p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signaling to p38 MAPK

Melissa A. Dechert1,2, Jennifer M. Holder2, and William T. Gerthoffer1,2

1 Cell and Molecular Biology Program and 2 Department of Pharmacology, School of Medicine, University of Nevada, Reno, Nevada 89557-0046


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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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.


    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). [gamma -32P]ATP was purchased from ICN Biomedicals (Costa Mesa, CA). alpha -PAK C-19 antibody, alpha -PAK N-20 antibody, alpha -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-alpha -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 with alpha -PAK C-19 antibody, which cross-reacts with all three PAK isoforms, at a dilution of 1:1,000. Blots were visualized with biotinylated goat anti-rabbit-conjugated secondary antibody at a dilution of 1:15,000, followed by alkaline phosphatase-conjugated streptavidin at a dilution of 1:20,000.

Cell 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 alpha -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 beta -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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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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Fig. 1.   Mammalian p21-activated protein kinase (PAK) isoform expression in tracheal smooth muscle. A: RT-PCR was performed on total RNA isolated from tracheal smooth muscle tissue (TSM) or tracheal smooth muscle cultured cells (TSMC) using isoform-specific primers. Brain served as a positive control for all 3 isoforms. Expected product sizes were 506, 620, and 487 bp for PAK1, PAK2, and PAK3, respectively. B: Western blotting was performed on total protein homogenates from tissue or lysates from cells using a PAK1 antibody that also recognizes PAK2 and PAK3. Primary immunoreactive bands of ~62, 65, and 68 kDa are visible.

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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Fig. 2.   Adenoviral-mediated expression of wild-type and kinase-dead (K299R) mutant human PAK1. A: TSM cells were infected at 50 multiplicity of infection (MOI) with either myc-tagged wild type (AdPAK1WT) or mutant PAK1 (AdPAK1K299R) adenoviruses and allowed to express the transgene for 3 days. Immunocytochemistry using anti-myc antibody and Napthol red staining was performed to detect transgene expression. Gray scale images are displayed. Transduction efficiency was ~90%. B: Western blotting using anti-myc or anti-alpha -PAK antibodies was performed on 15 µg of total protein after infection for 3 days at 50 MOI. Anti-alpha -PAK immunoreactive bands were quantified by densitometry and normalized to endogenous (noninfected) levels of PAK1 expression. Bar graphs show relative levels of expression determined from the blots shown. NI, noninfected.

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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Fig. 3.   Activation of PAK1 in tracheal smooth muscle cells by platelet-derived growth factor (PDGF). A: confluent TSM cells were stimulated with 10 ng/ml of PDGF over a 60-min time course. Endogenous PAK1 activity in immunoprecipitates from each time point was determined by in vitro kinase assays using myelin basic protein (MBP) as the substrate. Radioactive phosphate incorporation of MBP was normalized to the amount of MBP present in Coomassie-stained gels. B: cells were infected with AdPAK1WT at 50 MOI and were allowed to express the transgene for 3 days. Cells were then stimulated with PDGF, and immunoprecipitates were subjected to in vitro kinases assays as in A. Western blotting on 10-µl aliquots of the immunoprecipitates was performed to assure that equal amounts of PAK1 were present. Radioactive phosphate incorporation was measured by densitometry and normalized to Western blot controls. Results in A and B are expressed as fold increase in activation relative to basal (time = 0 min); n = 4 ± SE.

Immunoprecipitation of endogenous PAK1 from tracheal smooth muscle cells is difficult because its expression levels are so low. Furthermore, we were concerned that we were not able to detect endogenous PAK1 in aliquots of immunoprecipitates. To attempt to resolve this issue, we infected confluent cells with AdPAK1WT before stimulating with PDGF and performing in vitro kinase assays as described above. This time, PAK1 was detectable in aliquots of immunoprecipitates, and Western blotting confirmed that equal amounts of PAK1 were present (Fig. 3B). As with endogenous PAK, activity of overexpressed PAK1 increased over time, but maximal activation (3-fold) was observed at 30 instead of 20 min.

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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Fig. 4.   Wild-type and kinase-dead PAK1 reduce cell migration. Tracheal smooth muscle cells were infected at 50 MOI for 3 days with adenovirus encoding the wild type (AdPAK1WT), kinase-dead PAK1 mutant (AdPAK1K299R), or a green fluorescent protein vector control lacking an insert (AdGFP). Western blotting using anti-myc antibodies was performed on 5 µg of total cell protein to verify overexpression. Cells were then stimulated to migrate with 10 ng/ml of PDGF as described in MATERIALS AND METHODS. Cells from five 0.113-mm2 fields were counted, averaged, and divided into the total membrane area of 28.274 mm2. Duplicate assays were then averaged. Results are expressed as migrated cells per membrane; n = 5 ± SE; *significantly different from stimulated, vector control-infected cells; P < 0.05. Migration in stimulated, NI cells did not differ significantly from stimulated vector control cells.

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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Fig. 5.   Kinase-dead mutant PAK1 reduces p38 mitogen-activated protein kinase (MAPK) phosphorylation. NI tracheal smooth muscle cells (A) or cells infected with AdGFP vector control (B), AdPAK1WT (C), or AdPAK1K299R (D) were stimulated with 10 ng/ml of PDGF over a 30-min time course. Ten micrograms of whole cell lysates were separated by SDS-PAGE, and tyrosine/threonine phosphorylation of p38 MAPK was assessed by Western blotting using a phosphoselective anti-p38 MAPK antibody. Densitometry of the images of immunoreactive bands at each time point was quantified and normalized to basal levels of p38 MAPK phosphorylation (time = 0 min); n = 4-5 ± SE; *significantly different from stimulated, vector control-infected cells; P < 0.05.


    DISCUSSION
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ABSTRACT
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DISCUSSION
REFERENCES

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 (alpha -PAK), PAK2 (gamma -PAK), PAK3 (beta -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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