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INTRODUCTION |
The small GTP-binding protein Rho belongs to the larger Ras family
of small GTPases and has been demonstrated to regulate cytoskeletal
reorganization, stimulating the formation of stress fibers and focal
adhesions in cultured cells (1). Characteristic of all Ras family
members, Rho functions as a "molecular switch" in the cell (2-4),
cycling between inactive GDP-bound and active GTP-bound states. The
rate of activation and inactivation of this GTPase is regulated by a
number of proteins, including guanine nucleotide exchange factors,
GTPase-activating proteins, and guanine nucleotide dissociation
inhibitors (2). Although considerable insight has been gained into the
mechanisms regulating small GTPases such as Rho, many components along
the Rho signaling pathway remain to be identified.
Protein tyrosine phosphorylation and/or dephosphorylation events play a
major role in the Rho-mediated regulation of cytoskeletal reorganization. Evidence for this comes from the use of inhibitors of
protein-tyrosine kinases such as genistein (5, 6), erbstatin (6), and
tyrphostin 25 (7), which have been demonstrated to inhibit the
activation of Rho and prevent cytoskeletal assembly. Consistent with
this, studies using protein-tyrosine phosphatase (PTPase)1 inhibitors, such as
pervanadate (6, 8, 9) and phenylarsine oxide (PAO) (10, 11), have
demonstrated induction of stress fibers in serum-starved cells. Since
these inhibitors block the enzymatic activity of a wide range of
kinases and phosphatases, it is unclear which enzyme(s) are responsible
for Rho regulation in these instances. In addition, studies
artificially overexpressing soluble PTPases (through microinjection or
scrape loading) resulted in disassembly of stress fibers and focal
adhesions (12). Although there is increasing evidence for PTPases as
important players in the regulation Rho and cytoskeletal reorganization
(13-16), the identity of the enzyme(s) lying upstream of Rho
activation remains elusive.
The second messenger cAMP also exerts dramatic effects on cytoskeletal
architecture. Elevation of cAMP in a variety of cell types induces loss
of actin stress fibers and focal adhesions, rounding of cells, and in
some cases detachment from the underlying substratum (17-19).
Increases in intracellular cAMP also decrease the phosphorylation of
multiple proteins, including the tyrosine phosphorylation of focal
adhesion proteins paxillin (20) and pp125FAK (21) and the
phosphorylation of myosin light chain (22). Although the mechanism(s)
eliciting the effects of cAMP appear to be complex, recent studies
(23-27) point toward a role for this cyclic nucleotide in the
down-regulation of Rho. There may be multiple ways in which cAMP can
regulate cytoskeletal reorganization, and they may all participate to
some extent in the cytoskeletal disassembly associated with cAMP elevation.
One potential candidate that may play a role in the disassembly of
cytoskeletal structures is the ubiquitous thiol protease calpain. This
enzyme localizes to focal adhesions (28, 29), where it has been shown
to participate in the limited proteolysis of numerous structural and
signaling proteins associated with these adhesions (30-34). The
proteolytic actions of calpain have been postulated to destabilize
focal adhesions and sever the linkage between the extracellular matrix
and the contractile cytoskeleton of the cell (35, 36). Consistent
with this, several recent reports have confirmed a role for calpain in
the down-regulation of cell processes such as spreading (37), migration
(36), and platelet-mediated clot retraction (35, 38). The possible participation of calpain in events requiring remodeling of the cytoskeleton prompted us to investigate the role of this protease in
disassembly of the cytoskeleton. Surprisingly, we found that treatment
of cells with calpeptin, an inhibitor of calpain, was sufficient to
induce stress fiber formation, focal adhesions, and cell contractility.
This effect was specific for calpeptin and could not be mimicked by
other calpain inhibitors. Furthermore, calpeptin-induced formation of
stress fibers and focal adhesions was inhibited by botulinum toxin C3,
suggesting that the calpeptin target was upstream of Rho. We
report that calpeptin cross-reacts with another family of enzymes,
PTPases, which also possess a critical cysteine in their active site.
Using this knowledge, we provide evidence for the existence of a PTPase
activity, associated with the membrane fraction of cells, which acts
upstream of Rho.
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EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
Calpeptin, PAO, calpain inhibitor 1 (CI-1), recombinant calpastatin, and E64d were obtained from
Calbiochem-Novabiochem. Forskolin was purchased from Biomol Research
Laboratories (Plymouth Meeting, PA). Dibutyryl cAMP and Protein
A-Sepharose were from Sigma. Glutathione-Sepharose 4B was obtained from
Amersham Pharmacia Biotech (Uppsala, Sweden). Transfer polyvinylidene
difluoride membranes were obtained from Millipore Corp. (Bedford, MA).
