From the Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02115 and the § Department of
Molecular Biology, Yokohama City University School of Medicine, 3-9, Fuku-ura, Kanazawa-ku, Yokohama 236, Japan
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ABSTRACT |
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Adhesion of fibroblasts to extracellular matrices
via integrin receptors is accompanied by extensive cytoskeletal
rearrangements and intracellular signaling events. The protein kinase C
(PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To
characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using
pharmacological and genetic approaches. We found that inhibition of
classical and novel isoforms of PKC by down-regulation with
12-0-tetradeconoyl-phorbol-13-acetate or overexpression of
dominant-negative mutants of PKC significantly reduced extracellular
regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7
cells. Furthermore, overexpression of constitutively active PKC Adhesion of cells to extracellular matrices through integrin
transmembrane receptors initiates the assembly of an actin cytoskeletal complex at the inner surface of the membrane that is required for
filopodia, lamellipodia, focal adhesion, and stress fiber formation.
Multiple intracellular signaling molecules are stimulated following
integrin-dependent adhesion, some of which require assembly of these actin complexes for activation. Integrin targeted signaling molecules include members of the mitogen-activated protein kinase (MAPK)1 signaling pathways,
Rho family GTPases, nonreceptor tyrosine kinases such as focal adhesion
kinase (FAK) and Src, and members of the lipid signaling pathways such
as phosphatidylinositol 3-kinase (PI 3-K), and protein kinase C
(PKC) (reviewed in Refs. 1 and 2). How ligand binding to integrins
activates these signaling events and how activation of the different
molecules mediates integrin functions is still poorly understood.
The protein kinase C family of serine/threonine kinases can be
classified into three major subgroups (3). The classical PKCs consist
of PKC PKC has been shown to play an important role in cell-regulated events
such as secretion, differentiation, tumorigenesis, mitogenesis, signal
transduction (3), intracellular transport (4), gene expression (5), and
cytoskeletal regulation (6). Several in vivo PKC
targets have been identified; the best characterized being the
80-kDa phosphoprotein MARCKS (7). In addition to MARCKS, other
cytoskeletal proteins such as pleckstrin, talin, vinculin, annexins,
Several lines of evidence indicate that PKC may be important in
integrin-mediated adhesion and signaling events. First, treatment of
many different cell types with TPA causes increased adhesion, spreading, and migration of cells on extracellular matrices (19, 20).
Conversely, inhibition of PKC with pharmacological agents blocks cell
adhesion and cell spreading (21, 22) and have been reported to inhibit
cell migration (23), FAK phosphorylation (24, 25), and focal adhesion
formation (26). Second, several PKC isoforms have been implicated in
adhesion-dependent events. PKC Although the specific PKC isoforms involved in integrin-mediated events
are beginning to be defined, how PKCs regulate integrin-induced signaling events and what their targets are have not been fully explored. To characterize the role of PKC in
integrin-dependent signaling, we monitored the effects of
inhibiting PKC on fibronectin-induced signaling events in Cos7 cells
using pharmacological and molecular genetic approaches. Down-regulating
classical and novel PKC isoforms with TPA or over-expression of
dominant-negative mutants of PKC in Cos7 cells did not block
integrin-induced FAK or paxillin tyrosine phosphorylation; however,
fibronectin-induced Erk2 activation was significantly reduced.
Inhibition of PKC also greatly reduced fibronectin-induced MEK1, Raf-1,
and Ras activation as well as Shc tyrosine phosphorylation and Grb2
association. These results indicate that fibronectin-induced PKC
activation plays a role in modulating the MAPK pathway by regulating
early events upstream of Shc.
Cell Culture and Adhesion Assays--
Cos7 cells were maintained
in DMEM supplemented with 10% fetal bovine serum (Life Technologies,
Inc.), 50 units penicillin, 50 µg/ml streptomycin, and 2 mM glutamine. For adhesion assays, tissue culture plates
were coated with 5 µg/ml fibronectin (Collaborative Biomedical) in
PBS overnight at 4 °C and blocked with 1% bovine serum albumin
prior to use. Near confluent plates of cells were serum starved
overnight in DMEM containing 0.1% fetal bovine serum. Cells were
washed with PBS and trypsinized in 0.01% trypsin containing 5 mM EDTA for 5 min at 37 °C. Trypsinization was
terminated with 1 mg/ml soybean trypsin inhibitor (Life Technologies,
Inc.) in PBS containing 5 mM EDTA. Cells were collected,
washed in PBS/5 mM EDTA, resuspended in serum free DMEM,
and held in suspension at 37 °C for at least 30 min. Cells were
either left in suspension or placed on fibronectin-coated plates at a
density of 3-4 × 106 cells/10-cm plate for various
times. TPA (Calbiochem) at 25-100 ng/ml was added to plates 18-24 h
before plating on fibronectin. Suspension cells were lysed in 2× lysis
buffer as defined below and diluted with 1× buffer when necessary.
Adherent cells were washed once with DMEM prior to lysis to remove
nonadherent cells and lysed in 1× lysis buffer on the plate.
Antibodies--
Monoclonal antibodies for immunoblotting PKC
isoforms were purchased from Transduction Laboratories, except PKC Constructs and Transfections--
The pcDNA3-Flag-Raf-1
construct was generated by subcloning as described previously (34).
pSVL-HA-Erk2 was a gift from Mike Weber (University of Charlottesville,
VA). pcDNA3-HA-FAK was provided by Tony Hunter (Scripps, San Diego,
CA). pSRD plasmids containing wild type PKC Erk2, MEK1, and Raf-1 Kinase Assays--
Erk2, MEK1, and Raf-1
kinase assays were performed as described previously (36, 37). Extracts
were clarified by centrifugation at 13,000 × g for 10 min, and protein concentrations were determined using Coomassie Blue
Reagent (Bio-Rad). Erk2, MEK1, and Raf-1 substrates were 0.25 mg/ml
myelin basic protein (Life Technologies, Inc.), 30 µg/ml kinase dead
GST-MAPK (Upstate Biochemical), and 12.5 µg/ml kinase dead GST-MEK1
(Upstate Biochemical), respectively. Reactions were incubated at
30 °C for 15-30 min and terminated with 40 µl of 2× SDS Sample
buffer by heating to 95 °C for 5 min and analyzed by
SDS-polyacrylamide gel electrophoresis. Gels were transferred to
polyvinylidene difluoride (Bio-Rad), and after autoradiography and
quantitation by phosphoimaging (Fuji) blots were subjected to
immunoblotting for the respective kinases.
