From the School of Biological Sciences, Seoul National University, Seoul, 151-742, Republic of Korea
Received for publication, December 4, 2000, and in revised form, March 26, 2001
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ABSTRACT |
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The Rho family GTPases have emerged as key regulators that mediate
extracellular signaling pathways leading to the formation of polarized
actin-containing structures such as stress fibers, membrane ruffles,
lamellipodia, and filopodia. Besides changes in cytoskeletal
architecture, these GTPases mediate diverse biological events,
including stimulation of DNA synthesis, cellular transformation, and
signaling to the nucleus (for recent reviews, see Refs. 1 and 2).
Although direct association of serine/threonine kinases or signaling
proteins with the individual GTPases is critical for downstream
effects, the precise mechanism by which these GTPases regulate actin
cytoskeleton and exhibit these biological activities remains to be
determined (3, 4). Among these effectors, the binding partner of
Cdc42 and Rac, p21-activated kinase
(PAK)1 activity has been
linked to Cdc42 and Rac-mediated cytoskeletal changes. The important
roles that PAK plays as an effector of Cdc42/Rac signaling have been
established from genetic and biochemical studies in yeasts and
mammalian cells (reviewed in Refs. 5 and 6). The repertoire of
signaling pathways that are responsive to activation by Rho family
members is rapidly expanding, and many of these have shown apparent
multiplicity of PAK-mediated signaling pathways (7).
Recently, the PAK-interacting exchange factor (Pix/COOL/p85SPR)
family was cloned by several groups, including our laboratory (8-11).
The Pix family has Dbl homology and a flanking PH domain, which are
conserved in all of the guanine exchange factors for Rho GTPases. Pix
can induce membrane ruffling, with an associated activation of Rac1
(9); however, the extent and selectivity between Pix isoforms to
activate Cdc42 and Rac are not clear (10, 12). Pix interacts tightly
with the regulatory N terminus of PAK via its SH3 domain. As shown by
Daniels et al. (12), It was reported that Cdc42 and Rac also regulate the JNK/SAPK and p38
signaling pathway and the ability of PAK to regulate these same MAP
kinases pathways (16-18). There are several reports showing that
Dbl-related proteins, the activators of Rho GTPases, have roles in the
regulation of several biological activities, including transformation,
metastasis, cytoskeletal reorganization, and transcriptional activation
(7). However, relatively little was known of the exact roles of
these GEFs in the MAP kinase pathway, whereas Cdc42 and Rac were known
as potent SAPK and p38 activators (19-21). Despite the coincidence of
two signalings leading to actin reorganization and MAP kinase activity
by Cdc42/Rac/PAK in many cases, there were few reports about cross-talk
between these two signalings (22).
In the present study, we demonstrate that the overexpression of Cell Culture and Transfection--
All materials for culture
were purchased from Life Technologies, Inc. NIH3T3 cells were cultured
in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine
calf serum and maintained in 10% CO2 at 37 °C.
Transient transfection of cells with mammalian expression vectors was
performed using LipofectAMINEPLUS according to
manufacturer's instructions.
Expression Vectors--
The coding region of
Mismatches are indicated by lowercase letters. The mutation was
verified by automatic DNA sequencing (Applied Biosystems).
Immunocytochemistry--
35-mm dish-cultured cells were
transfected on 0.1% gelatin-coated coverslip. After 24 h, the
medium was removed, and the cells were starved in serum-free medium for
16 h before fixation. SB203580 (Calbiochem, Inc.) was treated for
2 h before fixation at 10 µM concentration. Cells
were fixed in 3.7% paraformaldehyde solution in phosphate-buffered
saline (PBS) for 10 min and permeabilized with 0.5% Triton X-100 in
PBS for 5 min. Cells were incubated in 10% goat serum (Vector
Laboratories, Inc.) and 3% bovine serum albumin-containing 0.1%
Triton X-100 in PBS for 1 h. Primary antibodies were diluted as
follows: Anti-FLAG M2 antibody (Sigma), 5000:1; anti-Myc 9E10 hybridoma
culture medium, 200:1; anti-phospho-specific p38 rabbit antibody (New
England Biolabs, Inc.), 500:1. Rhodamine-phalloidin (Molecular Probes)
was diluted at 2000:1 and treated for 15 min after 1 h primary
antibody incubation. Cells were then stained with fluorescein
isothiocyanate or rhodamine-conjugated secondary antibodies (100:1,
Jackson ImmunoResearch Laboratories, Inc.). 4,6-Diamidino-2-phenylindole (Molecular Probes) was added to
VECTASHIELD mounting solution (Vector Laboratories, Inc.) at 2 µg/ml concentration. Cells were observed under the fluorescence
microscope (Axioplan2, Zeiss) equipped with a 63× (1.4 NA)
Planapochromat objective lens. Fluorescence micrographs were taken on
T-max P3200 film (Kodak).
