Article |
Address correspondence to J. Thomas Parsons, Dept. of Microbiology, Box 800734, University of Virginia Health System, Charlottesville, VA 22908-0734. Tel.: (434) 924-5395. Fax: (434) 982-1071. E-mail: jtp{at}virginia.edu; or Andrew D. Catling at his present address Dept. of Pharmacology and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112-1393. Tel.: (504) 568-2222. Fax: (504) 568-2361. E-mail: acatli{at}lsuhsc.edu
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Abstract |
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Key Words: integrin; adhesion; focal adhesion kinase; Src; extracellular matrix
M. Zecevic's present address is Cancer Prevention Fellowship Program, Division of Cancer Prevention, National Cancer Institute, 6130 Executive Blvd., Suite 309, MSC 7361, Rockville, MD 20852.
* Abbreviations used in this paper: FN, fibronectin; MEK, MAPK kinase; MEKK, MEK kinase; PAK, p21-activated kinase; PI3K, phosphoinositide 3-kinase.
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Introduction |
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Adhesion of cells to the ECM stimulates a number of signaling pathways including FAK, Src, and those initiated by the small GTPase Rac (Parsons et al., 2000). Integrin binding to ECM proteins elicits the formation of focal complexes and the recruitment and activation of two protein tyrosine kinases, FAK and Src (Parsons et al., 2000). Integrin-induced activation of FAK results in autophosphorylation of FAK on tyrosine 397 (Y397), which correlates with increased catalytic activity of FAK (Lipfert et al., 1992; Calalb et al., 1995) and serves as a high affinity binding site for the SH2 domain of Src family kinases.
The small GTPase Rac mediates lamellipodia extension and focal complex formation initiated by integrin binding to ECM proteins (Nobes and Hall, 1995). Activation of Rac by cell adhesion stimulates membrane ruffling and the formation of peripheral focal complexes through signaling cascades involving the RacCdc42 effector p21-activated kinase (PAK; Bagrodia and Cerione, 1999). Localization of activated PAK to focal adhesions and membrane ruffles influences cytoskeletal dynamics through phosphorylation of LIM (Edwards et al., 1999) and myosin light chain kinases (Sanders et al., 1999). Signals initiated by Rac activation also influence the RasMAPK pathway by synergizing with Raf to activate MAPK (Frost et al., 1997), possibly by sensitizing MEK1 to activation by Raf (Coles and Shaw, 2002). Recently, we demonstrated that RacPAK signaling can enhance the association of MEK1 and MAPK and that this pathway is required for the formation of MEK1MAPK complexes and MAPK activation upon cellular adhesion (Eblen et al., 2002). Moreover, synergy between Rac and Raf promotes anchorage-independent growth of fibroblasts (Qiu et al., 1995).
Because the RasMEKMAPK signal transduction pathway serves as a point of convergence for the regulation of proliferation and migration by growth factors and ECM proteins, we examined the adhesion-dependent activation of both MEK and MAPK. Here, we provide evidence that phosphorylation of MEK1 on S298 by PAK is one point at which these two signaling pathways converge. We show that adhesion to fibronectin (FN) induces PAK1 phosphorylation of MEK1 on S298 and that MEK1 S298 phosphorylation is necessary for efficient activation-specific phosphorylation of MEK1 and subsequent MAPK activation. Adhesion-dependent phosphorylation of MEK1 on S298 is dependent in part upon FAK/Src signaling, consistent with the localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membraneproximal adhesion structures. Moreover, synergistic activation of MEK1 by growth factors and cell adhesion is diminished in cells expressing an MEK1 S298A mutant. We propose that FAK/Src-dependent PAK phosphorylation of MEK1 on S298 is central to the organization and localization of active RafMEK1MAPK signaling complexes and that formation of such complexes underlies the observed adhesion dependence of growth factor signaling to MAPK.
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Results |
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PAK regulates adhesion-dependent phosphorylation of MEK1 on S298 and subsequent MEK1 activation
To examine whether PAK regulates adhesion-dependent MEK1 S298 phosphorylation in vivo, COS-1 or REF52 cells expressing kinase-defective PAK1 (or vector control) together with HA-tagged wild-type MEK1 were placed in suspension and replated on FN for the indicated times and S298 phosphorylation was assessed by Western blotting. Expression of kinase-defective PAK1 significantly reduced FN-stimulated S298 phosphorylation of exogenously expressed MEK1 (Fig. 4 A, not depicted), indicating that PAK activation is necessary for adhesion-dependent MEK1 S298 phosphorylation. To determine if PAK activity is sufficient to mediate MEK1 S298 phosphorylation in vivo, MEK1 S298 phosphorylation was examined in cells overexpressing activated PAK (T423E). REF52 cells expressing activated PAK together with wild-type MEK1 exhibited elevated MEK1 S298 phosphorylation whether in suspension or plated on FN (Fig. 4 B). Lastly, endogenous PAK1 immunoprecipitated from REF52 cells was activated by adhesion to FN (see Fig. 8 B), whereas endogenous PAK2 and 4 were not (not depicted). Together, these data indicate that PAK1 activity is both necessary and sufficient to stimulate MEK1 S298 phosphorylation and that PAK1-mediated phosphorylation of MEK1 on S298 is regulated by cell adhesion.