Immunoprecipitations and Immunoblotting--
Cells adherent to
fibronectin or left in suspension were lysed in RIPA buffer (10 mM Tris, pH 7.2, 158 mM NaCl, 1 mM
EDTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton-X, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 100 units/ml aprotinin, and 10 µg/ml
leupeptin), passed through a 25ga needle, and clarified by
centrifugation at 13,000 × g for 10 min. Protein
concentrations were determined using the BCA assay (Pierce).
Immunoprecipitations (IP) were incubated for 2-4 h at 4 °C with
protein A-conjugated agarose beads (Pierce) to capture the complexes.
All IP were washed three times with RIPA, resuspended in 2× SDS sample
buffer, boiled, and analyzed by SDS-polyacrylamide gel electrophoresis.
Gels were transferred to a polyvinylidene difluoride membrane and
probed by immunoblotting. After blocking in 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 and incubating with primary antibody, blots were incubated with horseradish
peroxidase-conjugated secondary antibody and visualized with
chemiluminescence reagent (NEN Life Science Products). Blots were
stripped in 2% SDS at 65 °C for 30 min, rinsed extensively, and
reprobed as indicated in the figure legends.
Cell Fractionation--
Cells were lysed in hypotonic cytosolic
buffer (10 mM Tris, pH 7.4, 0.5 mM EDTA, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 units/ml
aprotinin) for 20 min on ice after washing with PBS. Cells were
collected and broken open by Dounce homogenization. The soluble
cytosolic fraction (S30) was collected after centrifugation at
30,000 × g for 30 min. The pellets (P30) were further
fractionated into membrane and Triton X-100-insoluble fractions by
solubilizing in cytosolic buffer containing 1% Triton X-100. The
soluble membrane fraction (P30Mem) was recovered after centrifugation
at 30,000 × g for 30 min. The Triton X-100-insoluble
pellets were resuspended in RIPA, and supernatants (P30Ins) were
collected after centrifugation at 14,000 × g. The ratio of total cytosolic protein:membrane protein:Triton-insoluble was
calculated, and that ratio was maintained when extracts were loaded
onto SDS gels. Both soluble and insoluble membrane extracts were pooled
for immunoprecipitation of PKC Ras GTP Loading Assay--
The amount of GTP bound to Ras was
measured according to the protocol of Vaillancourt et al.
(38), which was modified as described by Zheng et al. (39)
and Clark and Hynes (40). Briefly, confluent, serum starved cells were
washed with phosphate-free DMEM and incubated with 0.25 mCi of
32PO4/plate for 4-5 h. Cells were washed,
trypsinized, and placed in suspension as described above, except that
TBS was substituted for PBS. Suspended cells were incubated in
phosphate-free DMEM and 1 mCi/ml 32PO4 for
1 h at 37 °C. Cells were plated onto fibronectin coated plates
for 20-30 min, until cells were adherent and beginning to spread.
Plated cells were washed with PBS, lysed, processed, and
immunoprecipitated as described (39). GTP/GDP was eluted from the Ras
immunoprecipitates with 25 µl 0.75 M
KH2PO4 (pH 3.4) at 68 °C for 10 min. TLC was
carried out on polyethyleneimine-cellulose plates. GTP and GDP
unlabeled standards were run in parallel and visualized under UV light.
The percentage of GTP bound relative to the ratio of GTP/GDP was
quantitated on a Fuji phosphoimaging system. The ability of GTP-Ras to
bind the effector region in Raf-1 was monitored as described previously
(41).
PKC Expression in Cos7 Cells--
As a first step toward defining
the role of PKC in fibronectin-induced signaling in Cos7 cells, we
examined which PKC isoforms are expressed in Cos7 cells. Immunoblot
analysis of Cos7 cell extracts with antibodies specific for eleven
different PKC isoforms revealed that Cos7 cells express eight PKC
isoforms (Fig. 1A): classical
PKC
Prolonged treatment of cells with TPA results in degradation and loss
of expression of TPA-responsive PKCs, effectively resulting in a cell
that is null for those PKCs. Of the PKC isoforms in Cos7 cells, only
PKC Stimulation of PKC Following Adhesion--
The best characterized
mechanism leading to in vivo activation of classical and
novel PKCs following receptor stimulation involves an increase in
intracellular diacylglycerol levels, which is mediated by PLC isozymes
(42). The observations that PLCs translocate to integrin complexes and
that adhesion of epithelial cells to collagen induces PLC
TPA-responsive PKCs translocate from the cytosolic fraction into
detergent-soluble membrane fractions during activation. To determine
whether adhesion of Cos7 cells to fibronectin causes an increase in
membrane-associated PKCs, cells were subjected to biochemical
fractionation as outlined under "Experimental Procedures." Cells
were fractionated into cytosolic (S30) and membrane fractions (P30).
The membrane fraction was further fractionated into Triton X-100-soluble (P30Mem) and Triton X-100-insoluble (P30Ins) fractions, and each was analyzed by immunoblotting with different PKC isoform antibodies. Adhesion to fibronectin resulted in translocation of a
small fraction of PKC Effect of PKC Inhibition on Adhesion and
Spreading--
TPA-induced PKC down-regulation did not adversely
affect the ability of Cos7 cells to adhere to fibronectin (Fig.
3A). However, prolonged TPA
treatment did result in delayed spreading on fibronectin (Fig.
3B). Spreading in untreated cells could be seen as early as
10 min after plating on fibronectin (60%), but TPA-treated cells were
poorly spread at 10 min (5%). By 45 min TPA-treated cells had reached
the same level of spreading as untreated cells at 10 min (58%). At 60 min after plating only 64% of the TPA-treated cells had spread
compared with 90% for untreated cells. Thus inhibition of PKC function
in Cos7 cells by long term TPA treatment affects cell spreading but not
cell adhesion.
Effect of PKC Inhibition on Tyrosine Phosphorylation--
We next
investigated which integrin-induced downstream signaling events are
affected by loss of novel or classical PKC isoform function. Adhesion
to extracellular matrices induces tyrosine phosphorylation of two focal
adhesion-associated proteins, FAK and paxillin (46). To determine
whether inhibition of PKC affects fibronectin-induced FAK or paxillin
phosphorylation, FAK and paxillin were immunoprecipitated from cells
plated on fibronectin for various times and probed by immunoblotting
with anti-phosphotyrosine antibody. Adhesion to fibronectin induced
robust tyrosine phosphorylation of both FAK and paxillin (Fig.
4A, P-tyr blot).
TPA-induced PKC down-regulation did not block fibronectin-induced FAK
or paxillin phosphorylation, although it did reduce tyrosine
phosphorylation at very early (10 min) stages of cell adhesion.