Radiochemicals were from NEN Life Science Products. A cyclic AMP
3H assay system was purchased from Amersham International
(Buckinghamshire, United Kingdom). Antiphosphotyrosine polyclonal,
antiphosphotyrosine PY20, and anti-paxillin monoclonal antibodies were
purchased from Transduction Laboratories (Lexington, KY). Anti-mouse
peroxidase-conjugated IgG was from Jackson Laboratories (West Grove,
PA). Fluorescein-conjugated rabbit IgG and rabbit anti-mouse IgG were
from Chemicon (Temecula, CA). Rhodamine-conjugated phalloidin was from
Molecular Probes, Inc. (Eugene, OR). Fluorescein-conjugated
anti-human IgG for microinjection was from ICN/Cappel (Aurora, OH). GST
constructs for the recombinant PTPases and botulinum toxin C3 were
generous gifts from various research laboratories. The catalytic domain
of PTP-1B (PTP-37K-1B) (Dr. J. Dixon, University of Michigan),
cytoplasmic domain of PTP
(Dr. M. Thomas, Washington University, St.
Louis, MO), PTEN (Dr. N. Tonks, Cold Spring Harbor, NY), and C3
exotransferase (Dr. L. Feig, Tufts University, Boston, MA).
Cell Culture--
Swiss 3T3 fibroblasts and REF-52 fibroblasts
were maintained in DMEM as described previously (6). Swiss 3T3
fibroblasts were serum-starved by incubation in DMEM lacking serum for
at least 24 h.
Drug Treatment--
In some experiments, cells were preincubated
with vehicle alone (Me2SO) (0.1%, v/v) or one of the
following compounds: calpeptin (0.1-1.0 mg/ml), E-64d (100 µM), CI-1 (10 µM), pervanadate (50 µM), EGTA/MgCl2 (1 and 2 mM,
respectively), or PAO (1 µM). Cells were incubated with
these compounds for 30 min at 37 °C, unless otherwise indicated.
Immunofluorescence Microscopy--
Immunofluorescence microscopy
was performed as described previously (6).
Preparation of Whole Cell Lysates and Subcellular
Fractions--
Whole cell lysates were prepared as described
previously (6). For isolation of cytosol and membrane fractions,
adherent cells were scraped into resuspension buffer (5 mM
Tris, pH 7.6, 0.25 M sucrose, 2 mM EDTA, 10 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride), sonicated six times (8 s each), and
then centrifuged at 1,000 × g to remove intact cells and nuclei. Cytosol and membrane fractions were isolated by
centrifugation at 100,000 × g for 30 min. The membrane
pellet was resuspended in Triton X-100-containing buffer. Measurement
of protein concentration was determined using the Coomassie Protein
Assay Reagent, with bovine serum albumin as a standard, following the
manufacturer's instructions (Pierce).
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
of paxillin or phosphotyrosine-containing proteins was performed as
described previously (6). Cell lysates or immunoprecipitated samples
were separated by 10% SDS-polyacrylamide gel electrophoresis (39)
under reducing conditions and then transferred to polyvinylidene
difluoride membranes. Western blots were performed as described by
Towbin et al. (40). Blots were developed using SuperSignal
Substrate for Western blotting (Pierce).
Preparation of Pervanadate--
Vanadate stock solution was
prepared essentially as described (41). Pervanadate (50 mM)
was prepared by combining 100 µl of vanadate, 88 µl of
Tris-buffered saline, and 12 µl of 30% hydrogen peroxide. The
pervanadate solution was used within 5 min of preparation.
Measurement of Intracellular cAMP Levels--
Intracellular cAMP
levels were measured as described by Yuan et al. (42), using
a 3H-labeled cyclic AMP assay system according to the
manufacturer's instructions (Amersham Pharmacia Biotech).
Growth and Purification of GST Fusion Proteins--
The
recombinant catalytic subunit of PTP-1B (PTP-37K-1B), the cytoplasmic
tail of PTP
, PTEN, and C3 exotransferase were expressed as GST
fusion proteins in Escherichia coli (BL21) as described previously (43).
Microinjection of C3 Exotransferase or Calpastatin--
Cells
were grown on coverslips and injected with either 100 µg/ml C3
exotransferase or 2 mg/ml recombinant calpastatin (diluted in
microinjection buffer (5 mM Tris, pH 7.6, 150 mM NaCl, 5 mM MgCl2 and 0.1 mM dithiothreitol)). Cells were coinjected with fluorescein-conjugated anti-human IgG in order to detect injected cells. Cells were returned to the tissue culture incubator to allow for
recovery (30-60 min).
In Vitro PTPase Assays--
Whole cell lysates or subcellular
fractions from forskolin-treated (10 µM, 30 min) REF-52
or serum-starved (24 h) Swiss 3T3 fibroblasts were prepared as
described previously (44). [32P]poly(Glu:Tyr) substrate
was prepared according to the method of Burridge and Nelson (45). All
assays were performed within the linear range with respect to
protein-tyrosine phosphatase activity. Samples were diluted in
phosphatase assay buffer (50 mM Tris, pH 7.0, 1 mg/ml BSA,
0.3%
-mercaptoethanol, 50 µM EDTA) to a final volume
of 100 µl. Reactions were started by the addition of
[32P]poly(Glu:Tyr) (approximately 40,000 cpm) and
incubated at 30 °C for 5-20 min. Reactions were terminated by the
addition of an equal volume of 20% trichloroacetic acid. Tubes were
incubated on ice for 10 min and vortexed, and the precipitated protein
was pelleted by centrifugation in a microcentrifuge at 12,000 rpm for 2 min. The supernatant was removed and added to 2 ml of scintillation fluid. Phosphatase activity was determined by the amount of free label
released into the supernatant measured by scintillation counting, minus
the amount of label released in the absence of lysate. Results are
expressed as the mean of several independent experiments.
