From the Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037
Received for publication, August 17, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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A variety of intracellular signaling
pathways are linked to cell surface receptor signaling through their
recruitment by Src homology 2 (SH2)/SH3-containing adapter
molecules. p21-activated kinase 1 (PAK1) is an effector of Rac/Cdc42
GTPases that has been implicated in the regulation of cytoskeletal
dynamics, proliferation, and cell survival signaling. In this study, we
describe the specific interaction of PAK1 with the Grb2 adapter protein
both in vitro and in vivo. We identify the site
of this interaction as the second proline-rich SH3 binding domain of
PAK1. Stimulation of the epidermal growth factor receptor (EGFR)
in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2,
although the PAK1-Grb2 association is itself independent of this
stimulation. A cell-permeant TAT-tagged peptide encompassing the
second proline-rich SH3 binding domain of PAK1 simultaneously blocked
Grb2 and activated EGFR association with PAK1, in vitro and
in vivo, indicating that Grb2 mediates the interaction of
PAK1 with the activated EGFR. Blockade of this interaction decreased
the epidermal growth factor-induced extension of membrane lamellae.
Thus Grb2 may serve as an important mechanism for linking downstream
PAK signaling to various upstream pathways.
Growth factors, cytokines, and many other hormones signal through
specific cell surface receptors that contain intrinsic tyrosine kinase
activity (1, 2). Activation of these receptors stimulates autophosphorylation at tyrosine residues within the cytoplasmic receptor tail. Many of these residues are contained in specific motifs
that, when phosphorylated, recruit various signaling molecules and/or
SH21/SH3-containing adapter
proteins (3-5). These adapter molecules, including Nck and Grb2, serve
as scaffolds to recruit and/or stimulate additional downstream
signaling pathways, leading to cellular responses including
cytoskeletal rearrangement and proliferation (3-5). Grb2 is a 25-kDa
adapter protein that associates with activated receptor tyrosine
kinases via its single SH2 domain (6). Grb2 also contains two SH3
domains flanking the SH2 domain on both sides that serve to recruit
additional signaling molecules to the receptor to form multimeric
signaling complexes (7). The role of the Grb2 in the Ras signaling
pathway has been well established (8, 9).
The p21-activated kinases (PAKs) are serine/threonine kinases that
serve as important mediators of Rac- and Cdc42 GTPase, and possibly
sphingolipid, signaling (10-13). The catalytic activity of PAK is
regulated by the binding of active GTPases and/or sphingolipids to the
conserved p21 binding motif in the N terminus (12, 13), leading to
relief of an autoinhibitory interaction with the C-terminal catalytic
domain (14-16). The kinase activity of PAK has been implicated in both
cytoskeletal regulation and proliferative signaling by growth factor
receptor tyrosine kinases (17-20). The PAK1 N terminus also contains
several putative SH3 binding motifs that are thought to be involved in
the interaction of PAK with regulatory proteins, including
PAK-interacting exchange factor (PIX) (21). It has been shown
previously (22, 23) that the adapter Nck binds via its second SH3 motif
to the first PAK1 PXXP domain beginning at amino acid
residue 13. There appears to exist a pool of cellular PAK1
constitutively associated with Nck (22, 23), although this interaction
is modulated by cell adhesion (24) and can be dissociated when PAK is
phosphorylated at Ser-21 (25). The binding of Nck to PAK acts to
recruit and couple PAK to signaling by the T cell receptor (26), the
platelet-derived growth factor receptor (22, 23), the Fc In this study, we describe the specific interaction of PAK1 with the
Grb2 adapter protein both in vitro and in vivo.
We identify the site of this interaction as the second proline-rich SH3
binding domain of PAK1. Stimulation of the EGF receptor (EGFR) in HaCaT cells greatly enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. Evidence is presented that Grb2 mediates the coupling of
PAK1 to the activated EGFR. Grb2 may thus serve as an important mechanism for linking downstream PAK signaling to various upstream pathways that assemble signaling modules containing the Grb2 adapter protein.
