1 Cell and Molecular Biology Program and 2 Department of Pharmacology, School of Medicine, University of Nevada, Reno, Nevada 89557-0046
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ABSTRACT |
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Cell migration contributes to many physiological processes and requires dynamic changes in the cytoskeleton. These migration-dependent cytoskeletal changes are partly mediated by p21-activated protein kinases (PAKs). At least four closely related isoforms, PAK1, PAK2, PAK3, and PAK4, exist in mammalian cells. In smooth muscle cells, little is known about the expression, activation, or ability of PAKs to regulate migration. Our study revealed the existence of three PAK isoforms in cultured tracheal smooth muscle cells (TSMCs). Additionally, we constructed adenoviral vectors encoding wild type and a catalytically inactive PAK1 mutant to investigate PAK activation and its role in TSMC migration. Stimulation of TSMCs with platelet-derived growth factor (PDGF) increased the activity of PAK1 over time. Overexpression of mutant PAK1 blocked PDGF-induced chemotactic cell migration. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) in cells overexpressing wild-type PAK1 was similar to vector controls; however, p38 MAPK phosphorylation was severely reduced by overexpression of the PAK1 mutant. Collectively, these results suggest a role for PAK1 in chemotactic TSMC migration that involves catalytic activity and may require signaling to p38 MAPK among other pathways.
isoforms; platelet-derived growth factor; adenovirus; p38 mitogen-activated protein kinase
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INTRODUCTION |
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CELL MIGRATION UNDERLIES a number of physiological and pathological processes in many different cell types, including wound healing, embryonic development, immune response, and tumor metastasis (20). In smooth muscle, cell migration contributes significantly to physiological and pathological hyperplasia of the vasculature and the airway. Vascular smooth muscle cells are recruited during angiogenesis and contribute to the pathology of atherosclerosis after angioplasty (24, 31). In the respiratory system, an increased thickness in bronchial smooth muscle is observed in asthmatic individuals, which, in addition to hypertrophy, is largely attributable to hyperplasia (21, 41).
Dynamic changes in the cytoskeleton are required for cell migration. After an acquired spatial asymmetry and polarized morphology, cells extend protrusive filopodia and lamellipodia at the leading edge of the plasma membrane, which are governed by the polymerization of actin. The formation of adhesive complexes helps to stabilize these protrusions and anchor the cell to the substratum at fixed sites, creating points of traction for the body of the cell to travel over and forward (20, 25).
Considerable interest in the signaling mechanisms dictating the formation of these protrusions and adhesions has led to the emergence of the Rho family GTPases, Rho, Rac, and Cdc42, as key regulators of actin cytoskeletal dynamics. Previous studies have established that activation of Rho in Swiss 3T3 fibroblasts either by growth factors or use of constitutively active mutant proteins leads to the formation of stress fibers and focal adhesions (35). Meanwhile, the formation of lamellipodia and membrane ruffles in the same cell type is governed by Rac activation, and activation of Cdc42 results in peripheral filopodia (32, 36). More recent investigations have demonstrated that Rac is required for formation of lamellipodia during wound-induced migration and that Cdc42 is necessary for determining cell polarity during movement. Rho is essential for maintaining adhesion during cell migration, but migration does not require Rho-induced stress fibers and focal adhesions (33). Furthermore, the GTPases mediate the construction of their actin-based structures differently. While Rho induces stress fiber formation by bundling preexisting actin filaments, Rac stimulates the rapid polymerization of monomeric actin into lamellipodia and membrane ruffles (27).
Rho family GTPases clearly mediate the development of migratory actin structures, but the mechanisms that regulate these structures are not fully elucidated. In their GTP-bound state, these proteins are able to bind and signal to downstream effector molecules. Effectors may allow for specific control of actin cytoskeletal dynamics and are thus of considerable interest. Among the effectors identified for Rac and Cdc42 are the p21-activated kinases, or PAKs. At least four mammalian isoforms, PAK1 (68 kDa), PAK2 (62 kDa), PAK3 (65 kDa), and PAK4 (68 kDa), of the yeast Ste20 homologue, have been detected. These serine/threonine kinases interact with GTP-bound Rac and Cdc42 through a conserved binding domain in the NH2-terminal regulatory region. PAKs subsequently autophosphorylate on several residues within the regulatory and COOH-terminal catalytic region, leading to activation (28). PAK activity is also induced by cytokines, lipids, G protein-coupled receptors, recruitment to the cell membrane, and the migratory stimulus, platelet-derived growth factor (PDGF) (5, 9, 13, 23). Activation of PAK may not require binding to Rac or Cdc42 (9, 13, 23, 26, 45).
Once activated, PAK1 participates in regulation of the cytoskeleton and in cell migration. Overexpression of a constitutively active PAK1 mutant in fibroblasts resulted in an increased number of stress fibers and membrane ruffles at one edge of the cell, and the mutant showed marked colocalization with F-actin (40). Formation of these structures and F-actin colocalization was also observed for endogenous PAK1 in PDGF-stimulated fibroblasts (9). In cell migration studies, stimulation of MCF7 breast cancer cells with heregulin resulted in an increase in PAK activity and subsequent actin reorganization and cell motility (2). Directional cell migration was also increased in fibroblasts expressing inducible wild-type or activated PAK1 constructs; fibroblasts expressing kinase-deficient PAK1, although motile, were unable to migrate directionally (38).