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RESULTS |
Activation of the thiol protease calpain has been demonstrated to
result in the down-regulation of a number of cellular processes including cell spreading, migration, and contraction (35-38). These processes are all dependent on reorganization of the cytoskeleton. In
addition, calpain is localized to focal adhesions, where its activation
has been shown to correlate with the proteolysis and loss of various
structural and signaling proteins (30-34) from these sites and the
disruption of integrin-cytoskeletal linkages (35, 36). These studies
imply a potentially important role for calpain in the disassembly of
cytoskeletal architecture. To investigate this possibility, we searched
for cell models that could easily be manipulated to induce cytoskeletal
disassembly. We chose a rat fibroblast cell line (REF-52) as one of our
cell models, since these cells have been demonstrated to rapidly and reversibly lose stress fibers and focal adhesions in response to agents
that elevate intracellular levels of cAMP (e.g. forskolin, dibutyryl cAMP) (17, 46). With this cell model, we set out to determine
whether calpain was involved in the disassembly of stress fibers and
focal adhesions induced by elevated cAMP levels.
Calpeptin Blocks the Disassembly of Stress Fibers--
In order to
study the role of calpain inside the cell, it was necessary to perturb
the function of this protease. Several synthetic inhibitors have been
produced that block calpain activity (47, 48). One such inhibitor is
calpeptin (49), a dipeptide aldehyde that was designed to bind
specifically to the critical cysteine residue in the active site of
calpain, preventing the binding and subsequent proteolysis of calpain
substrates (48). Therefore, we began our examination of the role of
calpain in regulating the disassembly of stress fibers in our chosen
cell model, using calpeptin. Under normal culture conditions, REF-52 fibroblasts displayed a striking array of thick actin stress fibers (Fig. 1a, A). This
organization was dramatically lost in response to treatment with 10 µM forskolin (elevation of cAMP) (Fig. 1a, B). However, when these cells were treated with the calpain
inhibitor calpeptin (100 µg/ml), prior to forskolin treatment, the
cells retained their normal actin organization and did not lose their stress fibers (Fig. 1a, C). We found this effect
to be dose-dependent, with noticeable effects at
concentrations of calpeptin as low as 20 µg/ml (data not shown). To
ensure that the effects of calpeptin were not unique to forskolin, we
also performed similar studies using the cAMP derivative, dibutyryl
cAMP. Consistent with forskolin treatment, we found that calpeptin
could prevent the dibutyryl cAMP-induced loss of stress fibers in
REF-52 fibroblasts (data not shown). As a further control, we measured
intracellular cAMP levels using a 3H-labeled cAMP assay
system and confirmed that calpeptin was not merely acting by lowering
cAMP levels inside the cell (data not shown). These results suggest
that calpeptin was able to block the disassembly of the cytoskeleton
induced by intracellular cAMP elevation.

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Fig. 1.
Calpeptin induces the formation of stress
fibers. REF-52 and Swiss 3T3 fibroblasts were cultured in complete
DMEM and plated onto coverslips. a, REF-52 fibroblasts were
preincubated with vehicle (Me2SO) alone (A and
B) or 100 µg/ml calpeptin (C) for 30 min. Cells
were then treated with vehicle (Me2SO) alone (A)
or 10 µM forskolin (B and C) for 30 min. b, Swiss 3T3 fibroblasts were placed in serum-free
medium and starved for 24 h. Cells were then treated with vehicle
(Me2SO) alone (A) or 100 µg/ml calpeptin for
30 min (B). Both REF-52 and Swiss 3T3 cells were fixed and
processed for fluorescence microscopy as described under
"Experimental Procedures." Cells were visualized by staining with
rhodamine-conjugated phalloidin.
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The calpeptin-mediated effects on cAMP-induced disassembly could be due
to blocking disassembly or alternatively promoting assembly of stress
fibers. To explore the possibility that calpeptin might promote stress
fiber assembly, we examined the effect of calpeptin on serum-deprived
Swiss 3T3 cells. When Swiss cells are starved of serum for up to
24 h, their actin stress fibers will disassemble (Fig.
1b, A). Previous studies have demonstrated that
the addition of serum or serum components such as lysophosphatidic acid
will rapidly induce the formation of stress fibers in starved cells via
the activation of Rho (1). Strikingly, when calpeptin was added to the
serum-free culture medium, Swiss 3T3 cells rapidly (within 30 min)
reformed their stress fibers (Fig. 1b, B), a
response reminiscent of serum or lysophosphatidic acid addition. These results are consistent with those obtained using REF-52 fibroblasts and
suggest that calpeptin can promote the formation of stress fibers.
