsp72 could interfere with
caspase-3. This does not appear to be likely because hsp70 has been
shown to act upstream (16) or downstream (45) of caspase-3 without
directly affecting enzyme activity in vitro (40). In
addition, we has shown that purified hsp72 did not interfere with
caspase-3 activity in whole cell lysates obtained from ATP-depleted
renal cells (Fig. 9) (5). Also, preincubation of hsp72 with caspase-3
prior to the addition of FAK actually reduced the protective effect of
hsp72 on FAK degradation (Fig. 7A). Finally, purified hsp72
did not alter caspase-3 activity (Fig. 9). As an alternative mechanism,
hsp72 could compete with FAK for degradation by caspase-3. However,
this hypothesis is not supported by the observation that caspase-3
failed to degrade purified hsp72 in vitro (Fig. 10). This
may be due to the fact that human hsp72 (NCBI BLAST Protein
Database accession number XP_175177) does not have a DQTD cleavage
sequence targeted by caspase-3 (27).
Taken together, these data support the hypothesis that cytoprotection
of FAK requires interaction with hsp72. hsp72, like other members of
the hsp70 family, releases non-native proteins in an
ATP-dependent manner (46-49). The observation that
exogenous ATP decreased protection of FAK by hsp72 (and removing ATP
increased protection) (Fig. 7B) implies that hsp72-FAK
binding is required to prevent caspase-3-mediated FAK proteolysis. This
hypothesis is strengthened by the observation that the hsp72
EEVD
mutant failed to inhibit FAK degradation during ATP depletion (Fig.
6A). Deletion of the substrate-binding domain also
sensitizes cells to apoptosis caused by thermal stress (13, 18, 50),
suggesting that this domain mediates cytoprotection. The ability of ATP
to affect protection of FAK by hsp72 could be the result of changes in
either hsp72 or FAK. In addition to directly altering the binding affinity of hsp72 for FAK, exogenous ATP could also change the conformation of FAK by affecting its degree of phosphorylation (51).
Either mechanism (or both) could explain the enhanced interaction
between FAK and hsp72 observed during ATP depletion in the absence of
changes in hsp72 content (Fig. 7).
This study shows that caspase-3-mediated FAK degradation occurs early
in the course of ATP depletion and clearly precedes morphologic
evidence of apoptosis (3, 17). It is therefore possible that
pharmacologic intervention could inhibit this deleterious event. hsp72
is poised to be a potent anti-apoptotic protein because it acts at
multiple sites in the cell death cascade. hsp72 suppresses the
activation of stress kinases that participate in the caspase-8 pathway
(13), prevents the release of cytochrome c from mitochondria and the subsequent activation of caspase-3 (5, 14, 18), and reduces
caspase-independent cell death by apoptosis-inducing factor (15). Both
heat stress and selective hsp72 overexpression have recently been shown
to decrease primary mitochondrial membrane injury and to partially
inhibit caspase-3 activation in ATP-depleted renal epithelial cells (5,
17). Interaction between FAK and hsp72 prevents FAK degradation by
activated caspase-3 and may represent a secondary cytoprotective
pathway. These two points of intervention ensure that FAK cleavage is
prevented. This study identifies a new mechanism by which hsp72 could
prevent cell death by apoptosis.
 |
ACKNOWLEDGEMENTS |
We appreciate the expert advice regarding the
use of hsp70 adenoviruses from Drs. M. Y. Sherman, V. L. Gabai, and J. Yaglom (Boston University). We also thank Dr. J. L. Guan for the generous gift of purified FAK that was used in
preliminary experiments.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK-53387 (to S. C. B.) and DK-52898 (J. H. S.).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: Evans Biomedical
Research Center, Renal Section, Rm. 547, 650 Albany St., Boston, MA
02118-2518. Tel.: 617-638-7330; Fax: 617-638-7326; E-mail: sborkan@bu.edu.
Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M300126200
 |
ABBREVIATIONS |
The abbreviations used are:
FAK, focal adhesion
kinase;
OK, opossum kidney;
Ad, adenovirus;
GFP, green fluorescent
protein;
CMV, cytomegalovirus;
tTA, tetracycline-controlled
transactivator;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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