The downstream effectors mediating PAK-dependent cell migration are not well defined. PAKs activate the stress-related members of the mitogen-activated protein kinase (MAPK) family, c-jun NH2-terminal kinase (JNK) and p38, and to some extent, extracellular signal-related kinase (ERK) MAPK (4, 7, 12, 26, 47). ERK, JNK, and p38 MAPKs are expressed in smooth muscle and may contribute to a variety of physiological processes, including proliferation, synthesis of cytokines, and cell migration. Both ERK and p38 are activated by muscarinic stimulation, and activation of ERK leads to phosphorylation of the thin-filament contractile protein, caldesmon, in vivo (18). p38 MAPK is also activated by growth factors and inflammatory mediators and regulates smooth muscle cell migration by signaling to the 27-kDa heat shock protein (HSP27) (17). Both pathways contribute to cytokine production in smooth muscle cells (19). The ability of PAKs to regulate MAPKs in other cell types suggests that PAKs may be upstream of one or more MAPKs in smooth muscle, and MAPKs may be effectors of the effects of PAKs on actin remodeling and cell migration. In this investigation, we identify the mammalian PAK isoforms present in airway smooth muscle and test whether expression of a kinase-deficient PAK1 mutant affects chemotactic cell migration. We demonstrate that PAK1 is activated over time by the migratory stimulus PDGF and that cell migration is inhibited in cells that overexpress the kinase-dead mutant. Furthermore, we show that this PAK1 mutant prevents p38 MAPK phosphorylation in response to PDGF. Collectively, our data support the notion that PAK1 might at least partially regulate actin remodeling and migration of smooth muscle cells by signaling to the p38 MAPK pathway.
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MATERIALS AND METHODS |
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Materials.
Adult mongrel dogs of either sex were killed by barbiturate overdose.
The trachea was removed and placed in cold physiological salt solution
composed of 2 mM 3-(N-morpholino)propanesulfonic acid, pH
7.4, 140 mM NaCl, 4.7 mM KCl, 1.2 mM Mg2SO4,
2.5 mM CaCl2, 1.2 mM Na2HPO4, 0.02 mM EDTA, and 5.6 mM D-glucose. Tracheal smooth muscle was
dissected free, dispersed, and grown to 90% confluence in medium 199 (M199; Life Technologies, Grand Island, NY) supplemented with 10%
newborn calf serum. Restriction enzymes were purchased from Promega
(Madison, WI). Oligonucleotide primers were purchased from
Bio-Synthesis (Lewisville, TX). [-32P]ATP was
purchased from ICN Biomedicals (Costa Mesa, CA).
-PAK C-19 antibody,
-PAK N-20 antibody,
-PAK N-20 agarose conjugates, and p38 MAPK
antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Phospho-p38 MAPK (Thr-180/Tyr-182) antibody was purchased from New
England Biolabs (Beverly, MA). IgG alkaline phosphatase-conjugated
antibody was purchased from Promega. Biotin-conjugated goat anti-rabbit
IgG antibody and alkaline phosphatase-conjugated streptavidin were
purchased from Tropix (Bedford, MA). Anti-myc 910E
monoclonal antibody, human recombinant platelet derived growth factor-AB (PDGF-AB), and myelin basic protein (MBP) were purchased from
Sigma-Aldrich (St. Louis, MO).
Generation of recombinant human PAK1 adenoviruses and cell
infection.
pCMV6M plasmids containing myc-tagged, wild-type human PAK1
(hPAK1) or K299R hPAK1 mutant inserts were kindly provided by Dr. Wange
Lu (Harvard University, Boston, MA) and have been previously described
(26). For convenience, the myc-tagged hPAK1
inserts were cut with SalI and EcoRI and
subcloned into SalI-EcoRI-cut pBluescript
KS+ (Stratagene, La Jolla, CA). Recombinant adenoviruses
were constructed according to the methods of He et al.
(16). Briefly, the pBluescript KS+ plasmids
containing wild-type or K299R mutant hPAK1 inserts were cut with
NotI and SalI, and the inserts were ligated into
the NotI and SalI sites of the
pAdTrack-cytomegalovirus (CMV) shuttle vector, which contained the
green fluorescent protein (GFP) behind a CMV promoter
(16). The control pAdTrack-CMV shuttle vector contained no
transgene and was referred to as the AdGFP control. Wild-type and
mutant PAK1 vectors were referred to as AdPAK1WT and
AdPAK1K299R, respectively. Recombinant vectors were
digested with PmeI to linearize the plasmid and transfected
into the Escherichia coli host strain BJ5183 containing the
adenoviral backbone plasmid pAdEasy-1 (16). Successful
GFP/pAdEasy-1 or hPAK1/pAdEasy-1 recombinants were selected by
kanamycin resistance. To produce adenoviruses, 2 µg of
GFP/pAdEasy-1, wild type, or K299R mutant hPAK1/pAdEasy-1 recombinant
DNA was digested with PacI and transfected into the 293 packaging cell line (Microbix, Toronto, Ontario, Canada) using
Effectene transfection reagent according to the manufacturer's
instructions (Qiagen, Valencia, CA). Adenoviruses were harvested,
plaque purified, and titered by an agarose overlay plaque assay as
described previously (15). Cells were infected with
recombinant adenoviruses at a multiplicity of infection (MOI) of 50 plaque-forming units/cell. Medium was removed, and 4.0 × 105 cells/well of a 24-well plate or 1.0 × 106 cells/60 × 15-mm dish were infected with 200 µl
or 1 ml, respectively, of virus diluted in low-serum media (0.1%
newborn calf serum) for 60 min. Infected cells were then incubated in
low serum for 2 days and in serum-free medium for 24 h and used in
the cell migration assay or biochemical assays as described below.
Transduction efficiency was assessed by Sigma Fast Red TR/Napthol AS-MX
immunocytochemistry using anti-myc monoclonal antibodies
(1:500) according to the manufacturer's instructions. Fold increase in
transgene expression was detected by Western blotting using
anti--PAK C-19 polyclonal antibodies (1:1,000) and 15 µg/lane of
whole cell protein.