Calpain Is Not the Target for Calpeptin-mediated Inhibition of
Stress Fiber Disassembly--
Although calpeptin has proven to be an
effective inhibitor of the calpain-mediated proteolysis of several
calpain substrates (30-34), like many other inhibitors its specificity
at higher concentrations is questionable. To confirm that calpain was
the protein targeted for inhibition by calpeptin, we tested a number of
other calpain inhibitors widely used in the literature. These
inhibitors were used at concentrations previously demonstrated to block
calpain activity (35, 38). Surprisingly, preincubation of REF-52
fibroblasts (Fig. 2) or Swiss 3T3
fibroblasts (data not shown) with E64d (100 µM) (Fig.
2a, D) or CI-1 (10 µM) (Fig.
2a, E) did not prevent the forskolin-induced
disassembly of stress fibers. Since calpain is a
calcium-dependent protease, we tried chelation of calcium, which is an effective method of inhibiting calpain activation. However,
consistent with CI-1 and E64d, incubation of REF-52 fibroblasts with a
combination of EGTA (1 mM) and Mg2+ (2 mM) (Fig. 2a, F) had no effect on the
forskolin-induced disassembly of stress fibers. Finally and most
convincingly, the introduction of recombinant calpastatin (the
endogenous and highly specific inhibitor of calpain) into these cells
did not block forskolin-induced events (Fig. 2b). These
results suggested that calpain was not the target of calpeptin in these
studies and thus not responsible for the disassembly of stress fibers
under conditions of elevated intracellular cAMP or deprivation of
serum.

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Fig. 2.
Calpeptin-mediated induction of stress fibers
is not mediated through inhibition of calpain. REF-52 fibroblasts
cultured in complete DMEM were plated onto coverslips. a,
cells were preincubated with vehicle (Me2SO) alone
(A and B), 100 µg/ml calpeptin (C),
100 µM E64d (D), 20 µM CI-1
(E), or 1 mM EGTA and 2 mM
MgCl2 (F) for 30 min. Cells were then treated
with vehicle (Me2SO) alone (A) or 10 µM forskolin (B-F) for 30 min. b,
cells were injected with recombinant calpastatin (2.0 mg/ml) along with
fluorescein-conjugated anti-human IgG to identify injected cells. Cells
were then treated with vehicle (Me2SO) (A and
B) or forskolin (10 µM) for 30 min
(C and D). c, all cells were treated
with 10 µM forskolin for 30 min (A-D).
Following this, 100 µg/ml calpeptin was added, and cells were
incubated for 0 (A), 10 (B), 20 (C),
or 30 (D) min. Cells were visualized by staining with
rhodamine-conjugated phalloidin, as described under "Experimental
Procedures."
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Further evidence ruling out calpain as the target of calpeptin in these
experiments came from studies in which we demonstrated that calpeptin
could reverse stress fiber disassembly, even after forskolin treatment
had been administered. REF-52 fibroblasts were incubated for 30 min
with forskolin, conditions that would allow for the complete disruption
of stress fibers (Fig. 2c, A). Calpeptin was then
added to the culture medium, in the continued presence of forskolin,
and the cells incubated for various time periods (Fig. 2c,
B-D). Reappearance of stress fibers was evident as early as
10 min after calpeptin addition, and full recovery of stress fibers was
achieved within 30 min. This result is inconsistent with the notion
that calpeptin is inhibiting the activation of calpain, since
proteolytic cleavage of calpain substrates cannot be "reversed."
Furthermore, new protein synthesis could not be responsible for the
restoration of stress fibers by calpeptin, since this process would not
be significant in the time scale of this experiment. Taken together,
these observations indicate that calpain is not the responsible
protease regulating stress fiber disassembly and suggest that calpeptin
is inhibiting some other protein(s) responsible for cytoskeletal remodeling.
Calpeptin Mediates Its Effects via the Inhibition of
PTPases--
Calpeptin is a dipeptide aldehyde whose mode of
inhibition is via binding the active site cysteine of calpain (48, 49). Since our results suggested that calpeptin was not promoting stress fiber formation through inhibition of calpain, it was necessary to
determine other potential targets for the inhibitor. One other group of
enzymes with a critical cysteine residue in their active site is the
PTPases (50). To investigate the possibility that calpeptin may inhibit
PTPases, we initially examined the tyrosine phosphorylation pattern in
both REF-52 and Swiss 3T3 fibroblasts, under conditions where calpeptin
had previously been demonstrated to induce stress fiber formation (see
Fig. 1, a and b). We found that the tyrosine
phosphorylation of several proteins was enhanced by calpeptin treatment
(Fig. 3a). In particular, we
noted a dramatic increase in tyrosine phosphorylation of a protein
migrating as a broad band above the 64-kDa molecular mass marker. By
performing immunoprecipitations of REF-52 cell lysates, we were able to
identify this protein as paxillin (Fig. 3b), a 68-kDa
adapter protein localized to focal adhesions. Paxillin has previously
been demonstrated to undergo rapid dephosphorylation in response to
intracellular cAMP elevation (20). Consistent with this, we also found
that paxillin was rapidly dephosphorylated upon treatment with
forskolin in REF-52 fibroblasts (Fig. 3c). Furthermore, in
correlation with immunofluorescence studies demonstrating a
calpeptin-mediated reversal of stress fiber disassembly in
forskolin-treated cells (Fig. 2c), the dephosphorylation of
paxillin was completely reversed upon the addition of calpeptin (Fig.