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Adhesion-mediated activation of FAK results in the stimulation of Src after binding to FAK phosphorylated on Y397 (Cobb et al., 1994; Schaller et al., 1994, 1999; Reiske et al., 1999). Because Y397F FAK was unable to restore MEK1 S298 phosphorylation, we assessed the role of Src in regulating adhesion-dependent MEK1 S298 phosphorylation using the Src family kinase inhibitor, PP2. REF52 cells were suspended in serum-free media in the presence or absence of PP2, and replated on FN in the continued presence or absence of PP2 (Fig. 7 A). PP2 treatment decreased the extent and delayed the time course of S298 phosphorylation after FN stimulation (Fig. 7 A), similar to the observations for FAK-null cells (Fig. 6 A). MEK1 S298 phosphorylation in cells stimulated to adhere to FN for 5 min in the presence of PP2 was 28% the level of untreated cells when normalized to MEK1 loading control. Overexpression of activated PAK (PAK1 T423E) rescued the decrease in MEK1 S298 phosphorylation induced by PP2 treatment (Fig. 7 B). In addition, active PAK-stimulated MEK1 S218/S222 phosphorylation on wild-type MEK1 and to a lesser degree on MEK1 S298A. Finally, cells deficient for Src, Yes, and Fyn also showed decreased and delayed MEK1 S298 phosphorylation upon plating on FN (Fig. S2). These results indicate that Src or a Src family kinase is involved in regulating the pathway(s) leading to MEK1 S298 phosphorylation.
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Because Src-dependent MEK1 S298 phosphorylation is required for adhesion-mediated activation of MEK (Fig. 5) and subsequent MAPK activation (Eblen et al., 2002), we examined the effect of Src inhibition on phospho-MAPK localization in REF52 cells. Cells were suspended for 1 h and allowed to spread on FN for 1 h in the continued presence or absence of PP2 and immunostained for phospho-MAPK (red) and paxillin (green). Consistent with a previous report (Fincham et al., 2000), inhibition of Src activity had no effect on the localization of phospho-MAPK to focal adhesions (Fig. 9, arrowheads). However, PP2 decreased the extent of phospho-MAPK staining in peripheral structures reminiscent of Rac-induced adhesion complexes. Indeed, only 22% of the cells treated with PP2 exhibited peripheral adhesion complexes containing phospho-MAPK, whereas almost all (98%) of the untreated REF52 cells displayed peripheral adhesion complexes rich in phospho-MAPK staining (Fig. 9, arrows). These observations indicate that, in addition to decreasing MAPK activation, inhibition of Src activity reduces the pool of activated MAPK recruited to newly formed adhesion complexes.
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Discussion |
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The adhesion-dependent PAK1 phosphorylation of MEK1 on S298 indicates that MEK1, through its functional coupling of Raf and MAPK, might serve as a sensor for the adhesive status of cells or cellular compartments (e.g., protrusions at the leading edge). In addition, our work provides a molecular framework for the observation that stimulation of cells in suspension with physiologic concentrations of growth factor fails to activate MEK and MAPK despite activating Ras and Raf. These observations suggest that anchorage-independent activation of PAK and consequent phosphorylation of MEK1 might contribute to increased migration and anchorage-independent proliferation during malignancy.