PAK isoform detection by RT-PCR and Western blotting. Total RNA was isolated from 50 to 100 mg of untreated tracheal smooth muscle tissue or a 10-cm2 area of monolayered smooth muscle cells in 1 ml of TRIzol reagent (Life Technologies) according to the manufacturer's instructions. First-strand cDNA was synthesized from 2 µg of total RNA at 42°C using 250 ng of random oligonucleotides. First-strand synthesis buffer contained 50 mM Tris · HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 0.125 mM dATP, dTTP, dGTP, and dCTP, and 1 unit of SuperScript II RT (Life Technologies). Two units of RNAase H were then added for 20 min at 37°C to remove complementary RNA. PAK isoforms underwent 35 cycles of amplification by PCR in a Perkin Elmer GeneAmp PCR System 2400 thermal cycler. Fifty-microliter reaction mixtures contained 60 mM Tris · HCl, pH 8.5, 15 mM (NH4)SO4, 1.5 mM MgCl2, 0.25 mM dATP, dTTP, dGTP, and dCTP, template cDNA, 5 units Taq polymerase (Promega), and 100 ng of each forward and reverse primer (for PAK1: forward primer 5'-TTG AAT GTG AAG GCT GTG TC-3' and reverse primer 5'-AAT GTT TGG GTT CTT GTT TTC-3'; for PAK2: forward primer 5'-CGC GAC CGG ATC ATA CAA A-3' and reverse primer 5'-GGG CAC AGA AAC CAA AGT CAG T-3'; for PAK3: forward primer 5'-CAC TCA ACC ACA GCT CCA AAC CAC-3' and reverse primer 5'-AGG CGG GGG CTC ATT ATC ATC-3'). Isoform-specific primers were designed based on sequence accession numbers U24152 for PAK1, U25975 for PAK2, and U33314 for PAK3. Annealing temperatures were 53.9°C, 54.2°C, and 57.1°C for PAK1, PAK2, and PAK3 primer pairs, respectively. Brain template cDNA was used as a positive control.
Twenty-five micrograms of untreated tracheal smooth muscle tissue or cell extracts or 10 µg of control brain tissue extracts were separated by SDS-PAGE on 9% acrylamide gel and transferred to nitrocellulose in 25 mM Tris, 192 mM glycine, and 10% methanol buffer. Blots were probed withCell migration assay. Cell migration was assayed using 10 ng/ml of PDGF as the chemoattractant, as described previously, except that 4.0 × 104 cells were plated on the upper side of the collagen-treated polycarbonate membrane (8.0-µm pore; Costar, Cambridge, MA) (17). The number of cells that migrated to the lower face of the membrane was counted in five fields under ×45 magnification and averaged. The total number of migrated cells per membrane was then determined by dividing the area of the membrane (28.274 mm2) by the area of the magnified field (0.113 mm2) and multiplying this number by the average number of migrated cells per field. Assays were performed in duplicate and were repeated five times using cells from different animals.
hPAK1 immunoprecipitation and in vitro kinase assay.
Trachea smooth muscle cells were grown to confluence on six-well plates
in M199 that contained 10% newborn calf serum. To assess endogenous
PAK1 activation, cells were stimulated with 10 ng/ml of PDGF over a
60-min time course. To assess activation of overexpressed PAK1,
confluent cells were infected with recombinant, wild-type hPAK1
adenovirus (AdPAK1WT) and incubated before treatment as
described above. Infected cells overexpressing the wild-type hPAK1
transgene were then stimulated with 10 ng/ml of PDGF over a 60-min time
course. Both noninfected and AdPAK1WT-infected cells were
extracted in RIPA buffer, pH 7.4, containing 50.0 mM HEPES, 150.0 mM
NaCl, 1.0 µM leupeptin, 1.0 mM Na3VO4, 10.0 mM NaF, 0.5% Triton X-100, 0.5% Nonidet P-40, and 10.0% glycerol. Whole cell lysates were sonicated in an ice-bath solution for 10 min,
centrifuged at 10,000 g for 15 min, and quantified by bicinchoninic acid assay (Pierce, Rockford, IL). Three hundred fifty
micrograms of total protein from noninfected cells or 40 µg of total
protein from AdPAK1WT-infected cells were then
immunoprecipitated in a volume of 500 µl for 2 h at 4°C using
-PAK N-20 agarose conjugates. Samples were washed twice with 400 µl RIPA buffer and once with 400 µl kinase buffer containing 20.0 mM HEPES, 20.0 mM MgCl2, 25.0 mM
-glycerophosphate, 0.1 mM Na3VO4, and 2.0 mM DTT. Samples were then
subjected to an in vitro kinase assay, as described previously, using 5 µg of MBP substrate in a 30-µl reaction volume (47). Reactions were terminated by the addition of 20 µl of concentrated SDS sample buffer containing 0.5 M Tris · HCl, pH 6.8, 20% SDS, 10% glycerol, 4 mM DTT, and 0.5% bromphenol
blue. One half of the sample (25 µl) was then separated by
SDS-PAGE on 10% acrylamide gel. The amount of incorporated
radioactivity was analyzed by phosphorimaging and densitometry using
the volume analyze feature of Molecular Analyst software (Bio-Rad).
Data were normalized to unstimulated control samples. Experiments were
repeated four times using cells from different animals.
p38 MAPK phosphorylation. Tracheal smooth muscle cells were cultured and infected with either AdPAK1WT, AdPAK1K299R, or AdGFP control adenoviruses as described above. Cells were then stimulated with 10 ng/ml of PDGF over a 60-min time course and extracted in MAPK extraction buffer containing 20 mM Tris · HCl, pH 6.8, 100 µM leupeptin, 10 mM EGTA, 1 mM Na2EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 mM NaF, 2% SDS, and 10% glycerol. Whole cell lysates were sonicated, clarified by centrifugation, and quantified as described above. Ten micrograms of total cell protein was separated by SDS-PAGE on 12% acrylamide gel and transferred to nitrocellulose as described above. Blots were probed with anti-phospho-p38 MAPK (Thr-180/Tyr-182) antibody at a dilution of 1:1,000 or a nonselective anti-p38 MAPK antibody at a dilution of 1:500. Blots were then visualized with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody at 1:10,000 dilution. The intensity of the phospho-p38 MAPK band was quantified by densitometry using the volume analyze feature of Molecular Analyst software. Data were normalized to unstimulated control samples. Experiments were repeated four to five times using cells from different animals.