3c). These results are consistent with inhibition of a
tyrosine phosphatase. Our attempts to determine the identity of other
proteins migrating around 36-40 and 110-130 kDa that displayed
changes in tyrosine phosphorylation (albeit minor) were not successful.
We examined the tyrosine phosphorylation status of pp125FAK
and p130cas, which have previously been demonstrated to change
in correlation with the regulation of stress fibers and focal adhesions
(8, 51-57). We could not detect any significant changes in the
tyrosine phosphorylation of either of these proteins in response to
calpeptin. Therefore, it appears at least in this system that paxillin,
but not p130cas or pp125FAK, displays changes in
tyrosine phosphorylation during the calpeptin-mediated induction of
stress fibers.

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Fig. 3.
Calpeptin enhances tyrosine phosphorylation
of cellular proteins, including paxillin. REF-52 and Swiss 3T3
cells were cultured in complete DMEM. REF-52 cells were pretreated with
vehicle (Me2SO) alone (A) or 100 µg/ml
calpeptin (B) for 30 min, followed by incubation with 10 µM forskolin for 30 min. Swiss cells were serum-starved
for 24 h and then incubated with vehicle (Me2SO) alone
(C) or 100 µg/ml calpeptin (D) for 30 min.
Whole cell lysates were prepared as described under "Experimental
Procedures." a, equal amounts of protein were analyzed by
10% SDS-polyacrylamide gel electrophoresis, followed by immunoblotting
with an antiphosphotyrosine antibody (PY20). b, equal
amounts of proteins from REF-52 cell lysates were used to
immunoprecipitate paxillin, as described under "Experimental
Procedures." Immunoblot analysis of immunoprecipitates was performed
using an anti-phosphotyrosine antibody (PY20). A lower band of
approximately 55 kDa present in both lanes represents the mouse IgG
heavy chain. c, REF-52 fibroblasts were incubated with 10 µM forskolin for the indicated times. Following 20 min of
forskolin incubation, 100 µg/ml calpeptin was included in the cell
medium. After the completion of the time course, whole cell lysates
were prepared as described previously, and equal amounts of lysate were
analyzed by immunoblotting using an anti-phosphotyrosine monoclonal
antibody, PY20.
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To confirm the ability of calpeptin to act as a PTPase inhibitor, we
next examined its ability to inhibit PTPase activity in in
vitro assays using an exogenous substrate. Whole cell lysates from
Swiss 3T3 and REF-52 fibroblasts were prepared as described under
"Experimental Procedures" and assayed for their ability to remove
the radiolabeled phosphate from [32P]poly(Glu:Tyr). The
addition of cell lysates to the assay resulted in the release of
radiolabel from the substrate (Fig. 4,
a and b, DMSO). However, when
calpeptin was included in these assays, PTPase activity in both Swiss
3T3 and REF-52 fibroblast cell lysates was reduced by a small yet
significant level (10-20%) (Fig. 4, a and b,
100 µg/ml). It is important to note that calpeptin was unable to
fully inhibit the phosphatase activity in these cell lysates, even at
higher concentrations (Fig. 4, a and b, 1.0 mg/ml). This suggests that calpeptin may not be a very efficient
inhibitor of PTPases or, alternatively, that it may only target a
subset of phosphatases in the whole cell lysate.

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Fig. 4.
Calpeptin directly inhibits whole cell lysate
PTPase activity in vitro. Swiss 3T3 and REF-52 fibroblasts
were cultured in complete DMEM. Whole cell lysates were prepared from
forskolin-treated REF-52 fibroblasts (a) or serum-starved
Swiss 3T3 cells (b) as described under "Experimental
Procedures." In vitro PTPase assays were performed on
equal amounts of protein from cell lysates as described, in the
presence of vehicle (Me2SO (DMSO)) alone or 100 µg/ml or 1.0 mg/ml calpeptin. All assays were performed within the
linear range with respect to PTPase activity. Phosphatase activity is
expressed as the amount of 32P label released from
[32P]poly(Glu:Tyr) and reflects the mean ± S.D.
from several independent experiments.
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Calpeptin Preferentially Targets Membrane-associated
PTPases--
PTPases can be divided into two broad categories, the
cytosolic enzymes and the transmembrane receptor type PTPases (58, 59).