Previous studies demonstrated that inhibition of PKC blocked
integrin-induced FAK phosphorylation (22, 25). Because TPA-induced PKC
down-regulation did not completely inhibit PKC
Analysis of immunoprecipitated HA-tagged FAK by immunoblotting with
anti-phosphotyrosine antibody following 30 min of adhesion of
transfected Cos7 cells to fibronectin indicated that co-expression of
either dominant-negative mutant of PKC Effect of PKC Inhibition on Erk2 Activation--
Erk2 is activated
following integrin-mediated adhesion (1). Because TPA-induced
activation of PKC has been shown to stimulate Erk2 activation, we
examined whether PKC plays a role in fibronectin-induced Erk2
activation. Adhesion of Cos7 cells to fibronectin results in a 14-fold
stimulation of Erk2 activity, which peaks at 20-30 min after adhesion,
as measured in an immune complex kinase assay with myelin basic protein
(Fig. 5A; see also Fig. 7A). Inhibition of PKC by
TPA-induced down-regulation reduced fibronectin-induced Erk2 activity
by 60% (see Fig. 7A). These results suggest that maximal
integrin-induced activation of Erk2 requires PKC.
To determine whether the effects of TPA-induced PKC down-regulation
were due to specific effects on PKC and not due to nonspecific effects
of TPA, we over-expressed dominant-negative mutants of PKC and
monitored their effect on Erk2 activation in response to fibronectin.
Cos7 cells were transiently transfected with plasmids expressing PKC
mutants and HA-tagged Erk2, and Erk2 activation was measured in an
immune complex kinase assay after immunoprecipitating with HA tag
antibody. Over-expression of the regulatory domain of PKC
Transfection of Cos7 cells with either wild type PKC Effect of PKC Inhibition on MEK1 and Raf-1 Activation--
To
define the step in the Erk pathway that is sensitive to PKC inhibition,
we explored the effect of inhibiting PKC on upstream signaling
components of the Erk/MAPK pathway. Erk2 activation by fibronectin in
Cos7 cells requires Ras (34); therefore, we initially concentrated on
the signaling components known to lie between Ras and MAPK, namely
Raf-1 and MEK1. MEK1 and Raf-1 immune complex kinase assays were
performed on extracts from cells plated on fibronectin. Fibronectin
induced a 20-fold stimulation of MEK1 activity and a 2.7-fold
stimulation of Raf-1 activity (Figs. 6, A and B, and 7,
C and D). TPA-induced PKC down-regulation
inhibited MEK1 and Raf-1 activation by 56 and 65%, respectively. These
results indicate that PKC regulates fibronectin-induced MAPK activation at a step upstream of Raf-1.
Effect of PKC Inhibition on Ras Activation--
To determine
whether PKC is important in regulating fibronectin-induced events
upstream of Ras, we measured the effect of inhibiting PKC activation on
fibronectin-induced Ras activation by monitoring GTP binding to Ras in
response to fibronectin. Ras was immunoprecipitated from
32P-labeled cells in suspension or plated on fibronectin,
and the amount of radioactive GTP relative to GDP was measured by thin layer chromatography. Fibronectin induced a 2.48 ± 0.22-fold
increase in GTP binding to Ras (Fig. 8,
Ras GTP Loading Assay). TPA-induced PKC down-regulation
caused a 33% reduction in the level of activation of Ras-GTP
(1.67 ± 0.19-fold). To confirm these findings we also measured
Ras activation by monitoring the ability of Ras to bind its effector
Raf-1. The ability of Ras to bind to the Ras-binding domain (RBD) in
Raf-1 requires that GTP be bound to Ras (41). This method measures Ras
effector function and has the advantage of not requiring the use of
radioisotopes, which can be toxic to cells. Adhesion of Cos7 cells to
fibronectin induced Ras binding to GST-RBD beads, and TPA-mediated
down-regulation of PKC nearly abolished binding (Fig. 8, Ras-RBD
Assay) without reducing the total level of Ras (data not shown).
Thus TPA-induced PKC down-regulation suppresses the ability of
fibronectin to induce Ras activation. These results indicate that PKC
is able to regulate fibronectin-induced Erk2 activation by interceding
in the MAPK signaling pathway at a point upstream of Ras.
Effect of PKC Inhibition on Shc Phosphorylation--
Activation of
Erk2 through integrin receptors involves tyrosine phosphorylation of
the adaptor protein Shc (47-49). Phosphorylated Shc recruits the
Grb2·SOS complex, which enhances Ras-GTP binding through the GTP
exchange activity of SOS (50, 51). To determine whether inhibition of
PKC affects fibronectin-induced Shc phosphorylation, Shc was
immunoprecipitated from cells plated on fibronectin for various times
and probed by immunoblotting with anti-phosphotyrosine antibody.
Adhesion to fibronectin induced robust tyrosine phosphorylation of Shc
(Fig. 9A). Long term treatment
of cells with TPA did not affect the total levels of Shc (Fig.
9A, Shc blot) but dramatically reduced fibronectin-induced
Shc phosphorylation. The reduction in Shc tyrosine phosphorylation
observed in PKC down-regulated cells similarly resulted in reduced
association of Grb2 with immunoprecipitated Shc (Fig. 9B,
Grb2 blot). Thus Shc tyrosine phosphorylation represents at least one
PKC-dependent event upstream of Ras that is responsible for
integrin-induced Erk2 activation.
Using both pharmacological and genetic approaches we have
demonstrated that maximal Erk2 activation by fibronectin receptors in
Cos7 cells is dependent on activation of classical or novel PKCs.
Constitutively active mutants of three different PKC isoforms failed to
induce agonist-independent Erk2 activation, but activated mutants of
PKC PKC has been linked to activation of the Erk/MAPK pathway through
plasma membrane receptors in several cell types. G-protein coupled
receptors such as the M1 muscarinic receptor that couple to
Gq and Go activate Erk in a
PKC-dependent manner (52). EGF-induced Erk activation in
keratinocytes also requires PKC (14). However, in other cell types,
such as Rat-1 cells, PKC is not involved in EGF-induced Erk activation
(53). In Swiss 3T3 cells stimulated with platelet-derived growth
factor, the dose of platelet-derived growth factor influences the
extent to which PKC is required for Erk activation (54). Thus the
nature of the stimulus and strength of the signal may determine the
requirement for PKC in Erk activation.