Statistical analysis. Results are presented as the means ± SE. Hypothesis testing between two groups was performed using a two-tailed Student's t-test for unpaired, parametric data or Mann-Whitney's rank sum test for nonparametric data. P < 0.05 was accepted as a significant difference.
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RESULTS |
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Expression of PAK isoforms in differentiated tracheal smooth
muscle.
Four primary isoforms of PAK have been detected in mammalian tissues,
including brain (29), heart, kidney, liver, and skeletal and intestinal smooth muscle (1, 30). Whereas PAK1 and
PAK3 are primarily expressed in brain, the PAK2 and PAK4 isoforms are expressed in more peripheral tissues (1, 30). To determine which isoforms were expressed in tracheal smooth muscle tissue and
cultured cells, RT-PCR and Western blotting were conducted. RT-PCR
using isoform-specific primers resulted in three products of expected
size for PAK1 (506 bp), PAK2 (620 bp), and PAK3 (487 bp) in total RNA
isolates from both tracheal smooth muscle tissue and cells (Fig.
1A). BLAST analysis of
the resultant products from tissue and cell cDNA yielded between 84 and
94% homology with mouse and rat PAK1, 89-91% homology with PAK2,
and 87-92% homology with PAK3 (data not shown). An antibody
raised against the COOH-terminal portion of PAK1, which will also
cross-react with the PAK2 and PAK3 isoforms, was immunoreactive with
three protein bands of ~62, 65, and 68 kDa in both tissue and cell
extracts (Fig. 1B). The three bands correlate well with the
known molecular weights of PAK2, PAK3, and PAK1, respectively. These
results indicate that all three PAK isoforms are expressed in tracheal
smooth muscle tissue and cultured cells at both the mRNA and protein
level.
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Adenoviral-mediated expression of wild-type and mutant PAK1.
The use of recombinant adenoviral vectors is an efficient means of
exogenous gene delivery, and we have had previous success using
recombinant adenoviruses to deliver and overexpress genes of interest
in smooth muscle (17). To better study the activation of
PAK1 and its role in smooth muscle cell migration, recombinant adenoviral vectors encoding myc-tagged wild-type or
kinase-dead mutant K299R were constructed according to the methods of
He et al. (16). The K299R mutant contains a single amino
acid change within the catalytic region that renders the PAK1 protein
catalytically inactive toward substrates (26). When
infected at 50 MOI, >90% of tracheal smooth muscle cells expressed
the transgene after 3 days (Fig.
2A). Western blot analysis on
5 µg of total cell protein using an anti-myc antibody
demonstrated an increase in transgene expression over time. Western
blot analysis using anti-PAK1 antibodies demonstrated a parallel
increase in transgene expression. Endogenous PAK1 levels were detected
in 15 µg of total cell protein and were used to determine the fold
increase in expression of the transgene. After 3 days, an approximate
seven- to eightfold increase in expression was observed (Fig.
2B). Because the day 3 time point yielded a
sufficient increase in expression, it was chosen for further studies of
PAK1 activation and tracheal smooth muscle cell migration.
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Activation kinetics of PAK1 by PDGF.
PAK1 activation was first described by using it as a substrate for
GTP-bound Rac1 and Cdc42 in in vitro kinases assays (28). Since then, a number of stimuli have been shown to activate PAK1, including the mitogen PDGF. In response to PDGF stimulation, PAK1 localizes to the activated PDGF receptor in rat myoblasts, and PDGF has
been shown to activate PAK1 over time in fibroblasts (9,
13). To better understand the PDGF-induced activation kinetics
of PAK1 in tracheal smooth muscle cells, a previously described in
vitro kinase assay was used (47). To detect endogenous PAK1 activity, confluent cells were stimulated with PDGF (10 ng/ml) over a 60-min time course. PAK1 immunoprecipitates from cell extracts were then used to phosphorylate MBP in vitro. Kinase reactions were
stopped by the addition of concentrated SDS sample buffer, and
phosphorylated MBP was resolved by SDS-PAGE. Western blotting on
10-µl aliquots of the immunoprecipitates was performed to assure that
equal amounts of PAK1 were present in the kinase reactions; however,
protein levels were below the detection of the anti-PAK1 antibodies
(data not shown). Instead, the densitometry of radioactive MBP was
first normalized to the amount of MBP present in Coomassie-stained gels. The amount of radioactive phosphate incorporated by MBP at each
time point was then reported as fold increase over unstimulated levels.
The results illustrated in Fig.
3A show an increase in endogenous PAK1 activity over time, with maximal activation (2-fold) occurring at 20 min.
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PAK1 affects smooth muscle cell migration. PDGF is an established inducer of cell motility in fibroblasts and vascular smooth muscle cells and is known to promote actin remodeling (reviewed in Ref. 3). Evidence suggests a role for PAK1 in actin cytoskeletal remodeling, as overexpression of activated PAK1 mutants in fibroblasts has been shown to induce the formation of polarized membrane ruffles and focal complexes that are indicative of a motile phenotype (40). When fully activated, PAK1 is phosphorylated on multiple residues within its catalytic domain. Presumably, PAK1 can then phosphorylate an undefined set of effector proteins, which may be important for PAK1 to elicit its effects on the cytoskeleton. The K299R mutant, however, is unable to phosphorylate effectors and may affect motility.