We examined the possibility that calpeptin may preferentially inhibit
the activity of one of these categories by performing in
vitro PTPase assays on cytosolic and membrane fractions prepared from REF-52 fibroblasts. Interestingly, calpeptin appeared to exert a
greater inhibitory effect on the phosphatase activity present in the
membrane fraction of REF-52 fibroblasts (79% inhibition) when compared
with the cytosolic fraction (35% inhibition) (Fig. 5a). This suggests that
calpeptin preferentially inhibits either transmembrane PTPases or,
alternatively, PTPases that are localized to the membrane through their
association with membrane proteins. Consistent with an action on
transmembrane PTPases, we found that the effect of calpeptin on the
phosphatase activity of GST-PTP
(Fig. 5b, II),
a transmembrane PTPase, was greater than its effect on the cytosolic
PTPase GST-PTP-1B (Fig. 5b, I). We also tested the ability of this inhibitor to block the activity of a phosphatase with dual specificity, PTEN, and found that calpeptin exerted a
moderate inhibitory effect on PTEN (Fig. 5b,
III). In addition, preliminary kinetic studies performed on
the mode of calpeptin-mediated inhibition of PTP
suggested that
inhibition is most likely competitive in nature (data not shown). This
is similar to the mechanism of its inhibition of calpain and consistent
with an active site-directed inhibitor.

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Fig. 5.
Calpeptin exerts greater inhibitory effects
on membrane-associated PTPases in vitro.
Cytosolic (Cyt) and membrane (PM) fractions from
forskolin-treated REF-52 fibroblasts (a) and GST-PTP-1B
(I), GST-PTP (II), and GST-PTEN
(III) (b) were prepared as described under
"Experimental Procedures." In vitro PTPase assays were
performed in the absence or presence of calpeptin (100 µg/ml
(a) or 1.0 mg/ml (b)) as described for Fig.
4.
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Comparisons made between the calpeptin-mediated inhibition of PTPases
and that of two widely used PTPase inhibitors (pervanadate and PAO)
provided further evidence that calpeptin was selectively inhibiting a
subset of PTPases. Both pervanadate (8, 9) and PAO (10, 11) have
previously been demonstrated to induce stress fiber formation. In our
cell system, pervanadate and PAO both prevented the forskolin-mediated
disassembly of stress fibers in REF-52 fibroblasts (Fig.
6a, PV and
PAO) and increased tyrosine phosphorylation of cellular
proteins, including paxillin (Fig. 6b, PV and
PAO). Pervanadate is an irreversible and universal inhibitor
of PTPases (58), as demonstrated by its ability to induce an extremely
high level of tyrosine phosphorylation (Fig. 6, a and
b, PV). In contrast, PAO, which has been
suggested to selectively inhibit membrane PTPases (61), targets only
those PTPases containing vicinal thiol groups, and induces only a
modest increase in tyrosine phosphorylation (Fig. 6, a and
b, PAO). Calpeptin appeared to mimic the response
displayed by PAO (Fig. 6, a and b, CP)
and induced only a small increase in the tyrosine phosphorylation in
REF-52 fibroblasts.

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Fig. 6.
The effect of various PTPase inhibitors on
forskolin-induced disassembly of stress fibers and protein-tyrosine
phosphorylation. REF-52 fibroblasts were cultured in complete DMEM
on coverslips or 100-mm tissue culture dishes. Cells were pretreated
with vehicle (Me2SO) alone (control), 100 µg/ml calpeptin
(CP), 10 µM CI-1, 50 µM
pervanadate (PV), or 1 µM PAO for 30 min.
Cells were then treated with 10 µM forskolin for 30 min.
a, cells were visualized by double staining for actin and
phosphotyrosine using rhodamine-conjugated phalloidin and a polyclonal
antiphosphotyrosine antibody followed by a fluorescein-conjugated
anti-rabbit secondary antibody. b, whole cell lysates were
prepared as described under "Experimental Procedures," and equal
amounts of proteins were analyzed by immunoblot analysis using an
anti-phosphotyrosine antibody, PY20.
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We have used a number of experimental strategies in an attempt to
identify the calpeptin-sensitive membrane-associated PTPase. One of
these was the PTPase in-gel assay (45), which provides a method of
detecting phosphatase activity while identifying the molecular weight
of the enzyme. We have tried to modify this assay to identify the
calpeptin-sensitive PTPase by including calpeptin in the renaturation
procedure. This technique relies on effective renaturation of the
enzyme(s) following SDS-polyacrylamide gel electrophoresis, and
although it works well for cytoplasmic PTPases, it is unfortunately
ineffective for studying membrane PTPases, since they do not renature
sufficiently to detect any significant activity. Therefore, if
calpeptin is primarily inhibiting membrane PTPases, it is not
surprising that we did not detect changes in any of the bands appearing
in this assay.