Plasmids and Constructs--
PAK1 N-terminal fragment (aa
1-235), PAK1 N-terminal fragment (aa 1-74), and point mutants of
these N-terminal constructs were generated by polymerase chain reaction
and cloned into pET-28a (Novagen) for expression as N-terminal
His-tagged proteins. The point mutants included PAK1 P13A and PAK1
P42A; these mutations disrupt the PXXP SH3 binding motifs at
these sites (23). Proteins were isolated using nickel beads according
to the manufacturer's instructions (Qiagen) and gave predominantly
single bands on both silver- and Coomassie Blue-stained
SDS-polyacrylamide gels, with the exception of His full-length PAK1 N
terminus (aa 1-235), which degraded to yield several smaller fragments.
Preparation and Use of TAT-PAK1 Peptides--
PAK1 contains
several N-terminal proline-rich motifs that have the characteristic
PXXP (where X indicates a variable amino acid)
structure of SH3 binding domains (29). Two of these consist of the
sequences PPAPP (aa 12-16) and PLPPNP (aa 40-45). We prepared peptides encompassing these domains that were coupled to a
membrane-permeant TAT-derived sequence (YARAAARQARA) (30). These are as
follows: P1, YARAAARQARADKPPAPPM and P2, YARAAARQARASKPLPPNPEA.
A TAT peptide-only control was obtained from Dr. Steven Dowdy
(University of California, San Diego).
Antibodies--
A rabbit polyclonal antibody (R2124) raised
against amino acids 174-306 of PAK1 (as in Ref. 31) was used for PAK1
immunoprecipitation (1:50) and immunoblotting (1:1,000). Grb2
immunoprecipitation (1:50) and immunoblotting (1:5,000) was performed
with Grb2 mouse monoclonal antibody (G16720) from BD
Biosciences. Phosphotyrosine-containing proteins
(i.e. the stimulated EGFR) were detected using mouse monoclonal anti-phosphotyrosine 4G10 and Nck with rabbit polyclonal antibody 06-288 from Upstate Biotechnology. EGFR was detected using
mouse monoclonal antibody E12020 from BD Biosciences.
Cell Culture and Immunoprecipitation Analyses--
HaCaT cell
lines were maintained in Dulbecco's modified Eagle's medium
(Invitrogen) with 10% fetal calf serum, 10 mM
HEPES, 2 mM L-glutamine at 37 °C in an
atmosphere of 10% CO2. Jurkat T cells were maintained in
RPMI 1640 medium (Invitrogen) with 10% fetal calf serum, 10 mM HEPES, 2 mM L-glutamine at
37 °C in an atmosphere of 10% CO2.
For immunoblotting and immunoprecipitation studies, HaCaT cells were
plated in 10-cm tissue culture dishes and serum-starved for 18 h
prior to the start of experiment. As indicated, TAT-tagged peptides
were added at the time of plating (200 µg/ml) and left for the entire
time (18 h). The cells were then stimulated with EGF (200 ng/ml) for 2 or 30 min and then rapidly scraped from the dish into ice-cold lysis
buffer (as in Ref. 23). After 1 h on ice, the lysates were
pelleted for 10 min at 14,000 rpm at 4 °C, and the clarified
supernatants removed and used for immunoprecipitation and/or immunoblotting.
Aliquots (~1 mg of total protein) of cell lysates were incubated with
the indicated antibody overnight at 4 °C and then with 60 µl of a
1:1 slurry of protein A or protein G-Sepharose beads for 45-60 min.
Beads were pelleted and washed four times with 1 ml (each time) of
radioimmune precipitation assay buffer and then used for immunoblots.
Radioimmune precipitation assay buffer consisted of 50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium cholate, 0.1% SDS, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 50 IU/ml aprotinin.
Immunoblotting was done as follows: SDS-PAGE gels were transferred to
polyvinylidene difluoride membranes (Immobilon-P transfer membrane,
pore size 0.45 µm; Millipore) and incubated with blocking buffer (10 mM Hepes, pH 7.4, 0.5 M NaCl, 3% bovine serum
albumin, 10% goat serum) overnight at 4 °C, followed by incubation
with primary antibodies for 1 h at room temperature. After
extensive washing with Tris-buffered saline, 0.1% Triton X-100
membranes were incubated for ~1 h at room temperature with secondary
antibodies (anti-goat horseradish peroxidase) and visualized by
enhanced chemiluminescence (Pierce).