Cell Lysis, Immunoprecipitation, Immunoblotting, and Immune
Complex Kinase Assay--
To measure the activities of p38 and Erk,
cells on a 100-mm dish were transfected with pcDNA3FLAG-p38 or -Erk
together with other mammalian expression vectors of interest. At
40 h after transfection, cells were washed three times with
ice-cold PBS and lysed in immunoprecipitation buffer (1% Triton X-100,
50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 10 µM leupeptin, and 1.5 µM
pepstatin). Lysates were clarified by centrifugation at 15,000 × g for 10 min. Protein concentration in the supernatant was
determined using Bio-Rad protein assay reagent (Bio-Rad). Clarified
lysates of 500 µg of total protein were incubated with 4 µg of
anti-FLAG M2 antibody for 4 h, followed by additional incubation
with protein A-Sepharose (Amersham Pharmacia Biotech) for 1 h. The
immunoprecipitates were washed three times with immunoprecipitation
buffer, once with kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 0.2 mM dithiothreitol), and resuspended in 20 µl of kinase buffer containing 5 µCi of [ The effect of Pix (PAK-interacting exchange factor) is
a recently identified guanine nucleotide exchange factor for Rho family
small G protein Cdc42/Rac. The protein interacts with p21-activated
protein kinase (PAK) through its SH3 domain. We examined the effect of
Pix on MAP kinase signaling and cytoskeletal rearrangement in NIH3T3
fibroblast cells. Overexpression of
Pix enhanced the activation of
p38 in the absence of other stimuli and also induced translocation of
p38 to the nucleus. This
Pix-induced p38 activation was blocked by
coexpression of dominant-negative Cdc42/Rac or kinase-inactive PAK,
indicating that the effect of
Pix on p38 is exerted through the
Cdc42/Rac-PAK pathway and requires PAK kinase activity. The essential
role of
Pix in growth factor-stimulated p38 activation was evidenced
by the blocking of platelet-derived growth factor-induced p38
activation in the cells expressing
Pix SH3m (W43K) and
Pix DHm
(L238R,L239R). In addition, SB203580, a p38 inhibitor, and kinase-inactive p38 (T180A,Y182F) blocked membrane ruffling induced by
Pix, suggesting that p38 might be involved in mediating
Pix-induced membrane ruffling. The results in this study suggest
that
Pix might have a role in nuclear signaling, as well as in actin
cytoskeleton regulation, and that some part of these cellular functions
is possibly mediated by p38 MAP kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Pix stimulates PAK kinase activity
through exchange factor-dependent and -independent
mechanisms. Additionally, the guanine nucleotide exchange activity was
positively modulated by PAK binding. However, the exact role of Pix in
regulating PAK localization and function in the actin rearrangement
remains to be clarified. Pix was included in a protein complex
containing paxillin, PAK, Nck, and p95PKL (paxillin-kinase linker), a
member of ARF-GAP family, and this suggests a possibility for the
integration of the ARF and Rho family signal transduction at the
cytoskeleton (13, 14). It was also reported that the guanine nucleotide
exchange activity of Pix could be activated by direct association of
the p85 regulatory subunit of phosphatidylinositol 3-kinase (15).
Interaction of Pix with a variety of signaling proteins suggests that
Pix might have an important role in mediating the effects of
extracellular signals, e.g. growth factors, extracellular
matrix, stress, and cytokine, to cytoskeletal rearrangement.
Pix
in NIH3T3 fibroblast cells induces activation of p38 through a
Cdc42/Rac-PAK-MKK3/6-mediated pathway, and this activation of p38 is
necessary for the formation of membrane ruffles induced by
Pix overexpression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Pix was
subcloned into pFLAGCMV2 (Eastman Kodak Co.) and
pcDNA3.1MycHis (Invitrogen), respectively. pEBB/HA-PAK1 was from
Bruce J. Mayer. PAK inhibitory domain (PID: corresponding to 83-149 of
hPAK1) was amplified by polymerase chain reaction using
PIDforward (GGAATTCCACACAATTCATGTCGG) and
PIDreverse (CGTCTAGATGACTTATCTGTAAAGC) primers, cut with
EcoRI and XbaI, and inserted into the
EcoRI-XbaI site of HA-linked pcDNA3.