The exact step in the Erk/MAPK pathway where PKC is required to
stimulate Erk activation also depends on the stimulus and cell type
examined. In T lymphocytes stimulated through the T-cell receptor PKC
regulates the Erk/MAPK pathway at a step upstream of Ras (55, 56),
whereas PKC-mediated activation of Erk by the M1 receptor or the EGF
receptor appears to act at a point downstream of Ras that is required
for activation of Raf-1 (14, 57). A recent report suggests that in
platelet-derived growth factor-stimulated cells, PKC can act downstream
of MEK1, possibly by inhibiting an Erk phosphatase (58). Our results
indicate that maximal activation of Erk2 in Cos7 cells following
stimulation with fibronectin requires PKC activity at a step upstream
of Ras because Ras-GTP binding was reduced in PKC down-regulated cells.
Activation of Ras by many receptors involves tyrosine phosphorylation
of the adaptor protein Shc, which recruits the Ras exchange factor SOS
to the membrane. Recruitment of SOS to Shc is mediated by the
SOS-associated adaptor protein Grb2, which binds to tyrosine phosphorylated Shc through the Grb2 SH2 domain. Integrin receptor activation leads to tyrosine phosphorylation of Shc, and this event is
required for integrin-induced Erk2 activation (47, 49). In this report,
we show that integrin-induced tyrosine phosphorylation of Shc and
association with Grb2 in Cos7 cells is dependent on PKC activity,
indicating that PKC regulates a protein tyrosine kinase or protein
tyrosine phosphatase (PTP) that regulates tyrosine phosphorylation of
Shc. The evidence that acute TPA treatment also induces Shc tyrosine
phosphorylation in some cells (59-62) supports the possibility that
PKC can modulate other signaling molecules that affect tyrosine
phosphorylation in Shc.
Several integrin-activated tyrosine kinases have been implicated in Shc
phosphorylation following engagement of integrin receptors. Giancotti
and co-workers (47, 63) have shown that integrin-induced Shc
phosphorylation is mediated by caveolin-associated Fyn, a Src family
tyrosine kinase. Other studies have implicated Src or Fyn kinase and
FAK in integrin-regulated Shc phosphorylation (49, 64, 65). In NIH-3T3
cells attached to fibronectin, PKC inhibition reduced Src kinase
activity and Erk2 activation, possibly implicating Src kinases in some
aspect of PKC regulation of Shc phosphorylation (49). The effect of PKC
on Fyn activation has not been reported. However, Fyn, like Src, may be
inhibited by inhibition of PKC. Tyrosine phosphorylation of Shc has
also been shown to be dependent on PKC in TCR stimulated T-lymphocytes where Fyn and Lck are critical for Shc activation (66). Thus PKC may be
required for Shc phosphorylation mediated by Src family kinases, either
through caveolin-dependent or FAK-dependent pathways.
What are the possible mechanisms whereby PKC could activate Src family
kinases? PKC and Src can physically interact with each other and
directly affect each other's activity. Association of PKC Alternatively or in addition, PKC might regulate Shc tyrosine
phosphorylation by inhibiting a PTP(s) that dephosphorylates Shc.
Treatment of HeLa cells with TPA or stimulation of neutrophils induces
serine phosphorylation of PTP-PEST or SHP1, respectively, and inhibits
their activity (15, 72). Furthermore, PTP-PEST can bind directly to
Shc, an event that is up-regulated by TPA or carbachol (73), and
PTP-PEST localizes to focal adhesions. Thus PKC could target tyrosine
phosphatases and inhibit their activity and thereby indirectly promote
Shc tyrosine phosphorylation.
PKC may also regulate Ras activation by modulating other regulatory
factors, like p120rasGAP. TPA stimulation causes an
increase in GAP phosphorylation and inhibition of GAP activity toward
Ras, thus allowing Ras to maintain higher levels of GTP and to be more
active (55). Overexpression of GAP blocks the ability of TPA to
stimulate Erk, indicating that the ability of PKC to regulate Erk
activation may require inhibition of GAP (74, 75). It is also possible
that PKC isoforms may regulate events downstream of Ras
(e.g. Raf-1 and MEK1) as in other cell types. However, the
additional contribution of these downstream steps to the Erk/MAPK
pathway would be masked by inhibition at earlier stages of the pathways.
In our studies overexpression of constitutively active PKC was not
sufficient to activate Erk2 in suspension cells, nor did it enhance
Erk2 activation even after engagement of integrin fibronectin receptors. The inability of activated PKC to enhance Erk2 activation even under conditions of integrin engagement suggests that a downstream target(s) of PKC may be limiting. It is interesting to note that we
have also found that expression of a constitutively active p110 subunit
of PI 3-K is unable to induce or enhance Erk2 activity in suspension
cells or in cells stimulated by fibronectin, although its activity is
required for integrin-induced Erk2
activation.2
The inability of constitutively active PKCs to stimulate Erk2
activation in suspension cells suggests that PKC is not sufficient to
activate a pathway leading to Erk2 activation and indicates that
signaling events in addition to PKC are required for Erk2 activation.
Alternatively, it is possible that PKC-induced activation of Erk2 is
blocked in suspension. It has been shown that EGF-induced Erk2
activation is significantly inhibited in suspended cells due to a
requirement for an integrin-mediated event, possibly actin
polymerization, downstream of Ras that is necessary for Raf-1 or MEK1
activation (76, 77). Integrin engagement could be required for
organization of the cytoskeleton or for relieving a negative inhibitor
of a step in the Erk/MAPK pathway. Inhibition of actin polymerization
or actin-myosin contractility of adherent cells also blocks Erk2
activation, suggesting that actin polymerization is required for some
step in Erk2 regulation (49, 78). It is possible that signals that are
dependent on actin polymerization act in concert with PKC to regulate
Erk2 activation.
Other regulatory enzymes also modulate the level of activation of Erk2
in integrin pathways. We previously demonstrated that fibronectin-induced Erk2 activation in Cos7 cells is also regulated by
p85·PI 3-K (34). Unlike PKC, P85·PI 3-K is required for optimal activation of Raf-1, downstream of Ras. Therefore, it appears that PKC
and p85·PI 3-K are activated by different integrin-regulated events
that converge on the Erk/MAPK pathway at different points, but both are
required for maximal Erk2 activation. This possibility is supported by
the observation that inhibition of both PKC and PI 3-K reduces Erk2
activation more than either
alone.3 The ability of
fibronectin to induce PI 3-K activation, like Erk2, also requires actin
polymerization.2 Thus cooperation between PI 3-K and PKC
may require integrin-induced regulation of the actin cytoskeleton.