To more closely study the effect of PAK1 on cell migration, we overexpressed AdPAK1WT, the AdPAK1K299R mutant, or the AdGFP vector control in tracheal smooth muscle cells, and assayed migration in response to PDGF (10 ng/ml), as described in MATERIALS AND METHODS. In cells that were not infected with adenovirus, an average of 4,300 cells/membrane migrated in response to PDGF compared with basal migration levels of 1,400 cells/membrane, an approximate threefold increase. AdGFP-infected cells exhibited an ~2.6-fold increase in migration after stimulation with PDGF, with 3,300 cells/membrane migrating, compared with basal migration of 1,300 cells/membrane (Fig. 4). Differences in the number of cells migrated per membrane between noninfected tracheal smooth muscle cells and cells infected with the AdGFP control adenoviral vector were not significant. Cells infected with AdPAK1WT demonstrated a reduction in the number of migrating cells compared with AdGFP controls. When stimulated, 1,730 cells/membrane migrated, compared with 525 cells/membrane at basal (Fig. 4). In cells that overexpressed the AdPAK1K299R mutant, however, a formidable decrease in the number of migrating cells per membrane was observed, compared with both control and AdPAK1WT-infected cells. Stimulation with PDGF resulted in the migration of 330 cells/membrane, compared with a basal migration level of 170 cells/membrane (Fig. 4). Western blotting using an anti-myc antibody confirmed that wild-type and mutant PAK1 were overexpressed in AdPAK1WT- and AdPAK1K299R-infected cells (Fig. 4). These results suggest that PAK1 is important for spontaneous cell migration and PDGF-induced chemotaxis and that its catalytic activity toward substrates may be required.
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p38 MAPK phosphorylation.
Activation of p38 MAPK has been linked to PAKs in different cell types
(4). Furthermore, we have previously demonstrated that the
p38 MAPK pathway leading to phosphorylation of the small 27-kDa heat
shock protein significantly contributes to the regulation of tracheal
smooth muscle cell migration in response to PDGF (17). To
test the hypothesis that PAK1 might elicit its affects on cell migration by signaling to the p38 MAPK pathway, tracheal smooth muscle
cells were infected with either AdPAK1WT or
AdPAK1K299R and stimulated with PDGF over a 30-min time
course. Phosphorylation of p38 MAPK was then assessed by Western
blotting using a dual phosphospecific antibody, which will recognize
p38 MAPK when phosphorylated at Thr-180 and Tyr-182 in the regulatory
TGY motif (34). As illustrated in Fig.
5A, stimulation with PDGF
caused a transient activation of p38 MAPK in noninfected cells, with a
maximal fivefold increase in phosphorylation at 20 min. These
phosphorylation kinetics for p38 MAPK in response to PDGF were similar
to those observed previously (17). Additionally, Western
blotting using a nonselective anti-p38 MAPK antibody confirmed that
equal amounts of p38 MAPK were present in each sample. As a positive
control, cells stimulated with 200 µM sodium arsenite over the same
time course exhibited phosphorylation kinetics comparable with
PDGF-stimulated cells (data not shown). Patterns of p38 MAPK
phosphorylation in the AdGFP vector control-infected cells were similar
to those of noninfected cells, suggesting that viral infection or
expression of the GFP transgene does not interfere with signaling to
p38 MAPK (Fig. 5B). Overexpression of AdPAK1WT
resulted in little difference in p38 MAPK phosphorylation kinetics compared with the vector control, except that maximal activation occurred at 30 min instead of 20 min (Fig. 5C). By contrast,
p38 MAPK phosphorylation was considerably reduced in cells that
overexpressed the AdPAK1K299R mutant at all time points
after 1 min and failed to reach even a 1.5-fold activation at any time
point (Fig. 5D).
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DISCUSSION |
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Our investigation identifies the presence of three PAK isoforms in
airway smooth muscle tissue and cultured cells and demonstrates a role
for PAK1 in chemotactic cell migration. Four common mammalian isoforms
of the yeast Ste20 protein kinase have been described and given the
nomenclature PAK1 (-PAK), PAK2 (
-PAK), PAK3 (
-PAK), and PAK4.
These isoforms all contain an NH2-terminal regulatory domain and a COOH-terminal kinase domain and share 73% sequence identity. They differ slightly in their NH2-terminal
sequences, molecular weights, and tissue distributions
(39). These published sequence data enabled us to design
isoform-specific primers and detect the presence of the PAK1, PAK2, and
PAK3 isoforms in tracheal smooth muscle tissues and cells at the mRNA
level. At the protein level, our analysis revealed bands of ~68, 62, and 65 kDa, which correspond to the PAK1, PAK2, and PAK3 isoforms,
respectively. To our knowledge, this is the first description of PAK
isoform expression in airway smooth muscle. Using different methods,
Van Eyk et al. (44) identified the PAK2 and PAK3 isoforms
in guinea pig taenia coli and rat aorta smooth muscle. More recently,
the fourth PAK isoform, PAK4, was cloned from a Jurkat cell cDNA
library (1). Although we did not attempt to detect this
isoform in our tissues and cells, it is quite possible that this
isoform is also present given its wide distribution and presence in
smooth muscle-containing tissues (1).
PAKs are established effectors of the Rho family GTPases, Rac and Cdc42, which have been shown to modulate cytoskeletal actin remodeling, leading to the formation of organized actin structures. Consequently, the general hypothesis that PAKs might mediate at least some of these affects is being ardently pursued. Overexpression studies in different cell types have yielded data that support a function for PAKs in actin dynamics. Two major controversies surrounding this issue, however, are whether Rac/Cdc42-dependent activation and catalytic activity are required for PAKs to exert their effects. Binding of GTP-Rac or GTP-Cdc42 either in vivo or in vitro leads to robust activation of PAKs, yet strong activation is also observed independently of Rac/Cdc42 binding by sphingosine and related lipids (5). Additionally, membrane recruitment of PAK by myristoylation of the SH3-containing adaptor protein Nck activates PAK formidably (26). Nck binds to PAK1's first proline-rich domain in the NH2 terminus via its second SH3 domain and is recruited to the activated PDGF receptor after stimulation (6, 13). Our results demonstrate that PDGF activates both endogenous and overexpressed PAK1 in tracheal smooth muscle cells over time, as determined by increased phosphorylation of an MBP substrate in response to PDGF. This activation is similar to what has been observed in Swiss 3T3, although the kinetics of activation in tracheal smooth muscle appear to be slower (9). Additionally, this activation is likely mediated by PAK1 localization to the receptor by Nck, where, as proposed by other models, PAK1 then binds to GTP-Rac/Cdc42.