Role of Rac, PAK, and S298 phosphorylation in MAPK activation
Previous studies have shown that Rac signaling to PAK synergizes with Raf to regulate MAPK activity (Frost et al., 1997). The PAK effects on MAPK signal transduction appear to be mediated by phosphorylation of Raf on S338 (King et al., 1998; Chaudhary et al., 2000). PAK-mediated phosphorylation of c-Raf has been reported to stimulate its activity (King et al., 1998). Furthermore, adhesion of COS7 cells overexpressing c-Raf mutants has been reported to stimulate c-Raf S338 phosphorylation through a phosphoinositide 3-kinase (PI3K)dependent mechanism (Chaudhary et al., 2000). We did not observe stimulation of endogenous c-Raf S338 phosphorylation (not depicted) or endogenous c-Raf activity (Fig. 8 A) when cells were replated on FN. Moreover, inhibiting PI3K activity with LY294002 had no effect on MEK1 S298 phosphorylation (unpublished data). These discrepancies could reflect differences in the pathways used by different cell types or an alteration in pathway usage when proteins are overexpressed. Indeed, overexpression of active PAK1 led to a modest stimulation of the activation of MEK1 S298A as judged by increased phosphorylation of S218/S222. This observation suggests the possibility that overexpression of activated PAK1 may obviate normal signaling constraints by phosphorylating Raf on S338, thus enhancing MEK activation in a S298-independent manner. This is consistent with observations showing adhesion-dependent Raf activation after PAK phosphorylation of Raf-S338 in COS7 cells overexpressing c-Raf mutants (Chaudhary et al., 2000). Based on these observations, we suggest that the RafMEKMAPK pathway may be exquisitely sensitive to the level of PAK1 activation and in the case of adhesion of REF52 cells to FN, PAK1 is primarily a modulator of MEK1 activation.
An alternative possibility to explain PAK-Raf synergy is that PAK-dependent phosphorylation of MEK1 on S298 leads to enhancement of MAPK by modulating both the physical and functional interactions between Raf, MEK, and MAPK. Frost and co-workers showed that MEK1 with mutations of T292 and S298 to alanine bound c-Raf less efficiently than did wild-type MEK1 (Frost et al., 1997). Indeed, recent evidence has shown that PAK phosphorylation of S298 sensitizes MEK1 to activation by Raf (Coles and Shaw, 2002). Here, we show that MEK1 S298 phosphorylation is dependent on cell adhesion (Fig. 3 A) and that adhesion-dependent phosphorylation of MEK1 on S298 was required for phosphorylation and activation of MEK1 in response to FN stimulation (Fig. 5). However, MEK1 activation occurred in the absence of a measurable increase in c-Raf activity. One possible explanation is that adhesion-dependent activation of MEK1 is regulated by phosphorylation from MEK kinases other than Raf, including MEK kinase (MEKK) 1, 2, and 3, which have been implicated in stimulating the MAPK pathway (Ellinger-Ziegelbauer et al., 1997; Xia et al., 1998). We cannot rule out the possibility that other MEK kinases are activated by adhesion to FN, however, PAK1 phosphorylation of MEK1 would be required for MEKK to activate MEK1 because MEK1 S298A was not phosphorylated on the activating sites upon plating on FN (Fig. 5). An alternative possibility is that adhesion-dependent PAK1 activation and subsequent phosphorylation of MEK1 on S298 is sufficient to stimulate MEK1 phosphorylation and activation by low levels of c-Raf activity merely by enhancing the interaction between c-Raf and MEK1. In both scenarios, adhesion-dependent PAK1 phosphorylation sensitizes MEK1 for activation from the upstream activator. Previous observations demonstrated that MEK1 was constitutively phosphorylated on S298 in the presence or absence of serum (Catling et al., 1995), signifying that MEK1 S298 phosphorylation is regulated uniquely by cell adhesion. Furthermore, in a separate study we show that adhesion-dependent phosphorylation of MEK1 on S298 is necessary for the formation of MEK1MAPK complexes and subsequent MAPK activation (Eblen et al., 2002). Therefore, we suggest a unique activation mechanism by which cell adhesion stimulates MAPK activity by sensitizing MEK1 to activation by basal levels of c-Raf activity or a different MEKK and by promoting the assembly of MEK1MAPK signaling complexes.
In addition to regulating the amplitude of MAPK activity, PAK1 likely influences the spatial regulation of MAPK by stimulating localized assembly of MEK1-signaling complexes in focal adhesions and peripheral focal complexes. Activated PAK1 localizes to focal adhesions and membrane ruffles (Sells et al., 2000). Indeed, PAK1 activity, reflected by phospho-S298 MEK1 staining, was observed in peripheral membrane structures 10 min after replating onto FN (Fig. 3 C). We propose that MEK1 phosphorylated on S298 stimulates MAPK activity in peripheral membrane structures by promoting activation complexes containing Raf and MAPK. MAPK, once activated, might down-regulate components of its activation pathway (Brunet et al., 1994; Dong et al., 1996), which could result in the dissociation of MEK from MAPK, allowing MEK to translocate to additional cellular compartments as was observed at later time points after replating (Fig. 3 C, 60 min FN).