We also found that PKC is important for cell spreading. A role for PKC
in regulating integrin-mediated cell spreading has been observed in
other cell systems. For example, inhibition of PKC activity blocks
spreading of HeLa cells on collagen-coated surfaces (30), and
down-regulation of PKC Our ability to delineate exactly which PKC isoform is involved in
integrin-induced Erk2 activation has been hampered by the inability of
any PKC isoforms to enhance integrin-induced Erk2 activation and by
what appears to be a lack of specificity of the dominant inhibitory
mutants of PKC. We did observe integrin-induced membrane translocation
of PKC In Cos7 cells, TPA-mediated down-regulation of PKC did not block FAK or
paxillin tyrosine phosphorylation, whereas calphostin C caused a
significant inhibition as reported by others (22, 25). In those same
experiments, calphostin C significantly inhibited cell attachment and
completely blocked cell spreading (25). These effects contrast with the
inhibitory activity seen with TPA-mediated down-regulation and suggest
that calphostin C has broader inhibitory activities. Alternatively,
TPA-induced PKC down-regulation may not be completely effective.
Prolonged TPA treatment did not completely eliminate PKC In summary, we have characterized the role of PKC in regulating
integrin-mediated signaling events in Cos7 cells. These studies show
that integrin modulation of PKC isoforms belonging to the classical
and/or novel subclasses are required for efficient activation of Ras,
Raf-1, MEK1, and Erk2 by fibronectin. PKC is involved in regulating
integrin-induced Erk/MAPK pathway signaling by modulating events
upstream of Ras, most likely through regulation of Shc phosphorylation.
In addition, PKC is important for efficient cell spreading on
fibronectin. PKC may be important for integrin-induced tyrosine
phosphorylation of FAK and paxillin, but these events do not appear to
be regulated by classical or novel PKC isoforms.
,
PKC
, or PKC
was sufficient to rescue
12-0-tetradeconoyl-phorbol-13-acetate-mediated
down-regulation of Erk2 activation, and all three of these PKC isoforms
were activated following adhesion. PKC was required for maximal
activation of mitogen-activated kinase kinase 1, Raf-1, and Ras,
tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC
inhibition does not appear to have a generalized effect on integrin
signaling, because it does not block integrin-induced focal adhesion
kinase or paxillin tyrosine phosphorylation. These results indicate
that PKC activity enhances Erk2 activation in response to fibronectin
by stimulating the Erk/mitogen-activated protein kinase pathway at an
early step upstream of Shc.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
I,
II, and
, which are
Ca2+/lipid-dependent kinases. The novel PKCs,
PKC
,
,
, and
are Ca2+-independent but require
lipid for activation. The third class, atypical PKCs, consists of
PKC
and
/
, which are neither Ca2+- nor
lipid-dependent. Finally, PKCµ, although similar to novel PKCs, contains a membrane-spanning domain and is often placed in a
separate class. PKCs are activated in cells following stimulation with
a wide variety of agonists, including growth factors, antigens, cytokines, and neurotransmitters. In addition, phorbol esters such as
12-0-tetradecanoylphorbol 13-acetate (TPA) are direct stimulators of classical and novel PKCs. Acute treatment with TPA
causes translocation of classical and novel PKCs from the cytosol to
the membrane, an event that is necessary for activation of at least
some PKC members. However, prolonged treatment with TPA results in
degradation and loss of expression of some TPA-responsive PKCs.
- and
-adducin, Src suppressed protein kinase C substrate,
and paxillin are potential PKC substrates in vivo (6,
8-12). Possible in vivo PKC targets involved in
receptor-induced signaling events include the serine/threonine kinase
Raf-1 (13, 14), tyrosine phosphatase SHP1 (15), G
12
subunit (16), and the EGF and insulin receptors (17, 18).
and PKC
are associated
with focal adhesions (27, 28), and PKC
and PKC
translocate to the
membrane following integrin activation (29, 30). Third, integrin
engagement leads to increased phospholipase C (PLC) activity, increased
diacylglycerol levels, and arachidonic acid production, pathways
involved in PKC activation (21, 29, 31, 32).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
PKC
I, and PKC
II isoform-specific polyclonal antibodies, which
were from Santa Cruz. Polyclonal antibodies against Grb2, FAK, PKC
,
MAPK, MEK1, Raf-1, and Ras used for immunoprecipitation assays were purchased from Santa Cruz. Polyclonal Shc and Ras antibodies and monoclonal MAPK, FAK, and paxillin antibodies were purchased from Transduction Laboratories. Monoclonal PLC
-1 antibody was purchased from Upstate Biochemical. HA tag antibody was generated in cell culture
from 12CA5 hybridoma cells (33), which were kindly provided by Dr. Jeff
Settleman (Massachusetts General Hospital, Charlestown, MA).
Anti-phosphotyrosine 4G10 antibody was provided by Dr. Tom Roberts
(Dana Farber Cancer Institute, Boston, MA).
and kinase dead PKC
(K
) have been described previously (35). The regulatory
domain truncation mutant of PKC
(pSRD-PKC
RD) encodes the
N-terminal region of mouse PKC
from amino acid residues 1-298
connected to C-terminal amino acids 655-674. The regulatory domain
truncation mutant PKC
(pSRDHis-PKC
RD) encodes the N-terminal
region of rabbit PKC
from amino acid residues 1-385 and is tagged
with His6/T710 tag at the N terminus. The kinase domain
truncation mutant of mouse PKC
(pSRD-PKC
KD) encodes amino acid
residues 348-674 and is preceded by Met. The kinase domain truncation
mutant of rabbit PKC
(pSRDHis-PKC
KD) encodes amino acid residues
386-736 preceded by the His6/T710 tag. The kinase domain
truncation mutant of rabbit PKC
(pSRD-PKC
KD) encodes amino acid
residues 298-672. Cos7 cells were transfected at 2 × 106 cells/10-cm plate with 8-10 µg of total DNA using
the LipofectAMINE procedure as described by the manufacturer (Life
Technologies, Inc.). 48 h after transfection, cells were used in
adhesion assays as described above.
from membrane fractions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, PKC
I, and PKC
II, novel PKC
and PKC
, atypical PKC
and PKC
as well as PKCµ.
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Fig. 1.
Expression of different PKC isoforms in Cos7
cells and effect of long term TPA treatment. A, whole
cell lysates from Cos7 cells were probed by immunoblotting with
isoform-specific antibodies to the different classes of PKC
(lanes Cos). Rat brain extracts (lanes c) were
used as positive controls for antibody recognition, except for PKC
and PKC
, which were MDCK and Jurkat cell extracts, respectively. The
asterisks denote the PKC-specific bands present in Cos
cells. B, Cos7 cells were left untreated or treated with 100 ng/ml TPA for 24 h, placed in suspension (S), or plated
on fibronectin (FN) for various times. Whole cell lysates
were analyzed by immunoblotting for the presence of different PKC
isoforms. The change in mobility shift observed for PKCµ is due to a
tear in the gel and does not represent a mobility shift due to
fibronectin-mediated adhesion.