Imaging--
HaCaT cells suspended in either medium alone or
medium containing indicated concentrations of the TAT peptide were
seeded on 25-mm glass coverslips coated with 10 µg/ml fibronectin and allowed to spread for 18 h. The coverslips were placed into
Auttoflor live cell chambers (Molecular Probes), and time-lapse imaging was carried out at 30-s intervals using an Olympus IX70 microscope fitted with a Princeton MicroMax 5-MHz 12-bit cooled CCD camera. The
cells were stimulated on the microscope stage with EGF (200 ng/ml).
Images were analyzed for total cell area using ISEE software from Inovision.
Identification of Grb2 as a PAK1-interacting Protein--
We
utilized various purified His-tagged PAK1 N-terminal constructs in an
effort to affinity purify potential PAK1-interacting proteins from
Jurkat T cell lysates. Along with a number of other protein bands
detected by silver staining, both full-length PAK1 N terminus (aa
1-235) and a shorter version of the PAK1 N terminus (aa 1-74)
specifically bound an ~25-kDa protein (Fig.
1A, lanes 2 and
3). This 25-kDa protein band was not observed in control incubations with nickel beads (Fig. 1A, lane 1)
or when beads to which the PAK1 C terminus (aa 236-545) was coupled
were used (data not shown).
Analysis of PAK1 structure using the ScanSite program (32) predicted
that the 42PPNP sequence was solely likely to bind to
the SH3 domain of the 25-kDa adapter protein Grb2. We therefore tested
whether the PAK1-associated protein was indeed Grb2 by immunoblotting
the band pulled down out of Jurkat lysates with a specific Grb2
antibody (Fig. 1B). The bound protein strongly reacted with
the Grb2 antibody, confirming its identity as Grb2. To further
characterize the binding of Grb2 to the PAK N terminus, we prepared
mutations of the first (aa P13A) and second (aa P42A) proline-rich SH3
binding motifs. As shown in Fig. 1C, Grb2 bound effectively
to wild-type N-terminal aa 1-74 and aa 1-235 constructs and to a
slightly lesser extent to the P13A mutant of these fragments. The
latter may indicate some weak association of Grb2 with the first PAK1
proline-rich motif. In contrast, mutation of P42A resulted in a
dramatic reduction in binding of Grb2 to PAK1 (p < 0.003). These data indicate that Grb2 interacts most effectively with
the PAK1 N terminus via the second PXXP-SH3 motif interaction.
To confirm a specific interaction of Grb2 with the second PAK1 SH3
binding domain beginning at position 42, we examined the ability of
TAT-tagged peptides that encompassed both the first (aa 10-17 = DKPPAPPM) and second (aa 38-45 = SKPLPPNP) PAK1 proline-rich domains to block binding of Grb2 to PAK1. As shown in Fig.
1D, the peptide containing the second PXXP motif
substantially inhibited Grb2 binding to PAK1. In contrast, there was a
weak inhibitory effect observed with the first PXXP site
peptide. We verified that PAK1 interacts directly with Grb2 using
purified protein components. Fig. 1E shows that the
His-tagged PAK1 1-74 fragment bound specifically to GST-Grb2 beads
(lanes 2 and 4) but not to control beads
(lanes 1 and 3).
PAK1 Interacts with Grb2 and EGFR in Intact Cells--
We examined
the interactions of endogenous Grb2 and PAK1 in several cell types. We
observed that Grb2 could be specifically co-immunoprecipitated from
cell lysates with a PAK1 antibody (Fig. 2A). Conversely, with a Grb2
antibody we co-immunoprecipitated PAK1 (Fig. 2B). The
association of Grb2 with PAK1 was not significantly increased upon
treatment of HaCaT cells with EGF (see below). These data indicate that
endogenous Grb2 interacts constitutively with a pool of PAK1 in various
cell types, including Jurkat and HaCaT cells.
Adapter proteins such as Grb2 are known to be important in the coupling
of various growth factor receptors to downstream signaling pathways.
Grb2 has been shown to associate with tyrosine-phosphorylated EGFRs via its SH2 domain (6). Activity of PAK1 is stimulated by
EGF (22). To assess the physiological relevance of the Grb2-PAK1 interaction, we decided to examine this interaction in a cell line that
responds well to EGFR stimulation. The human keratinocyte HaCaT cell
line exhibits a dramatic stimulus-dependent 13-15-fold increase in the amount of tyrosine-phosphorylated EGFR over a 30-min
time course (Fig. 3A).