Site-directed mutagenesis was performed using a
QuikChangeTM site-directed mutagenesis kit (Stratagene).
The following mutagenic primers were used: pFLAGCMV2-
Pix (W43K),
5'-GTGGAGGAAGGAGGCaaGTGGGAGGGCACACA C-3' and
5'-GTGTGTGCCCTCCCACttGCCTCCTTCCTCCAC-3'; pFLAGCMV2-
Pix (L238R,L239S), 5'-GTCTCTCCAGCTCCTTTgaCaGTGTGGGGTACTT GT-3' and 5'-ACAAGTACCCCACA CtGtcAAAGGAGCTGGAGAGAC-3'; pEBB/HA-PAK1 (K299R), 5'-CAGGAGGTGGCCATTAgGCAGATGAATCTTCAG-3' and
5'-CTGAAGATTCATCTGCcTAATGGTCACCTCTG-3'; pcDNA3/FLAG-p38
(T180A,Y182F), 5'-CAGATGATGAAATGgCAGGCTtCGTGGCCACTAGGTG-3' and
5'-CACCTAGTGGCCACGaAGCCTGcCATTTCATCATCTG-3'; pcDNA3/HA-PID (L107F),
5'-AGCAGTGGGCCCGCTTGtTTCAGACATCAAATATC-3' and
5'-GATATTTGATGTCTGAAaCAAGCGGGCCCACTGCT-3'.
-32P]ATP (PerkinElmer Life Sciences), 100 µM ATP, and 4 µg of glutathione S-transferase-ATF2 (for p38 assay) or MBP (for Erk kinase
assay) and incubated at 30 °C for 30 min. Reactions were stopped by
the addition of SDS-sample buffer (100 mM Tris, pH 6.8, 2%
SDS, 10% glycerol, 5%
-mercaptoethanol, 0.25% bromphenol blue).
After SDS-polyacrylamide gel electrophoresis, proteins were transferred onto a polyvinylidine difluoride membrane (Millipore), and the membrane
was subjected to autoradiography. The same membrane was then washed
with 0.1% Triton X-100 containing PBS and immunoblotted using
anti-FLAG M2 antibody. After visualization of kinase proteins by ECL
reagent (Amersham Pharmacia Biotech), the membrane was stained with
Coomassie Brilliant Blue to normalize the amount of substrates. To
visualize active MKK3/6 protein, phosphorylation-specific MKK3/6-specific antibody (500:1) and MKK3 antibody (500:1) were used,
respectively (New England Biolabs, Inc.). Horseradish
peroxidase-conjugated secondary antibodies were purchased from Jackson
ImmunoResearch Laboratories, Inc.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Pix on MAP kinases was examined using NIH3T3
fibroblast cells that were double transfected with FLAG-tagged MAP
kinase and Myc-tagged
Pix. Transfectants were subject to immune
complex kinase assay and phosphorylation of each substrate was
visualized by autoradiography. As shown in Fig.
1A,
Pix augmented p38
activity about 3.5 times. We observed similar results in the immunoblotting analysis using phosphorylation-specific p38 antibody (data not shown).
Pix had no effect on Erk kinase activity in several separated experiments (Fig. 1B), and JNK activity
was slightly increased upon
Pix overexpression (data not shown). Under these conditions, strong activation of Erk kinase by PDGF was
observed. Next, we examined the effect of
Pix overexpession on the
p38 activation in a single cell level using phosphorylation-specific p38 antibody. In
Pix-overexpressing cells, we could observe
increased phospho-p38 signal, especially in the nucleus, compared with
untransfected cells in the same field that had no or very weak signal
(Fig. 2, upper panels). The
treatment of SB203580, a p38-specific inhibitor, blocked
Pix-induced
p38 activation (Fig. 2, lower panels).
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Fig. 1.