FAK and Src as regulators of MAPK
FAK and Src positively regulate phosphorylation of MEK1 on S298 by PAK1. Cells deficient in FAK expression displayed decreased MEK1 S298 phosphorylation and a delayed time course of phosphorylation after FN stimulation (Fig. 6 A), which could be rescued by activated PAK (not depicted). The observation that wild-type FAK, but not FAK Y397F, which is incapable of interacting with Src, stimulated MEK1 S298 phosphorylation in FAK-deficient cells strongly indicates that these kinases function coordinately to regulate downstream signals to Rac and PAK. This is supported by similar observations in Src/Yes/Fyn-deficient cells and the observation that PP2 was an effective inhibitor of MEK1 298 phosphorylation. No change in MEK1 S298 phosphorylation was observed in cells treated with LY294002 (unpublished data), suggesting that PI3K, which also binds FAK phosphorylated on Y397 (Reiske et al., 1999), is not required for MEK1 S298 phosphorylation.
Inhibition of Src family kinases with PP2 not only inhibited MEK1 S298 phosphorylation, but also decreased phosphorylation of both MEK and MAPK on their respective activating sites in response to adhesion. Furthermore, PP2 inhibited the appearance of activated MAPK at the cell periphery. PP2 might inhibit Raf-mediated activation of MEK1 by reducing MEK1 S298 phosphorylation, or alternatively might reduce Raf activity directly, because Src phosphorylation of Raf has been shown to increase its activity (Fabian et al., 1993; Marais et al., 1995). However, adhesion to FN failed to stimulate Raf activity. Interestingly, Raf activity was actually increased after PP2 treatment even though phosphorylation of its downstream substrate MEK1 was suppressed. In contrast, adhesion-dependent PAK1 activation was decreased by PP2 treatment consistent with the hypothesis that FN-stimulated MEK1 activation is regulated primarily by PAK1 phosphorylation of MEK1 on S298. These observations, together with data showing that phospho-MAPK staining was decreased in peripheral focal complexes after PP2 treatment, indicate that the activation and localization of active MAPK to Rac-like focal complexes is regulated by Src-mediated activation of PAK1 and subsequent phosphorylation of MEK1.
Integrin activation of PAK
Integrin engagement leads to PAK activation and the targeting of PAK to adhesion complexes (Manser et al., 1998; del Pozo et al., 2000; West et al., 2001). The small GTPases Rac and Cdc42 are potent activators of PAK and are activated in response to integrin ligation. The molecular pathways leading to RacCdc42 and PAK activation are likely numerous, however, the data presented in this paper indicate that in the setting of cell adhesion to FN, FAK, and Src play a significant (but not exclusive) role in the activation of PAK1. Previous studies have implicated PI3K in the integrin-dependent activation of PAK1, Raf, and MAPK (Chaudhary et al., 2000). In our work, the PI3K inhibitor LY294002 had little effect on the adhesion-induced phosphorylation of MEK1 on S298, consistent with activation via the integrin FAK/Src-directed pathway. Of interest is the recent observation that DOCK180, when combined with its binding partner ELMO, is an efficient activator of Rac (Brugnera et al., 2002). This suggests the possibility that FAK/Src-dependent activation of p130 Crkassociated substrate and Crk may lead to the activation of DOCK180/ELMO and the subsequent activation of Rac and PAK.
Biological importance of adhesion signaling to MEK
Active MAPK and pS298 MEK1 were found proximal to immature focal complexes and focal adhesions after FN stimulation. As pS298 MEK1 is a surrogate for active PAK, these findings support a role for these kinases in the regulation of focal adhesion dynamics and cell migration. MAPK and PAK activities have both been found to be important for cell migration (Klemke et al., 1997; Kiosses et al., 1999; Sells et al., 1999). Moreover, Rac overexpression enhances MAPK-dependent migration of cells stimulated with low levels of EGF while having little effect on migration in response to high concentrations of growth factor (Leng et al., 1999). PAK phosphorylation of MEK1 on S298, which is required for MEK1 activation-specific phosphorylation in the absence of inducible Raf activity, provides a mechanism whereby suboptimal Raf activation likely achieved with shallow gradients of growth factor stimulation could promote maximal MAPK activity in a localized manner.