Endogenous PAK1, Grb2, and Nck protein levels do not change during EGFR
stimulation over this period (Fig. 3A). We examined the EGFR
activation-dependent interactions among EGFR, Grb2, and
PAK1. As shown in Fig. 3B, we observed that there was a pool
of Grb2 that appeared to be constitutively associated with endogenous
PAK1, and this did not change significantly with EGFR stimulation. The
association of PAK1 with the adapter Nck has similarly been shown to be
largely independent of growth factor receptor stimulation (23). The
recruitment of Grb2 to the activated EGFR was assessed in
phosphotyrosine antibody immunoprecipitations of the receptor (Fig.
3C). We observed that Grb2 was co-precipitated with the
stimulated, but not the unstimulated, EGFR. Similarly, the recruitment
of PAK to the EGFR was dependent upon receptor stimulation (Fig.
3D).
The Interaction of PAK1 with the Phosphorylated EGFR Is Dependent
upon Grb2 Adapter Function--
The results of Fig. 3 suggest that
Grb2, which has been shown to interact directly with stimulated EGFR,
serves as an adapter for the PAK1-EGFR association. To test this
hypothesis, we made use of the TAT-tagged PAK1 domain peptides that
specifically blocked the PAK1-Grb2 interaction in vitro (see
Fig. 1D). The polybasic sequence derived from the human
immunodeficiency virus TAT protein has been shown to mediate the
transport of peptides and proteins through cell membranes into the
cytoplasm (30), enabling us to use these as a means to investigate the
PAK1-Grb2 interaction in intact cells. As shown in Fig.
4A, the TAT-PAK P2 domain
peptide effectively blocked the association of endogenous Grb2 with
PAK1, whereas the TAT-PAK P1 domain peptide or a control TAT peptide both exhibited only slight (non-statistically significant) inhibitory effects. A similar pattern of inhibition was observed for the co-precipitation of the EGFR in PAK1 immunoprecipitates (Fig. 4B). In this case, however, for reasons that are not
evident, both the control TAT peptide and the P1 peptide significantly decreased to a similar extent EGFR co-precipitating with PAK1. This is
thus unlikely to reflect a specific inhibitory effect of the P1
peptide. Once again, however, the P2 peptide was significantly more
effective, completely abolishing the EGFR-PAK1 interaction. The
addition of TAT peptides did not affect cellular expression levels of
EGFR, Grb2, or PAK1, or the level or time course of EGFR tyrosine
phosphorylation in response to EGF (data not shown). These results
demonstrate that blocking of the second proline-rich region of PAK1
causes a simultaneous decrease in the association of PAK1 with Grb2 and
the EGFR. We interpret these data as evidence that the interaction of
PAK1 with Grb2 mediates the interaction of PAK1 with the activated
EGFR.
To establish that inhibition of the recruitment of PAK1 to the EGFR via
Grb2 had biological consequences, we examined cytoskeletal remodeling
induced by EGFR stimulation. As shown in Fig.
5A, stimulation of control
cells with EGF for 15 min induced the extension of broad lamellae-like
ruffles. This could be quantified as an increase in cell area (Fig.
5B). The TAT-P2 peptide completely prevented lamellar
extension, and the cells actually retracted slightly to give a decrease
in the initial surface area. As opposed to effects observed in the
EGFR-PAK1 co-precipitation experiments (Fig. 4B), no
significant inhibition was observed with TAT-P1 peptide or TAT control
peptide (Fig. 5B). PAK1 thus appears to be an important
downstream mediator of EGF-induced cytoskeletal remodeling.