Calpeptin Induces Cell Contractility--
The formation of
actin-rich stress fibers and the clustering of integrins and signaling
proteins to focal complexes are hallmark responses of activation of the
small GTPase Rho. Rho activation has also been shown to stimulate cell
contractility (62, 63). Since calpeptin was able to induce the
formation of stress fibers and focal adhesions, we were interested in
determining whether calpeptin could also induce cell contractility.
REF-52 fibroblasts were incubated with calpeptin for a time period of
30 min, and cells were monitored in real time using video microscopy
(Fig. 7a). Alternatively,
cells were fixed and stained at certain points throughout the same time
course to examine their actin stress fibers (Fig. 7b). Fig.
7 depicts sequential frames, each 15 min apart and covering a length of
30 min. As demonstrated in the control, where the vehicle alone
(Me2SO (DMSO)) (Fig. 7a,
A-C) was added to the medium, the morphology of the cells
did not change dramatically over the 30-min time period. However, when
calpeptin was added to the medium, a significant contraction of the
cells occurred (Fig. 7a, D-F), which was evident
even as early as 15 min. This contractile morphology is strikingly
similar to that observed in Swiss 3T3 fibroblasts microinjected with
constitutively activated recombinant Rho (64). Contractility of
fibroblasts was also observed by Rhodamine-phalloidin staining of cells
incubated with calpeptin at similar time points (Fig. 7b).
These results suggest that calpeptin not only induces the formation of
stress fibers but also regulates the contractile behavior of
cells, both of which are associated with activation of Rho.

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Fig. 7.
Calpeptin induces cell contractility.
REF-52 fibroblasts were cultured in complete DMEM on 60-mm tissue
culture dishes or coverslips. Cells were treated with vehicle
(Me2SO (DMSO)) alone or 100 µg/ml calpeptin
for 30 min. a, the morphology of live cells was monitored in
the presence of 25 mM Hepes, pH 7.3, and recorded using
video microscopy. b, cells cultured on coverslips were fixed
and processed for fluorescence microscopy. Cells were visualized by
staining for actin using rhodamine-conjugated phalloidin.
Panels depict cells after 0, 15, and 30 min of incubation
with Me2SO (A-C) or calpeptin
(D-F). Note the smaller "contracted phenotype" of the
cells at 15 and 30 min.
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Calpeptin Inhibits a PTPase Upstream of Rho--
Since the effect
of calpeptin on cells appeared to mimic Rho activation, we next
examined if the effects of calpeptin were being exerted upstream or
downstream of Rho. The Rho inhibitor, C3 exotransferase, was
microinjected into REF-52 fibroblasts, and cells were allowed to
recover for 60 min. Within this time, cells injected with C3 lost their
stress fibers as a consequence of Rho inactivation. Cells were then
treated with calpeptin. If acting at a site upstream of Rho, we would
expect that calpeptin would not be able to bypass C3-inactivated Rho
and induce stress fiber formation in injected cells. However, if
calpeptin was acting downstream of Rho activation, stress fiber
formation should be induced upon calpeptin treatment. As demonstrated
in Fig. 8 (C-F), calpeptin
was not able to reverse the disassembly of stress fibers induced by C3.
This suggests that calpeptin is acting on a PTPase upstream of Rho.
Studies are under way to identify the PTPase targeted by calpeptin.

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Fig. 8.
Calpeptin exerts its actions upstream of
Rho. REF-52 fibroblasts cultured in complete DMEM on coverslips
were microinjected with fluorescein-conjugated anti-human IgG
(A and B) or co-injected with
fluorescein-conjugated anti-human IgG and 100 µg/ml C3 exotransferase
(C-F), as described under "Experimental Procedures."
Cells were allowed to recover for 60 min and then treated with 100 µg/ml calpeptin for 30 min. Cells were visualized by staining for
actin using rhodamine-conjugated phalloidin. Injected cells were
identified by positive fluorescein staining. Note that cells injected
with C3 lack stress fibers even in the presence of calpeptin.
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DISCUSSION |
The Rho family of small GTPases play an essential role in actin
reorganization (3, 4). Therefore, their regulation has important
implications for processes such as embryonic development, cell
migration, and wound healing. The elucidation of signaling components
regulating Rho and its family members is consequently integral to a
better understanding of these basic cellular events. The studies
presented here define a novel role for calpeptin, a widely used
inhibitor of calpain, as an inhibitor of PTPases. In doing so, we have
identified a calpeptin-sensitive PTPase activity that acts as a
regulator of Rho and cytoskeletal reorganization. The
calpeptin-sensitive PTPase described in this report induces a
number of the characteristic responses associated with Rho activation, such as formation of stress fibers and focal adhesions, as well as the
induction of cell contractility. Furthermore, its actions are blocked
by the Rho inhibitor C3 exotransferase, suggesting that it is acting
upstream of Rho. Although numerous guanine nucleotide exchange factors
have been identified that act upstream of Rho to regulate its
activation state, signaling events linking cell receptor stimulation
and guanine nucleotide exchange factor activity are relatively unexplored.