PAKs are believed to exist in an autoinhibitory, intramolecular conformation before activation, where the NH2-terminal regulatory domain and the COOH-terminal catalytic domain interact. Activation subsequently leads to the disruption of this interaction and exposes numerous serines and threonines within both domains to phosphorylation (28). Zenke et al. (46) have proposed Thr-423, which resides in the activation loop of subdomain VIII of the catalytic domain, to be a key intermolecular phosphorylatable residue for complete enzymatic activation of PAK1. Although catalytic activity toward substrates is assumed to be a significant factor in PAK-mediated events, investigations using catalytically inactive PAK mutants yield conflicting results. Kinase-deficient PAK1 has been shown to inhibit oncogenic transformation of Ras GTPase in Rat-1 fibroblasts, rat Schwann cells, and neurofibrosarcoma cell lines, implying that catalytic ability is important in these cases (42, 43). PAK1 mediates neurite outgrowth from PC-12 cells and organized actin structures in Swiss3T3 fibroblasts, but these events are independent of catalytic activity (8, 38). Our results demonstrate that chemotactic cell migration may also depend on PAK1 kinase activity in smooth muscle cells. The data show a 3.3-fold increase in PDGF-stimulated migration of cells overexpressing wild-type PAK1 compared with their nonstimulated counterparts. This is an increase in stimulated migration compared with AdGFP controls, which migrated 2.6-fold above basal. Conversely, AdPAK1K299R-expressing cells migrated twofold above basal in response to PDGF. While this is a significant increase in migration compared with basal, an average of only 335 cells/membrane were stimulated to migrate in AdPAK1K299R-expressing cells, compared with 3,325 cells/membrane in AdGFP controls. Additionally, basal migration of 170 cells/membrane in AdPAK1K299R-overexpressing cells was dramatically lower than the basal migration of 1,295 cells/membrane in AdGFP-infected cells. Adenoviral-mediated expression of the kinase-deficient K299R mutant, therefore, severely reduced the number of both PDGF-stimulated and -unstimulated migrating cells compared with the vector control.
These results are supported by previously published cytoskeletal dynamics data, in which human microvascular endothelial and baby hamster kidney (BHK) cells were microinjected with PAK1 constructs containing the same K299R mutation (10, 22). Overexpression of PAK1-K299R in endothelial cells resulted in the formation of more focal adhesions and stress fibers compared with wild type, but these structures were essentially static and appeared to be fixed in one spot. In addition, any lamellipodia and ruffles that did form, formed around the entire periphery and failed to exhibit any polarity, which is necessary for migration (22). In BHK cells, the wild-type phenotype of dorsal ruffling and actin thickening was almost completely absent when the K299R mutant was overexpressed (10). The severe decrease in the number of basal and PDGF-induced migrating cells that overexpressed the catalytically inactive mutant in our experiments suggests that kinase activity may be involved in the initial formation of polarized structures required for directed migration. This is consistent with results from Sells et al. (38) in fibroblasts, which show that overexpression of any form of PAK1 can increase random cell movement that is independent of kinase activity, while haptotactic cell migration on an immobilized collagen gradient is reduced by the kinase-dead mutant.
Cells infected with AdPAK1WT exhibited an increase in PDGF-induced migration compared with basal. Surprisingly, however, we observed a decrease in the number of migrating cells overexpressing wild-type PAK, although this decrease was not nearly as dramatic as that observed in mutant-expressing cells. One possibility is that PDGF was unable to activate all of the overexpressed PAK1 present in the AdPAK1WT-infected cells. Indeed, PAK is thought to exist in an autoinhibited, inactivated state, and activation kinetics were slower for overexpressed PAK1 compared with endogenous PAK1. In fibroblasts, constitutively active forms of PAK1 increase haptotactic migration, but migration is slightly decreased by overexpression of wild-type PAK1 (38).
Cell migration requires not only the formation of actin structures such as lamellipodia and adhesions, but also their spatial and temporal orchestration. One explanation for our observations is that overexpression of the catalytically inactive PAK1 may interfere with this coordination of cytoskeletal dynamics by signaling to a number of pathways. One candidate pathway is that of p38 MAPK. Overexpression of PAK has been shown to activate p38 MAPK in nonmuscle cell types (4, 47). Furthermore, previous work in our laboratory has demonstrated that p38 MAPK plays a significant role in airway smooth muscle cell migration by signaling to HSP27 (17). We consequently addressed whether overexpression of either the wild-type or kinase-inactive mutant PAK1 influenced the phosphorylation of p38 MAPK. Noninfected cells, cells that overexpressed the GFP vector control, and cells that overexpressed wild-type PAK1 exhibited similar levels and kinetics of p38 MAPK phosphorylation when stimulated with PDGF over a 30-min time course. However, overexpression of the kinase-deficient mutant severely inhibited phosphorylation. These results suggest that PAK1 signals to p38 MAPK in PDGF-stimulated tracheal smooth muscle cells and may, at least in part, regulate PDGF-induced cell migration through this pathway. Furthermore, our results suggest a link between the PAK1-mediated p38 MAPK activation and the formation of actin structures observed independently in other cell types.
While our evidence implicates the p38 MAPK pathway, other signaling mechanisms are likely also important for cytoskeletal dynamics and cell migration in smooth muscle. A 3.9-fold increase in p38 MAPK phosphorylation was observed in AdGFP controls that correlated with a 2.6-fold increase in PDGF-stimulated migration. Concurrently, a 4-fold increase in p38 MAPK phosphorylation in AdPAK1WT-infected cells associated with a 3.3-fold increase in migration above basal levels. A 2-fold increase in migration above basal was still observed; in AdPAK1K299R-infected cells, however, there is less than a 1.5-fold increase in p38 MAPK phosphorylation. This suggests that other signaling pathways are activated and may work redundantly or in concert to influence cell migration. Furthermore, although the p38 MAPK pathway may be involved, there is not a one-to-one correlation with PAK-mediated activation of p38 MAPK and smooth muscle cell migration. This is to be expected, given PAK1's proximal position in the signaling pathway and its consequent opportunity to signal to multiple cascades.