In the work presented here, we utilized an affinity-based approach
in which the PAK1 N terminus was used as a probe to isolate specific
binding partners from Jurkat T cell lysates. Grb2 was identified as a
PAK1 N-terminal interacting protein. This was shown using both purified
recombinant proteins and with the endogenous proteins from several cell
lines. Grb2 specifically interacted with the second PAK1 SH3-binding
P42PNP motif. Using cell-permeant TAT peptides encompassing
the first and second SH3 binding regions of PAK1, we showed that we
could block more than 95% of the interaction between PAK1 and the
tyrosine-phosphorylated EGF receptor in HaCaT cells with the peptide
derived from the second SH3 binding domain. Although some nonspecific
inhibition of this interaction by other TAT peptides was observed in
the co-precipitation experiment for reasons that are not clear, these
peptides were clearly less effective. We further demonstrated that the
interaction of PAK1 with the activated EGFR mediated through the
Grb2-binding second proline-rich motif was required for EGFR-induced
cytoskeletal remodeling (Fig. 5). Extension of broad membrane lamellae
induced by EGF was specifically blocked by the P2 peptide, with no
significant inhibition by P1 or TAT control peptide. The EGFR thus
appears to primarily utilize Grb2 to recruit PAK, consistent with the
observation that dominant negative versions of Nck did not block PAK1
activation induced by EGFR stimulation in 293 cells (33). Although it
remains possible that the P2 peptide might be blocking the interaction
of an yet unidentified binding partner for the proline-rich SH3 binding motif of PAK1, the ScanSite analysis (32) of this region predicts that
Grb2 is the only known SH3-containing protein likely to bind to this
specific sequence.
Growth factor and cytokine tyrosine kinase receptors couple to
downstream signaling pathways leading to cell proliferation, cell
survival, and cytoskeletal rearrangements. Critical links in the
coupling of such receptors to signaling elements are SH2/SH3-containing adapter proteins, including Nck and Grb2 (reviewed in Refs. 1-5). PAK1
activity is stimulated by various tyrosine kinase receptors, including
the platelet-derived growth factor and EGF receptors, the insulin
receptor, interleukin-6 and interleukin-3, the T cell receptor, etc.
(34-36). Activation of PAK may thus be important in the proliferative,
cytoskeletal, and survival responses initiated by such receptors.
PAKs 1, 2, and 3 contain several conventional and one non-conventional
proline-rich SH3 binding domains in their regulatory N terminus (10,
11). It has been shown previously (22, 23) that PAK1 binds to the
adapter protein Nck via its first PAPP motif (aa 13-16) and that this
mediates coupling to certain tyrosine kinase receptors. Indeed, using a
strategy similar to that of the current study, Kiosses et
al. (37) demonstrated with TAT peptides based upon the PAK1 first
SH3 binding domain that the Nck-PAK1 interaction is important in
endothelial cell motility and angiogenesis. In conjunction with the
current study, these results demonstrate that TAT peptides containing
PAK1 proline-rich protein interaction motifs can be effectively used to
specifically block the interaction of PAK1 with SH3-containing adapter
molecules in intact cells.
Taken together, these data indicate that PAK1 can interact with both
the Nck and Grb2 adapter proteins for recruitment and activation by
various receptors and/or signaling molecules that utilize these
adapters. Including the molecular interactions mediated by
PAK-interacting exchange factor via the unconventional SH3 binding
motif (PRP) at aa 191-193, these results suggest that PAK1 may thus be
a widely utilized signaling component. It will be of interest to
determine whether the interaction of PAK with Grb2 can also be
regulated by the phosphorylation of PAK by exogenous kinases
(38),2 or by cell
adhesion (24), as has been demonstrated for the PAK1-Nck interaction.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
RII receptor
(27), and to integrins (24, 28). Proteins interacting with the other
PAK N-terminal SH3 binding sites remain to be identified.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of Grb2 as a PAK1 binding
partner in vitro. A, purified
His-tagged PAK1 N-terminal fragments 1-235 and 1-74 bound to nickel
beads were incubated with Jurkat T cell lysates, and pull-down assays
were performed as described under "Experimental Procedures." As
detected by silver staining, an ~25-kDa band specifically bound to
both the PAK1 1-235 and 1-74 fragments. This band was not detected
using either nickel beads in the absence of PAK1 N-terminal fragments
(Control) or with a PAK1 C-terminal fragment bound to the
nickel beads (data not shown). B, immunoblotting of PAK1
pull-downs from Jurkat T cell lysates using Grb2 monoclonal antibody at
1:5000 dilution. The blot shown is representative of three
separate experiments. C, pull-down assays were performed as
described for A. Equal amounts (5 µg of protein) of the
mutated versions of the N-terminal PAK1 1-235 and 1-74 fragments,
P13A and P42A, corresponding to the first and second proline-rich SH3
binding motifs, respectively, were used to pull down the 25-kDa Grb2
protein from Jurkat T cell lysate. The blots were immunostained with
anti Grb2 antibody as described earlier. The graph shows the
densitometric quantitation (in arbitrary units) of three similar
experiments, with the mean ± S.D. shown. The asterisks
indicate statistically significant differences as compared with
corresponding wild-type constructs. The decrease observed with the
1-74 p13 mutation was not statistically different from the wild-type
(p > 0.5), whereas that with the 1-235 p13 mutation
was statistically different at the p < 0.05 level. The
decrease observed with both the p42 mutants, on the other hand, were
highly significant (p < 0.003). D,
TAT-tagged peptides encompassing the first (P1) or the
second (P2) proline-rich domains of PAK1 were added to
pull-down assay incubations with HaCaT lysates at a concentration of
200 µg/ml. Control and TAT control indicate
controls in which lysates with either no peptide added or 200 µg/ml
of a control TAT peptide were added, respectively. Immunoblotting of
pull-down assays was at 1:5000 dilution with Grb2 monoclonal antibody.