Pix augmented p38 activity, but
not Erk kinase activity. NIH3T3 fibroblast cells were transfected
with empty vector or Myc-tagged
Pix together with FLAG-tagged p38 or
Erk. After serum starvation for 16 h in DMEM, cells were lysed,
and immune complex kinase assays of p38 (A) and Erk
(B) were performed as described under "Experimental
Procedures." For a positive control in B, cells were
treated with 20 ng/ml PDGF for 15 min before lysis (third
lane in B). After visualization of phosphorylated ATF2
or MBP by autoradiography (first panels), the same membranes
were probed with anti-FLAG M2 antibody (third panels) and
then stained with Coomassie Brilliant Blue to normalize the amount of
total ATF2 or MBP (second panels). The fourth
panels show the amount of transfected Myc-
Pix in 20 µg of
total cell lysate using anti-Myc 9E10 antibodies. Data shown are
representative of three independent experiments.
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[in a new window]
Fig. 2.
Effect of Pix
overexpression on p38 activity at a single cell level. NIH3T3
fibroblast cells were transiently transfected with Myc-tagged
Pix.
At 40 h after transfection, cells were treated without
(Control) or with 10 µM SB203580 for
1 h and then triple-immunofluorescence staining was carried out
using anti-Myc, anti-phospho-specific p38 antibodies, and
4,6-diamidino-2-phenylindole. Fluorescein
isothiocyanate-labeled anti-mouse and tetramethylrhodamine B
isothiocyanate-labeled anti-rabbit antibodies were used as
second antibodies for anti-Myc and anti-p38 antibodies, respectively.
Two representative examples of each result are presented. Three
independent experiments were performed and two examples representative
of all three experiments are presented.
We next generated two mutants of Pix,
Pix SH3m (W43K) and
Pix
DHm (L238R,L239S), whose SH3 domain and Dbl-homology domain were
changed, respectively. Coimmunoprecipitation assay and glutathione S-transferase-pulldown assay showed that
Pix SH3m cannot
bind to PAK (data not shown).
Pix DHm has been reported to have no exchange activity, while wild-type
Pix acted as a GEF for Rac1 in vivo (9). Unlike wild-type
Pix, overexpression of
these two mutants failed to activate p38 (Fig.
3), indicating that PAK or other SH3
domain-binding proteins and GEF activity of
Pix are essential for
transmission of the extracellular signaling to p38 MAP kinase
activation. p38 has been reported to be activated by several
extracellular stimuli, such as growth factors, cytokines, and stresses
(25, 26). We also examined the involvement of
Pix in PDGF-induced
p38 activation. In
Pix-expressing cells, p38 activity was increased
about four times upon PDGF treatment (Fig. 3B, lanes
Vector and WT). However, in cells expressing
Pix SH3m or
PIX DHm, PDGF-induced p38 activation was not evident (Fig. 3B, lanes W43K and L238R,L239S),
suggesting that
Pix might mediate p38 activation by PDGF in
vivo. However, it should be noted that in either case of
Pix
SH3m or DHm, expression of mutant
Pix still induced membrane
ruffling (data not shown). The extent of ruffling was not much
different from that of wild-type
Pix in case of
Pix SH3m
and reduced by 20-30% in case of
Pix DHm. In contrast, expression
of the
Pix SH3m-DHm double mutant failed to induce membrane ruffling
and also did block PDGF-induced membrane ruffling (data not shown).
Results suggest that disruptions of both PAK interaction (SH3) domain
and GEF activity are necessary to block
Pix-induced membrane
ruffling. Blocking the function of either domain was not
sufficient to inhibit membrane ruffling. This could be explained by
GEF-dependent and -independent PAK activation by Pix (12)
or by the role of PAK as a upstream activator of Rac (27). On the other
hand, inhibition of p38 by either
Pix SH3m or DHm (Fig. 3) indicates
that both PAK interaction (SH3) domain and GEF activity are required
for
Pix-induced p38 activation.
Pix-induced membrane ruffling in
the case of
Pix SH3m and DHm despite p38 inhibition is probably due
to incomplete inhibition of p38 by either mutant. Although
Pix SH3m
and DHm could block p38 activation by PDGF upstream, the basal p38
activity could concert with other Rac (and/or PAK) effector(s) to
induce membrane ruffling.
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Rho family GTPases regulate JNK and p38, as appeared in the previous
reports that the dominant-negative form of Cdc42 or Rac blocked p38
activity and transfection of active Cdc42 or Rac caused a large
enhancement of p38 activity (16-18). To elucidate the hierarchy of
Cdc42/Rac and Pix, the effect of dominant-negative (T17N) Cdc42 or
Rac1 on the p38 activation by
Pix were examined. T17N mutants of
Cdc42 and Rac1 completely inhibited
Pix-enhanced p38 activation as
expected (data not shown), indicating that Cdc42 and Rac1 act
downstream of
Pix, possibly being activated by the GEF activity of
Pix (9).