,
,
, and
should be affected by prolonged TPA exposure.
Treatment of Cos7 cells for 24 h with 100 ng/ml TPA eliminated
PKC
,
I, and
but only reduced PKC
by 70% as monitored by
immunoblotting of whole cell lysates (Fig. 1B). Higher doses
or longer TPA treatment did not further reduce PKC
levels (data not
shown). As expected, atypical PKC isoforms and PKCµ were unaffected
by long term TPA treatment. Plating cells on fibronectin did not change
the level of PKC expression either before or after TPA-mediated
down-regulation (Fig. 1B).
activation
through
1 integrin indicate that activation of PLC may be important
for integrin signaling events (31, 32, 43). To determine whether PLC
can be activated by fibronectin in Cos7 cells, PLC
1 was
immunoprecipitated from cell extracts following adhesion of Cos7 cells
to fibronectin, and the levels of tyrosine phosphorylation were
monitored by immunoblotting with anti-phosphotyrosine antibody, because
tyrosine phosphorylation of PLC
1 activates it. Plating cells on
fibronectin resulted in a rapid increase in tyrosine phosphorylation of
PLC
1 (Fig. 2A). No tyrosine
phosphorylation of PLC
1 was detected in suspension cells. Thus
adhesion of Cos7 cells to fibronectin activates PLC
1, which could
lead to activation of PKC through diacylglycerol production.
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Fig. 2.
Fibronectin-induced regulation of
PLC and different PKC isoforms in Cos7
cells. A, Cos7 cells were placed in suspension
(S) or plated on fibronectin (FN) for various
times. PLC
1 was immunoprecipitated (PLC
IP) from whole
cell lysates, and fibronectin-induced tyrosine phosphorylation was
monitored by immunoblotting with anti-phosphotyrosine antibody
(P-tyr blot). PI, preimmune serum. Total levels
of PLC
1 in the IP were measured by reprobing the blot with
anti-PLC
1 antibody (PLC
blot). B, Cos7
cells were placed in suspension or plated on fibronectin for 30 min.
Cells were fractionated into cytosolic (S30), detergent-soluble
membrane (P30Mem), and detergent-insoluble (P30Ins) fractions as
described under "Experimental Procedures." Lysates from each
fraction were probed by immunoblotting with isoform-specific PKC
antibodies. C, Cos7 cells were stimulated and fractionated
as described above. PKC
was immunoprecipitated (PKC
IP) from the membrane (P30) fractions, and tyrosine
phosphorylation was monitored by immunoblotting with
anti-phosphotyrosine antibody (P-tyr blot). Total levels of
PKC
in the IP were detected by reprobing the blot with anti-PKC
antibody (PKC
blot).
and PKC
I to the soluble membrane fraction,
both of which were predominately cytosolic in suspension cells (Fig.
2B). A corresponding increase in soluble membrane-associated PKC kinase activity was also observed (data not shown). Unlike PKC
I,
PKC
II was found to be primarily in the P30 pellet and fractionated
equally between the soluble membrane and detergent-insoluble fraction
in suspension cells. This distribution did not change following
adhesion to fibronectin (Fig. 2B). Approximately a third of
total PKC
was associated with the P30 pellet in suspension cells.
Following adhesion to fibronectin a small but reproducible increase in
the amount of PKC
in the soluble membrane fraction (P30Mem) occurred
concurrently with a reduction in mobility (Fig. 2B). Like
PKC
, approximately a third of the total PKC
was found in the P30
pellet, but PKC
distribution did not change following adhesion.
Growth factors and TPA have been shown to induce tyrosine phosphorylation of PKC
(44, 45). Adhesion of Cos7 cells to fibronectin also resulted in inducible tyrosine phosphorylation of
membrane-associated PKC
, as seen by immunoprecipitation of PKC
and immunoblotting with anti-phosphotyrosine antibody (Fig. 2C). Thus adhesion of Cos7 cells to fibronectin induces
activation of PLC
1, membrane association of PKC
,
I, and
,
and tyrosine phosphorylation of membrane-associated PKC
.
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Fig. 3.
Effect of PKC inhibition on cell adhesion and
spreading. A, Cos7 cells were left untreated or treated
with 100 ng/ml TPA for 24 h then placed in suspension or plated on
fibronectin (FN) for various times. The percentage of
adhesion was measured by assaying the amount of protein in adherent
cells relative to that in suspension. Results presented are from three
independent assays. B, Cos7 cells were treated as described
above, except that at various times after spreading cells were
photographed under 40× magnification and the amount of cell spreading
was quantitated by counting the percentage of spread cells
versus round cells in several fields. Cells were considered
spread if they lost nuclear refractility and membrane blebbing. Graph
legends are fibronectin ( ) and TPA-treated on fibronectin
(
).
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Fig. 4.
Effect of PKC inhibition on
fibronectin-induced FAK and paxillin tyrosine phosphorylation.
A, Cos7 cells left untreated or treated with 100 ng/ml TPA
for 24 h then placed in suspension (S) or plated on
fibronectin (FN) for various times. Paxillin or FAK was
immunoprecipitated (Paxillin IP and FAK IP) from
cell lysates, and the level of tyrosine phosphorylation analyzed by
immunoblotting with anti-phosphotyrosine antibody (P-tyr
blots). PI, preimmune serum. Total levels of paxillin
in the IP were measured by reprobing the blot with anti-paxillin
antibody (Paxillin Blot). B, Cos7 cells were
transfected with 3 µg of plasmid expressing HA-tagged FAK and 8 µg
of empty vector (vector) or plasmids expressing various
PKC mutants. Cells were left in suspension (S) or plated
on fibronectin (FN) for 20 min. FAK was immunoprecipitated
(HA IP) with HA antibody, and its phosphorylation was
monitored by immunoblotting with anti-phosphotyrosine antibody
(P-tyr blot). Total levels of FAK were measured in duplicate
immunoprecipitation reactions by immunoblotting with FAK antibody
(FAK blot). Whole cell extracts were probed by
immunoblotting with PKC antibodies to measure the level of
overexpressed PKC mutants (PKC blot). *, PKC bands.
expression, we
examined the effect of inhibiting PKC
on integrin-induced FAK
phosphorylation. Either of the two dominant-negative mutants of PKC
(PKC
K
or PKC
RD) was co-expressed with HA-tagged
FAK, and the ability of fibronectin to induce FAK tyrosine
phosphorylation was monitored. The PKC
mutants were expressed at
levels greater than 10-fold over endogenous levels (Fig. 4B,
PKC blot) as determined by immunoblotting of whole cell
extracts with PKC
antibody. The kinase inactive PKC
mutant
(PKC
K
) and the regulatory domain of PKC
(PKC
RD) have previously been shown to act as a dominant-negative mutants
(14, 35) and in Cos7 cells inhibited fibronectin-induced Erk2
activation (Fig. 5).