Involvement of PTPases is not a new concept in the area of Rho
regulation. Studies using various inhibitors of tyrosine kinases (genistein, erbstatin, tyrphostin 25) (5-7) or phosphatases
(pervanadate, PAO) (8-11) have implicated tyrosine phosphorylation
events both upstream and downstream of Rho activation. But information
regarding the responsible enzymes mediating this regulation remains to
be identified. Recent reports have provided some insight into the potential identity of some PTPases with the ability to regulate cell
migration and stress fiber and focal adhesion formation. The tumor
suppressor PTEN, a phosphatase with dual specificity, was identified as
a regulator of cell migration and spreading (15). 2-Fold overexpression
of PTEN down-regulated focal adhesions and stress fibers. However,
whereas we did not detect any significant changes in
pp125FAK tyrosine phosphorylation, a 60% decrease in the
tyrosine phosphorylation of this protein was observed in response to
PTEN expression, with no detectable changes in the tyrosine
phosphorylation of paxillin. In addition, more recent studies suggest
that the cellular effects of PTEN may not be the result of modulating
the enzyme's tyrosine phosphatase activity but may be attributed to
the phosphatidylinositol lipid phosphatase activity of PTEN (65-68).
Yu et al. (13) have reported that cells deficient in
functional SHP-2, a ubiquitous cytosolic PTPase, exhibit delayed
spreading, reduced cell migration, an increased number of focal
adhesions, and reduced pp125FAK dephosphorylation. However,
deletion of SHP-2 from cells appears to have no dramatic effects on the
tyrosine phosphorylation of paxillin, in contrast with the
calpeptin-sensitive PTPase described in this report. Finally, recent
studies have demonstrated that overexpression of PTP-1B, also a
cytosolic PTPase, in 3Y1 rat fibroblasts not only resulted in impaired
cell adhesion but also interfered with cell spreading, migration,
cytoskeletal architecture, and focal adhesion formation (14). However,
a separate study using different cell lines showed no effect on cell
morphology when overexpressing PTP-1B (69). Instead, this latter study showed decreased focal adhesions when an inactive form of
the PTPase was overexpressed.
A role for calpain in the regulation of PTPase activity has been
reported. Calpain has been shown to proteolyze the cytosolic PTPases
PTP-1B (32) and PTP-MEG (44), increasing their enzymatic activity.
Although we demonstrate reduced PTPase activity with the addition of
the calpain inhibitor calpeptin, several lines of evidence rule out
calpain activation as responsible for changes in phosphatase activity
in this report. First, we could not demonstrate a role for calpain in
forskolin-induced or serum deprivation-induced disassembly of stress
fibers, using any other calpain inhibitor available, including the
highly specific calpastatin. Second, chelation of calcium, which
inhibits calcium-dependent calpain activation, does not
mimic calpeptin-induced cellular effects. Third, forskolin treatment or
serum starvation of cells does not appear to induce the
characteristic cleavage of PTP-1B (32, 38) associated with
calpain activation.2
Therefore, we can conclude that changes in PTPase activity seen in our
studies are not the consequence of calpain activation.
The second important issue brought to light in this report concerns the
specificity of calpeptin. The original design of calpeptin as a calpain
inhibitor focused on manipulating the interaction formed between the
active site of the protease and its substrates (48, 49). As is the case
with other cysteine proteases, the protease domain of calpain contains
a cysteine residue (Cys108) that is essential for its
proteolytic activity. This cysteine residue participates in the
formation of a thioester intermediate between the protease and its
substrate. Other residues surrounding the active site cysteine may also
play some role in this process. This information was used to design an
inhibitor that would (in theory) recognize calpain but not other
cysteine proteases. Unfortunately, although calpeptin does exhibit
higher specificity toward calpain than other calpain inhibitors that
were produced in the past, when used at higher concentrations it has
also been demonstrated to inhibit other cysteine proteases such as
papain (48). We now report that calpeptin inhibits another species of
enzyme containing an active site cysteine, the PTPases. We find that
concentrations of calpeptin thought previously to be specific for
calpain (20-100 µg/ml) also appear to inhibit a subset of PTPases
both in vitro and in vivo. Preliminary kinetic
analyses suggest that calpeptin's mode of PTPase inhibition is most
likely competitive, similar to that of its inhibition of calpain. This
new information highlights the dangers of relying solely on the results
provided by a single pharmacological inhibitor.
In conclusion, we have identified a PTPase activity, associated with
the membrane fraction of cells, which appears to play an important role
upstream in the regulation of the small GTPase Rho. From our
preliminary characterizations of this enzyme activity, it appears to
effectively induce the formation of stress fibers, focal adhesions, and
cell contractility, similar to the actions of Rho activators such as
serum or lysophosphatidic acid. Although little is known about the
identity of this membrane-associated phosphatase, we hope to utilize
the novel application of calpeptin as a PTPase inhibitor to establish
its identity and define some of the signaling components that
lie upstream of Rho in the regulation of cytoskeletal reorganization.