The PAK family of protein kinases has been suggested as a main target
of the Cdc42/Rac1, and Pix tightly binds to a proline-rich motif of PAK
and regulates its kinase activity (9, 12). But it is also suggested
that PAK could act upstream of Rac1 mediating lamellipodia
formation through interaction with Pix (27). To address the role of PAK
in Pix-induced p38 activation, we utilized wild-type PAK1 or the
K299R mutant of PAK1 whose kinase activity was eliminated. Immune
complex kinase assay after triple transfection of FLAG-tagged p38,
HA-tagged wild-type, or mutant PAK1 and Myc-tagged
Pix showed that
kinase-inactive PAK1 blocked p38 activation induced by
Pix (Fig.
4A). To determine whether the
blocking of
Pix-induced p38 activation by kinase-inactive PAK1 was
due to loss of kinase activity or due to dominant-negative effects of
the expression of mutant PAK1 that could sequester interacting
proteins, we examined
Pix-induced p38 activation in cells expressing
the PID, which can block endogenous PAK kinase activity (Fig.
4B). Expression of PID abolished
Pix-induced p38
activation. Under the same conditions, inactive PID (PID L107F)
did not block
Pix-induced p38 activation. The results suggest that
PAK kinase activity is essential for
Pix-induced p38 activation. We
next investigated the effect of
Pix on MAP kinase kinases, MKK3 and
MKK6, which are known to phosphorylate and activate p38 MAP kinase,
using phosphorylation-specific MKK3/6 antibody.
Pix overexpression
led to a large enhancement of MKK3/6 activity (Fig.
5).
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It has been reported that PDGF stimulation of some adherent cells leads
to an induction of membrane ruffles in a Rac1
activity-dependent pathway (27). When we examined the
effect of Pix overexpression on actin structure by double
immunofluorescence assay, we found that actin structure modified by
Pix overexpression mimicked the
PDGF-induced membrane ruffles (Fig.
6).2 In porcine aortic
endothelial cells, PDGF treatment
enhanced chemotaxis in addition to
ruffling, and these effects were blocked by SB203580, suggesting that
PDGF-induced membrane ruffling and chemotaxis are dependent on p38
activity (25). In this regard, to test whether
Pix-induced membrane
ruffle is also mediated by p38 activity or not, we examined the effect
of a p38-specific inhibitor, SB203580. As shown in Fig. 6A,
under the
Pix-transfected condition, control cells formed enhanced
membrane ruffles, whereas 10 µM SB203580-treated cells
failed to make membrane ruffles. In addition, the localization of
FLAG-tagged
Pix was cytoplasmic in the SB203580-treated cells, in
sharp contrast with the membrane and edge-localized FLAG-
Pix in
control cells. We observed similar results in the cells expressing
kinase-inactive p38 (T180A,Y182F) (Fig. 6B,
p38AF/
Pix).
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There is now increasing evidence showing the involvement of p38 in
actin cytoskeleton regulation and/or cell migration (23). PDGF
activates p38 through a Ras-dependent pathway that is
important for actin reorganization and cell migration in porcine aortic endothelial cells (25). Activation of p38 MAP kinase pathway by growth
factors and inflammatory cytokines regulates smooth muscle cell
migration (24). In this report, p38 activates MAP kinase-activated
protein kinase-2 and -3 (MAPKAPK2 and -3), and these kinases
phosphorylate various substrates including heat shock protein 27 (HSP27). Phosphorylated HSP27 appears to modulate the polymerization of
actin and is proposed to play a role in actin cytoskeletal remodeling.
In our experiments, SB203580, at the concentration physiologically
blocking p38 activity, inhibited Pix-induced membrane ruffling. It
seems likely that p38 has a role in mediating Pix/PAK/Cdc42/Rac
signaling to the cytoskeletal rearrangement. It is reported that
activation of LIM-kinase by PAK1 couples Cdc42/Rac GTPase signaling to
actin cytoskeletal dynamics (28, 29). LIM-kinase is known to catalyze
phosphorylation of cofilin, thereby inactivating actin depolymerizing
activity and leading to accumulation of actin filaments (30, 31). We cannot rule out the possibility that
Pix also acts downstream of
p38, where it is proximal to actin structure regulation
(Pix-Cdc42/Rac/PAK-LIM kinase-cofilin-actin) and p38 activation is a
prerequisite for Pix/PAK/Cdc42/Rac action in actin regulation, rather
than downstream of
Pix.