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Fig. 5.
Effect of PKC inhibition on
fibronectin-induced Erk2 activation. A, Cos7 cells were
treated with 100 ng/ml TPA for 24 h and placed in suspension
(S) or allowed to adhere to fibronectin (FN). At
various times cells were lysed, and Erk2 activity was measured after
immunoprecipitation in an in vitro kinase assay using myelin
basic protein (MBP) as a substrate. PI, preimmune
serum. Total Erk2 levels present in the IP were measured by
immunoblotting with anti-Erk antibody (Erk blot).
B, Cos7 cells were transfected with 0.5 µg of plasmid
expressing HA-tagged Erk2 and 8 µg of empty vector
(Vector) or a plasmid expressing the PKC K
mutant. After transfection cells were either placed in suspension
(S) or plated on fibronectin (FN) for 20 min.
Erk2 activity was measured in an in vitro kinase assay as
described above. Total Erk2 levels present in the IP were measured by
immunoblotting with anti-Erk antibody (Erk blot). Total
levels of the PKC
K
mutant expressed in whole cell
lysates was measured by immunoblotting with PKC
antibody
(PKC
blot). C, Cos7 cells were transfected
with HA-Erk2 and plasmids expressing regulatory domain truncation
mutants of PKC
(PKC
RD) or PKC
(PKC
RD), and Erk2 kinase activity was measured 20 min after adhesion
as described above. ns, no substrate control. Total levels
of Erk2 in the IP were measured by immunoblotting with anti-Erk
antibody (Erk blot). Total levels of expression of each PKC
mutant in whole cell lysates was measured by immunoblotting with PKC
or PKC
antibody (PKC blot). *, PKC bands. D,
Cos7 cells were transfected with HA-Erk2 and plasmids expressing
different constitutively active PKC mutants. After transfection cells
were either left untreated or treated for 24 h with 25 ng/ml TPA.
Cells were either placed in suspension (S) or plated on
fibronectin (FN) for 20 min. Erk2 activity was measured as
described above. Total Erk2 levels present in the IP were measured by
immunoblotting with anti-Erk antibody (Erk blot). Total
levels of expression of each PKC mutant in whole cell lysates were
measured by immunoblotting with appropriate PKC antibody (PKC
blot). *, PKC bands; ns, no substrate control.
had no effect on the ability
of fibronectin to induce FAK phosphorylation (Fig. 4B). Similar results were observed with dominant-negative PKC
and PKC
mutants (data not shown). Furthermore, overexpression of the
constitutively active kinase domain of PKC
(PKC
KD) did not
induce or enhance FAK tyrosine phosphorylation (Fig. 4B). Expression of the dominant-negative PKC
(PKC
RD) in combination with TPA-mediated down-regulation of PKC also did not block
fibronectin-induced FAK tyrosine phosphorylation (data not shown).
These data indicate that members of the classical and novel PKCs are
not likely to be involved in regulating integrin-induced FAK tyrosine
phosphorylation in Cos7 cells.
(PKC
RD) and full-length kinase inactive PKC
(PKC
K
)
inhibited fibronectin-induced HA-Erk2 activation by 50 and 65%, respectively (Fig. 5, B and C; see also Fig.
7B). All PKC mutants were expressed greater than 10-fold
over endogenous levels as measured by immunoblotting with PKC antibody
(Fig. 5, B and C, PKC blot).
Over-expression of truncated PKC
(PKC
RD) also inhibited fibronectin-induced Erk2 activation by 45% (Fig. 5C).
Additionally, over-expression of constitutively active forms of PKC
or PKC
, mutants expressing only the catalytic domain (PKC
KD or
PKC
KD), were able to rescue Erk2 activation in cells where Erk2
activation was inhibited by TPA-mediated PKC down-regulation (Fig.
5D). Similar results were obtained with a PKC
KD mutant
(data not shown). Thus inhibition of PKC significantly blocks
fibronectin-induced Erk2 activation, indicating that PKC is an
important regulator of integrin-mediated Erk2 activation in Cos7 cells
and that several PKC isoforms are capable of regulating Erk2 activation.
(PKC
wild
type) or constitutively active forms of PKC (PKC
KD or PKC
KD)
did not induce a significant enhancement of fibronectin-induced Erk2
activation over that seen with vector alone. Nor did overexpression of
active PKC significantly increase basal levels of Erk2 activity. Co-expression of two or more constitutively active PKCs also failed to
enhance Erk2 activation (data not shown). Overexpression of the active
catalytic domain of PKC
(PKC
KD) inhibited cell growth and
greatly reduced the ability of the transfected cells to readhere to
fibronectin. Nevertheless, it was still able to rescue Erk2 activity in
those cells able to readhere (data not shown). Together these data
indicate that PKC is important for fibronectin-induced Erk2 activation
but that overexpression of constitutively active PKC is not sufficient
to induce Erk2 activation in suspension. In addition, overexpression of
PKC does not enhance fibronectin-induced Erk2 activation. These data
suggest that stimulation of PKC alone is not sufficient for Erk2
activation and that downstream targets of PKC in the Erk signaling
pathway may be limiting such that overexpression of PKC in the presence
of fibronectin is not further stimulatory.
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Fig. 6.
Effect of inhibition of PKC on
fibronectin-induced MEK1 and Raf-1 activation. A, Cos7
cells were treated with 100 ng/ml TPA for 24 h and placed in
suspension (S) or plated on fibronectin (FN) for
various times. MEK1 was immunoprecipitated and its activation measured
by an in vitro kinase assay with kinase inactive GST-Erk1 as
a substrate. Total levels of MEK1 in the IP were monitored by
immunoblotting with MEK1 antibody (MEK Blot). B,
Cos7 cells were transfected with 10 µg of plasmid expressing Raf-1
treated as described above except that Raf-1 was immunoprecipitated and
its in vitro kinase activity was measured using kinase
inactive GST-MEK as a substrate. Total levels of Raf-1 in the IP were
measured by immunoblotting with Raf-1 antibody (Raf
blot).
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Fig. 7.
Quantitation of Erk2, MEK1, and Raf-1 kinase
assays. Kinase assays were quantitated using a Fuji phosphoimager,
and the fold activation was calculated. Results presented are a direct
quantitation of autoradiographs shown in Figs. 5A
(A), 5B (B), 6A
(C), and 6B (D) and are representative
of at least three different experiments. A, C,
and D, , FN;
, TPA down-regulation on FN.