E, His-tagged purified PAK1 1-74 fragment was incubated
with GST beads alone (lane 1), GST-Grb2 (lane 2),
GST beads plus a protein A/protein G bead mixture, included as a
specificity control (lane 3), or GST-Grb2 beads plus a
protein A/protein G bead mixture (lane 4). Immunoblotting
with the Grb2 antibody reveals the direct binding of PAK1 to Grb2
(lanes 2 and 4 versus control
lanes 1 and 3). The results shown are
representative of at least three separate experiments.
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Fig. 2.
PAK1 interacts with Grb2 in cells.
A, immunoprecipitation of endogenous Grb2 with PAK1
antibody. The PAK1 R2124 rabbit polyclonal antibody prepared against aa
174-306 of PAK1 was used to immunoprecipitate (1:50) either Jurkat T
cell or HaCaT lysates. The Grb2 antibody was used for immunoblotting at
1:5000 dilution. Controls shown are a direct load of [1/50] volume of
the original Jurkat T cell lysate used for precipitations (Jurkat
lysate) and a precipitation using a nonspecific rabbit polyclonal
IgG antibody (IgG control). B,
immunoprecipitation of endogenous PAK1 with Grb2 antibody. The
monoclonal Grb2 antibody was used to immunoprecipitate (1:50) either
Jurkat T cell or HaCaT lysates. The PAK1 antibody used for
immunoblotting was R2124 (1:1,000), which detects multiple PAK
isoforms. Also shown are a direct load of [1/50] volume of the
original Jurkat T cell lysate used for precipitations (Jurkat
lysate) and a control precipitation using a nonspecific mouse
monoclonal antibody (IgM control). Note that the PAK doublet
detected represents PAK1 (upper band) and PAK2 (lower
band).
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Fig. 3.
PAK1 interacts with the
tyrosine-phosphorylated EGFR in HaCaT cells. A,
tyrosine phosphorylation of the EGFR in HaCaT cells was determined
after serum starvation for 3 h, 5 h, or overnight
(O/N) as indicated and stimulation for 0, 2, or 30 min with
200 ng/ml EGF. The lower panels show the expression levels
of EGFR, Nck, PAK1, and Grb2 in the HaCaT lysates under these
conditions. Immunoblots shown are representative of at least
three experiments. Antibodies used were as follows:
anti-phosphotyrosine mouse monoclonal 4G10 from Upstate Biotechnology
(1:1,000), anti-EGFR mouse monoclonal from BD Biosciences (1:1,000),
anti-Nck rabbit polyclonal from Upstate Biotechnology (1:5,000),
anti-PAK1 R2124 rabbit polyclonal (1:1,000), and anti-Grb2 mouse
monoclonal from BD Biosciences (1:5,000). B, PAK1 (R2124)
immunoprecipitation of HaCaT cell lysates lysed at 0 and 30 min
post-EGF stimulation (200 ng/ml), as described under "Experimental
Procedures." Immunoblotting was with Grb2 monoclonal antibody
(1:1000). C, anti-phosphotyrosine 4G10 immunoprecipitation
of HaCaT lysates lysed at 0 and 30 min post-EGF stimulation (200 ng/ml). Immunoblotting with Grb2 antibody was as in B. D, PAK1 (R2124) immunoprecipitation of HaCaT lysates lysed
at 0 and 30 min post-EGF stimulation (200 ng/ml). Immunoblotting was
done using 4G10 phosphotyrosine monoclonal antibody at 1:1000
dilution.