In many cases, the actin reorganization and MAP kinase activation arose
from the same extracellular stimuli, such as the ligands of several
receptor tyrosine kinases, and were mediated by the same cytoplasmic
signaling components during the signal transduction pathway (16, 19).
Cdc42 and Rac have been implicated in JNK/SAPK and p38, and to a lesser
degree, Erk kinase activation (16, 17, 32). We tested the
involvement of Pix in the MAP kinase pathway and found that
Pix
activates p38 significantly (Fig. 1). It is well established that
growth factor stimulation of Ras leads to the activation of the
Raf/MEK/Erk pathway, and Ras is also involved in signaling to the
cytoskeleton (25). In recent reports, Erk can be activated by Rho
family proteins, and Cdc42/Rac selectively interacts and activates MEK
kinases (32, 33). In our experiment,
Pix did not activate Erk kinase
activity, excluding the possibility that
Pix acts upstream of the
Erk-mediated mitogenic signaling pathway in NIH3T3 fibroblast cells. It
is still unclear whether
Pix has a role in the downstream of Ras.
Zhang et al. (18) proposed that Cdc42 and Rac might regulate
p38 activity through the downstream effector, PAK1. In our experiment,
kinase-inactive PAK1 blocked p38 activity, supporting the results of
others (Refs. 17 and 18 and Fig. 4). It is possible that PAK-Pix
interaction enhances both kinase activity of PAK and the GEF activity
of Pix, as suggested by others (5, 9). Pix might activate PAK,
MKK3/6 and p38 in two ways: by activating Cdc42/Rac1 (and effector PAK)
through its GEF activity and by interacting directly with the PAK
regulatory N terminus. The complexity of the signaling involving
Pix
might arise from the dual property of
Pix, which interacts with the
Cdc42/Rac downstream effector, PAK, and also acts as a GEF of
Cdc42/Rac1.
We propose a model of the Pix/PAK/Rho GTPase-mediated p38 activation
signaling pathway that is shown in Fig.
7. Pix might be activated by
extracellular stimuli such as PDGF, then activate Cdc42 and Rac through
its GEF activity. PAK can be activated by Cdc42/Rac binding and also by
the interaction with
Pix SH3 domain. PAK activates MAPK kinase, MKK3
and/or MKK6, leading to p38 activation. p38 activation has a role in
actin cytoskeletal change, such as lamellipodia formation and membrane
ruffling. It remains to be resolved what is the upstream activator of
Pix. Yoshii et al. (15) reported that
Pix is activated
by interaction with phosphatidylinositol 3-kinase and we also have
observed the association between
Pix and phosphatidylinositol
3-kinase in
vivo.3 We are now investigating other upstream
regulators of
Pix and extracellular stimuli that activate
Pix.
Our present study suggests that signaling pathways through
Pix, in
addition to Cdc42/Rac and PAK, are orchestrated to yield a coordinated
response of cytoskeletal and nuclear events to extracellular
stimuli.
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FOOTNOTES |
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* This work was supported by a grant from the 2000 Korean National Cancer Control Program, Ministry of Health & Welfare, Republic of Korea and also by a grant from Korea Science Foundation (KOSEF) through Center for Cell Signaling Research (1998G0202).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.
Both authors were supported by a BK21 fellowship from the Korean
Ministry of Education.
§ To whom correspondence should be addressed: School of Biological Sciences, Seoul National University, Kwanak-gu, Shinlim-dong, Seoul 151-742, Republic of Korea. Tel.: 82-2-880-5753; Fax: 82-2-872-1993; E-mail: depark@snu.ac.kr.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M010892200
2 S.-H. Lee, M. Eom, S. J. Lee, S. Kim, H.-J. Park, and D. Park, unpublished data.
3 M. Eom and D. Park, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PAK, p21-activated kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; MAP, mitogen-activated protein; DMEM, Dulbecco's modified Eagle's medium; PID, PAK inhibitory domain; HA, hemagglutinin; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; GEF, guanine nucleotide exchange factor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.
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