PAK-induced contraction of skinned taenia coli fibers correlates with an increase in caldesmon phosphorylation, and Foster et al. and Van Eyk et al. (11, 44) have recently identified the PAK phosphorylation sites on caldesmon. PAK-catalyzed phosphorylation of caldesmon reduces both caldesmon's affinity for calmodulin and its ability to inhibit actin-S1 ATPase activity (11). Therefore, PAK regulation of caldesmon phosphorylation might result in increased myosin II motor activation, which is known to be required for cell migration. There is also evidence of PAK-mediated cytoskeletal regulation involving myosin light chain kinase (MLCK) in nonmuscle cells. PAK2 can phosphorylate MLCK in vitro and reduces the development of MgATP-stimulated isometric tension in permeabilized endothelial cells (14). Overexpression of constitutively active PAK1 in BHK-21 and HeLa cells decreases MLCK activity toward myosin light chain and decreases cell spreading on a fibronectin substrate (37). By contrast, constitutively active PAK1 increases myosin light chain phosphorylation in both NIH/3T3 and human microvascular endothelial cells, correlating with an increase in cell migration. Interestingly, overexpression of the kinase-dead PAK K299R mutant in these two cell types had little or no effect on myosin light chain phosphorylation (22, 38), suggesting that other pathways are probably involved. Although the exact mechanism or mechanisms by which PAK1 exerts its affects on the cytoskeleton remains to be determined, our data demonstrate that inhibiting PAK activity prevents activation of p38 MAPK and cell migration in smooth muscle.
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ACKNOWLEDGEMENTS |
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We thank Michelle Deetken and Shanti Rawat for excellent technical assistance and the other members of the Gerthoffer laboratory for helpful discussion. We also thank Dr. Wange Lu (Harvard University) for providing us with wild-type and K299R mutant human PAK1 plasmids and the laboratory of Dr. Bert Vogelstein (Johns Hopkins University) for the pAdTrack-CMV and pAdEasy-1 plasmids.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. T. Gerthoffer, Dept. of Pharmacology/318, Univ. of Nevada School of Medicine, Reno, NV 89557-0046 (E-mail: wtg{at}med.unr.edu).
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.
Received 30 June 2000; accepted in final form 8 February 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abo, A,
Qu J,
Cammarano MS,
Dan C,
Fritsch A,
Baud V,
Belisle B,
and
Minden A.
PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia.
EMBO J
17:
6527-6540,
1998
2.
Adam, L,
Vadlamudi R,
Kondapaka SB,
Chernoff J,
Mendelsohn J,
and
Kumar R.
Heregulin regulates cytoskeletal reorganization and cell migration through the p21-activated kinase-1 via phosphatidylinositol-3 kinase.
J Biol Chem
273:
28238-28246,
1998
3.
Anand-Apte, B,
and
Zetter B.
Signaling mechanisms in growth factor-stimulated cell motility.
Stem Cells
15:
259-267,
1997
4.
Bagrodia, S,
Derijard B,
Davis RJ,
and
Cerione RA.
Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation.
J Biol Chem
270:
27995-27998,
1995
5.
Bokoch, GM,
Reilly AM,
Daniels RH,
King CC,
Olivera A,
Spiegel S,
and
Knaus UG.
A GTPase-independent mechanism of p21-activated kinase activation. Regulation by sphingosine and other biologically active lipids.
J Biol Chem
273:
8137-8144,
1998
6.
Bokoch, GM,
Wang Y,
Bohl BP,
Sells MA,
Quilliam LA,
and
Knaus UG.
Interaction of the Nck adapter protein with p21-activated kinase (PAK1).
J Biol Chem
271:
25746-25749,
1996
7.
Brown, JL,
Stowers L,
Baer M,
Trejo J,
Coughlin S,
and
Chant J.
Human Ste20 homologue hPAK1 links GTPases to the JNK MAP kinase pathway.
Curr Biol
6:
598-605,
1996[ISI][Medline].
8.
Daniels, RH,
Hall PS,
and
Bokoch GM.
Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.
EMBO J
17:
754-764,
1998
9.
Dharmawardhane, S,
Sanders LC,
Martin SS,
Daniels RH,
and
Bokoch GM.
Localization of p21-activated kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in stimulated cells.
J Cell Biol
138:
1265-1278,
1997
10.
Edwards, DC,
Sanders LC,
Bokoch GM,
and
Gill GN.
Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics.
Nat Cell Biol
1:
253-259,
1999[ISI][Medline].
11.
Foster, DB,
Shen LH,
Kelly J,
Thibault P,
Van Eyk JE,
and
Mak AS.
Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca2+ sensitivity of smooth muscle contraction.
J Biol Chem
275:
1959-1965,
2000
12.
Frost, JA,
Xu S,
Hutchison MR,
Marcus S,
and
Cobb MH.
Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.
Mol Cell Biol
16:
3707-3713,
1996[Abstract].
13.
Galisteo, ML,
Chernoff J,
Su YC,
Skolnik EY,
and
Schlessinger J.
The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1.
J Biol Chem
271:
20997-21000,
1996
14.
Goeckeler, ZM,
Masaracchia RA,
Zeng Q,
Chew TL,
Gallagher P,
and
Wysolmerski RB.
Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2.
J Biol Chem
275:
18366-18374,
2000
15.
Graham, FL,
and
Prevec L.
Methods for construction of adenovirus vectors.
Mol Biotechnol
3:
207-220,
1995[ISI][Medline].
16.
He, TC,
Zhou S,
da Costa LT,
Yu J,
Kinzler KW,
and
Vogelstein B.
A simplified system for generating recombinant adenoviruses.
Proc Natl Acad Sci USA
95:
2509-2514,
1998
17.