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Fig. 4.
Interaction of PAK1 with the
tyrosine-phosphorylated EGFR is Grb2-dependent.
A, lysates from HaCaT cells were stimulated with EGF (200 ng/ml) for 30 min in the presence or absence of the indicated TAT
peptides encompassing PAK1 proline-rich regions (as under
"Experimental Procedures"). TAT-tagged peptides were added to the
HaCaT cells at a concentration of 200 µg/ml overnight, followed by
EGF stimulation the next day, lysis, and immunoprecipitation with PAK1
(R2124) antibody. The first and fourth lanes
represent controls, with either no TAT peptide (lane 1) or a
TAT control peptide (200 µg/ml) added to cells. The Grb2
blot shown is representative of three experiments,
quantified in the graph by densitometry (in arbitrary units)
as mean ± S.D. The asterisk indicates statistically
significant difference at the p < 0.0001 level; the
P1 and Tat control peptides were not
significantly different from the no peptide control
(p > 0.5). The levels of total pEGFR/EGFR,
PAK1, and Grb2 were not affected by TAT peptide additions (data not
shown). B, lysates from HaCaT cells were stimulated with EGF
(200 ng/ml) for 0 min. (0' control) or 30 min (all
others lanes) in the presence or absence of the indicated
TAT-peptides encompassing PAK1 proline-rich regions. TAT-tagged
peptides were added to the HaCaT cells at a concentration of 200 µg/ml overnight, followed by EGF stimulation the next day, lysis, and
immunoprecipitation with PAK1 (R2124) antibody. Immunoblots were
immunostained with anti-phosphotyrosine antibody as described earlier.
The first and fourth lanes represent controls,
with either no TAT peptide (lane 1) or a TAT control peptide
(200 µg/ml) added to cells. The phosphotyrosine 4G10 antibody
blot shown is representative of three experiments.
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Fig. 5.
Grb2-dependent coupling of PAK1
with the tyrosine-phosphorylated EGFR mediates extension of membrane
lamellae. A, HaCaT cells in either medium alone
(control) or medium containing 200 µg/ml of the P2 peptide
(P2) were stimulated with 200 ng/ml EGF and imaged as
described under "Experimental Procedures." Contours of the cells
prior to stimulation (upper panels) were plotted
(dotted lines) and used to assess changes in cell area after
15 min of stimulation (lower panels). Arrows show
direction of lamellipodial extensions in EGF-stimulated control cells,
whereas retraction of the membrane observed in cells treated with the
P2 peptide are indicated by arrowheads. B, cells
were treated with the indicated concentrations of the TAT peptides and
stimulated with EGF as in A. Changes in the surface area of
cells after 15 min of stimulation were measured using ISEE software
from Inovision. Each value is a mean ± S.E. of 15-25 cells from
three separate experiments. Asterisks indicate statistically
significant (p < 0.001) difference compared with
control without peptide treatment.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We acknowledge Ben Bohl for technical advice and Lia Marshall for secretarial assistance. We thank Dr. Lewis Cantley (Harvard) for access to the ScanSite program and Dr. Steven Dowdy (University of California, San Diego) for providing the TAT control peptide. Dr. Edith Hintermann (The Scripps Research Institute) provided the HaCaT cells.
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FOOTNOTES |
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* This work was supported in part by Grants GM39434 and GM44428 from the National Institutes of Health (to G. M. B.). This is Publication IMM-15241 from The Scripps Research 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.
Present address: Dept. of Pharmacology, University of California,
San Diego, 9500 Gilman Dr., La Jolla, CA 92093.
§ To whom correspondence should be addressed: Depts. of Immunology and Cell Biology, The Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8217; Fax: 858-784-8218; E-mail: bokoch@scripps.edu.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M208414200
2 J. Field, C. C. King, and G. M. Bokoch, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SH, Src homology; PAK, p21-activated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; aa, amino acids; GST, glutathione S-transferase.
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