B,
, cells transfected with vector;
, kinase inactive
PKC
.
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Fig. 8.
Effect of inhibition of PKC on
fibronectin-induced Ras. Upper panel, Cos7 cells were
left untreated or treated for 18 h with 25 ng/ml TPA. Cells were
then labeled for 4 h with 32PO4, placed in
suspension (S), or plated on fibronectin (FN) for
30 min. Cells were lysed, Ras was immunoprecipitated, and the levels of
GTP and GDP were analyzed by thin layer chromatography. Middle
panel, the amount of Ras-associated radiolabeled GDP and GTP was
quantitated using a Fuji phosphoimager, and the percentage of GTP
relative to the total GTP plus GDP was calculated and expressed as fold
increase in GTP levels. Lower panel, Cos7 cells were left
untreated or treated for 18 h with 25 ng/ml TPA and placed in
suspension (S) or plated on fibronectin (FN) for
30 min. GTP-bound Ras was removed from cell lysates with GST-RBD bound
to glutathione beads, and the amount of Ras present was measured by
immunoblotting with anti-Ras antibody.
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Fig. 9.
Effect of inhibition of PKC on Shc tyrosine
phosphorylation and Grb2 association. A, Cos7 cells
were left untreated or treated with 100 ng/ml TPA for 24 h. Cells
were placed in suspension (S) or plated on fibronectin
(FN) for various times. Cells were lysed and Shc was
immunoprecipitated and probed by immunoblotting with
anti-phosphotyrosine antibody (P-tyr blot). Total levels of
Shc in the IP were measured by immunoblotting with anti-Shc antibody
(Shc blot). B, Cos7 cells were treated as above
and placed in suspension (S) or plated on fibronectin
(FN) for 20 min. Shc was immunoprecipitated, and the
presence of Grb2 was detected by immunoblotting with anti-Grb2 antibody
(Grb2 blot).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, PKC
, or PKC
were all able to rescue Erk2 activation after
TPA-induced PKC-down-regulation, suggesting that several PKC isoforms
are capable of regulating integrin-induced Erk2 activation. Maximal
activation of MEK1, Raf-1, and Ras was also dependent on PKC,
indicating that PKC regulates Erk2 activation at a step upstream of
Ras. Integrin-induced tyrosine phosphorylation of Shc and Grb2
association was also dependent on PKC. Thus PKC acts to stimulate Erk2
activation in response to fibronectin by stimulating the Erk/MAPK
pathway at a step upstream of Ras by regulating Shc phosphorylation.
with Src
increases the level of serine phosphorylation on Src and increases Src
activity (67, 68), but higher levels of Src activity can feed back and
inhibit PKC
(69). Additionally PKC and Src can both bind to the
PKC
binding protein RACK1. PKC
binding to RACK elevates its
kinase activity, whereas Src activity is inhibited by RACK binding
(70). Potentially, activation of PKC
could displace Src and relieve
this inhibitory interaction. This model is made even more attractive by
the recent finding that Rack1 can associate with
1 and
2 integrins in a PKC-dependent manner (71).
Alternatively, PKC could be required for activation of a PTP(s) that
dephosphorylates the C-terminal phosphotyrosine that negatively
regulates Src protein kinases, thus leading to activation of this
family of kinases.
and PKC
with antisense oligonucleotides in
vascular smooth muscle cells blocked cell spreading on fibronectin
(29). Additionally, overexpression of a dominant inhibitory mutant of
MARCKS, a well characterized PKC substrate, completely blocks cell
spreading of fibroblasts on fibronectin (79). Recent studies have
linked the ability of cells to migrate to the Erk/MAPK pathway
(80-82). However, a role for Erk in regulating cell spreading has not
been demonstrated, and in fact, Erk2 can be activated in the absence of
cell spreading, and inhibition of the Erk/MAPK signaling pathway does
not block cell spreading (83, 84). Thus PKC could be playing a role in
several integrin-mediated signaling events, only some of which are
dependent on Erk.
, PKC
I, and PKC
. Integrin-induced membrane translocation
was not seen for PKC
. However, we did detect increased tyrosine
phosphorylation of PKC
following adhesion to fibronectin. Thus, of
the five TPA-regulated PKCs expressed in Cos7 cells, we have detected
adhesion-induced regulation of four PKC isoforms. Inhibition of Erk2
activation was observed with dominant inhibitory mutants of two PKC
isoforms, and three constitutively active PKC isoforms were able to
rescue TPA-mediated down-regulated Erk2 activation. The
cross-inhibitory effects of dominant-negative mutants of different PKC
isoforms has been observed by others (35). Stimulation of MEK1 activity
by TPA and Raf-1 was inhibited by three different dominant mutants of
PKC, PKC
,
, or
, a situation analogous to fibronectin-induced
Erk2 activation. Additionally, several different PKC isoforms can
activate the Erk/MAPK pathway in different or in the same cell types
(14, 35, 85-88). One possible interpretation of these findings is that
several different PKC isoforms share some redundant functions in cells,
such as Erk activation, but also perform isoform-specific functions as
well. Another possibility is that specificity is lost under conditions
of overexpression employed in the approaches utilized in this study.
, and it is
possible that the remaining PKC
activity is responsible for
mediating some of these events. However, dominant-negative PKC
did
not block FAK phosphorylation under conditions where it blocked Erk2 activation. It is clear that these two agents may be acting in slightly
different ways, and further investigations into the role of PKC in
integrin-mediated events need to be addressed by using several
different approaches.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Michael Weber for HA-tagged Erk2, to Tony Hunter and David Schlaepfer for HA-tagged FAK, to Brian Drucker and Tom Roberts for the 4G10 monoclonal anti-phosphotyrosine antibody, and to Warren King for helpful discussions.
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FOOTNOTES |
---|
* This work was funded by grants CA 78773, CA27951 (J.S.B.) and CA72203 (C.K.M.) from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 617-432-3974; Fax: 617-432-3969; E-mail: Joan_Brugge{at}hms.harvard.edu.
2 W. King, unpublished results.
3 C. Miranti, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; PKC, protein kinase C; TPA, 12-0-tetradecanoyl-phorbol-13-acetate; Erk2, extracellular regulated kinase 2; MEK, mitogen-activated kinase kinase; FAK, focal adhesion kinase; PI 3-K, phosphatidylinositol 3-kinase; PLC, phospholipase C; EGF, epidermal growth factor; PTP, phosphotyrosine phosphatase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; RBD, Ras-binding domain; HA, hemagglutinin; IP, immunoprecipitation(s); GAP, GTPase-activating protein.
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REFERENCES |
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