Hedges, JC,
Dechert MA,
Yamboliev IA,
Martin JL,
Hickey E,
Weber LA,
and
Gerthoffer WT.
A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration.
J Biol Chem
274:
24211-24219,
1999
18.
Hedges, JC,
Oxhorn BC,
Carty M,
Adam LP,
Yamboliev IA,
and
Gerthoffer WT.
Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle.
Am J Physiol Cell Physiol
278:
C718-C726,
2000
19.
Hedges, JC,
Singer CA,
and
Gerthoffer WT.
Mitogen-activated protein kinases regulate cytokine gene expression in human airway myocytes.
Am J Respir Cell Mol Biol
23:
86-94,
2000
20.
Horwitz, AR,
and
Parsons JT.
Cell migration-movin' on.
Science
286:
1102-1103,
1999
21.
Jeffery, PK.
Morphology of the airway wall in asthma and in chronic obstructive pulmonary disease.
Am Rev Respir Dis
143:
1152-1158,
1991[ISI][Medline].
22.
Kiosses, WB,
Daniels RH,
Otey C,
Bokoch GM,
and
Schwartz MA.
A role for p21-activated kinase in endothelial cell migration.
J Cell Biol
147:
831-844,
1999
23.
Knaus, UG,
Morris S,
Dong HJ,
Chernoff J,
and
Bokoch GM.
Regulation of human leukocyte p21-activated kinases through G protein-coupled receptors.
Science
269:
221-223,
1995[ISI][Medline].
24.
Korpelainen, EI,
and
Alitalo K.
Signaling angiogenesis and lymphangiogenesis.
Curr Opin Cell Biol
10:
159-164,
1998[ISI][Medline].
25.
Lauffenburger, DA,
and
Horwitz AF.
Cell migration: a physically integrated molecular process.
Cell
84:
359-369,
1996[ISI][Medline].
26.
Lu, W,
Katz S,
Gupta R,
and
Mayer BJ.
Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck.
Curr Biol
7:
85-94,
1997[ISI][Medline].
27.
Machesky, LM,
and
Hall A.
Role of actin polymerization and adhesion to extracellular matrix in Rac- and Rho-induced cytoskeletal reorganization.
J Cell Biol
138:
913-926,
1997
28.
Manser, E,
Huang HY,
Loo TH,
Chen XQ,
Dong JM,
Leung T,
and
Lim L.
Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes.
Mol Cell Biol
17:
1129-1143,
1997[Abstract].
29.
Manser, E,
Leung T,
Salihuddin H,
Zhao ZS,
and
Lim L.
A brain serine/threonine protein kinase activated by Cdc42 and Rac1.
Nature
367:
40-46,
1994[ISI][Medline].
30.
Martin, GA,
Bollag G,
McCormick F,
and
Abo A.
A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20.
EMBO J
14:
4385,
1995[ISI][Medline].
31.
Newby, AC,
and
Zaltsman AB.
Molecular mechanisms in intimal hyperplasia.
J Pathol
190:
300-309,
2000[ISI][Medline].
32.
Nobes, CD,
and
Hall A.
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81:
53-62,
1995[ISI][Medline].
33.
Nobes, CD,
and
Hall A.
Rho GTPases control polarity, protrusion, and adhesion during cell movement.
J Cell Biol
144:
1235-1244,
1999
34.
Raingeaud, J,
Gupta S,
Rogers JS,
Dickens M,
Han J,
Ulevitch RJ,
and
Davis RJ.
Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine.
J Biol Chem
270:
7420-7426,
1995
35.
Ridley, AJ,
and
Hall A.
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell
70:
389-399,
1992[ISI][Medline].
36.
Ridley, AJ,
Paterson HF,
Johnston CL,
Diekmann D,
and
Hall A.
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70:
401-410,
1992[ISI][Medline].
37.
Sanders, LC,
Matsumura F,
Bokoch GM,
and
de Lanerolle P.
Inhibition of myosin light chain kinase by p21-activated kinase.
Science
283:
2083-2085,
1999
38.
Sells, MA,
Boyd JT,
and
Chernoff J.
p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts.
J Cell Biol
145:
837-849,
1999
39.
Sells, MA,
and
Chernoff J.
Emerging from the PAK: the p21-activated protein kinase family.
Trends Cell Biol
7:
162-167,
2000.
40.
Sells, MA,
Knaus UG,
Bagrodia S,
Ambrose DM,
Bokoch GM,
and
Chernoff J.
Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells.
Curr Biol
7:
202-210,
1997[ISI][Medline].
41.
Seow, CY,
Wang L,
and
Pare PD.
Airway narrowing and internal structural constraints.
J Appl Physiol
88:
527-533,
2000
42.
Tang, Y,
Chen Z,
Ambrose D,
Liu J,
Gibbs JB,
Chernoff J,
and
Field J.
Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts.
Mol Cell Biol
17:
4454-4464,
1997[Abstract].
43.
Tang, Y,
Marwaha S,
Rutkowski JL,
Tennekoon GI,
Phillips PC,
and
Field J.
A role for Pak protein kinases in Schwann cell transformation.
Proc Natl Acad Sci USA
95:
5139-5144,
1998
44.
Van Eyk, JE,
Arrell DK,
Foster DB,
Strauss JD,
Heinonen TY,
Furmaniak-Kazmierczak E,
Cote GP,
and
Mak AS.
Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle.
J Biol Chem
273:
23433-23439,
1998
45.
Westwick, JK,
Lambert QT,
Clark GJ,
Symons M,
Van Aelst L,
Pestell RG,
and
Der CJ.
Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways.
Mol Cell Biol
17:
1324-1335,
1997[Abstract].
46.
Zenke, FT,
King CC,
Bohl BP,
and
Bokoch GM.
Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity.
J Biol Chem
274:
32565-32573,
1999
47.
Zhang, S,
Han J,
Sells MA,
Chernoff J,
Knaus UG,
Ulevitch RJ,
and
Bokoch GM.
Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1.
J Biol Chem
270:
23934-23936,
1995