In addition to stimulating MAPK activity, cell adhesion also regulates growth factorinduced activation of MAPK (Renshaw et al., 1997). Without an appropriate ECM, serum growth factors stimulate Ras activity, however, cell adhesion is required to activate MAPK at the level of Raf or MEK (Lin et al., 1997; Renshaw et al., 1997). Furthermore, the adhesion requirement for growth factorinduced MAPK activation is lost in cells overexpressing activated FAK (Renshaw et al., 1999) or PAK (Howe and Juliano, 2000). Our findings that adhesion-dependent PAK1 activation is required for MEK1 activation by Raf and for MEK1MAPK coupling (Eblen et al., 2002) provide a mechanism to account for the disconnect between Raf and MEK in serum-stimulated suspended cells. In the absence of cell adhesion and MEK1 S298 phosphorylation, growth factorstimulated Raf activity resulted in reduced phosphorylation and activation of MEK, thereby rendering MAPK activation dependent on cell adhesion to ECM. Because sustained MAPK activity regulates adhesion-dependent growth (Assoian and Schwartz, 2001), anchorage-independent growth may result from continuous PAK phosphorylation of MEK1 on S298. Indeed, PAK has been implicated in regulating anchorage-independent growth (Tang et al., 1997, 1999; Vadlamudi et al., 2000). Furthermore, we find adhesion-independent MEK1 S298 phosphorylation in highly tumorigenic human prostate cell lines (unpublished observations). Therefore, the loss of adhesion-dependent regulation of PAK activity would have a profound impact on tumorigenesis and metastatic progression by affecting cellular proliferation and migration.
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Materials and methods |
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Generation and characterization of phospho-specific MEK1 antibody
The anti-MEK (pS298) antibody was prepared by Research Genetics. Rabbits were immunized with the KLH-conjugated phosphopeptide, R-T-P-G-R-P-L-pS-S-Y-G-M-D-S. Antibodies from pooled bleeds were affinity purified over a phosphopeptide column and passed over a nonphosphorylated peptide column to remove antibodies specific for the nonphosphorylated peptide. The purified antibody was tested for specificity both in ELISA assays, using bound phosphopeptide versus nonphosphopeptide, and on immunoblots of purified GST-MEK. Recombinant MEK1 was purified as described previously (Catling et al., 2001). Recombinant His-tagged PAK3 was a gift from S. Bagrodia and R.A. Cerione (Cornell University, Ithaca, NY). Purified MEK1 was incubated with or without recombinant PAK3 in 0.5 mM -[32P]ATP (
8,400 cpm/pmol), 10 mM MgCl2, 1 mM DTT, and 25 mM Hepes-NaOH, pH 7.4, for 20 min at 30°C. Reactions were terminated with SDS-PAGE sample buffer and resolved on 10% gels. Incorporation was analyzed by autoradiography and immunoblotting. Control reactions lacking MEK1 showed no incorporation or p-S298 MEK1 reactivity, and Cerenkov counting indicated that PAK3 rapidly phosphorylates MEK1 to stoichiometry under these conditions.
Plasmids and transfection assays
All constructs in this work used a cytomegalovirus promoter to drive expression of peptide epitopetagged protein. Wild-type MEK1, MEK1 T292A, MEK1 S298A, MEK1 T292A/S298A, and kinase-defective MEK1 (K97A) subcloned into pCMVHA (Catling et al., 1995), wild-type FAK, and the autophosphorylation mutant (Y397F) of FAK subcloned into pCMV-myc (Xiong and Parsons, 1997), and myc-tagged PAK1-K299R and PAK T423E subcloned into pRK5-myc (Weed et al., 1998).
To test the phosphorylation of MEK1 mutants (T292A, S298A, or T292A/S298A), two 150-mm dishes containing 2 x 106 REF52 were each transfected with 0.25 µg each MEK1 construct and 12.25 µg pCMVHA using Superfect. The effect of kinase-defective PAK on MEK1 S298 phosphorylation was tested by transfecting two 150-mm dishes containing 1.5 x 106 COS-1 cells each with 3 µg PAK1 K299R or empty vector together with 1.25 µg wild-type MEK1 using Lipofectamine. The effect of activated PAK on MEK1 phosphorylation was tested by transfecting two 150-mm dishes containing 1.5 x 106 REF52 cells each with 4 µg PAK1 T423E or empty vector together with 1 µg wild-type MEK1 using Superfect (QIAGEN). To assess the role of wild-type FAK or FAK Y397F on MEK1 phosphorylation, 4 x 106 FAK-deficient cells were plated on each of two 150-mm plates per construct and transfected each with 10.75 µg FAK, FAK Y397F, or empty vector together with 1.25 µg wild-type MEK1 using Polyfect (QIAGEN) according to the manufacturer's instructions. 24 h later, the cells were harvested, pooled from two dishes, suspended for 6090 min in serum-free media, and replated on dishes coated with 10 µg/ml FN (Sigma-Aldrich) for 530 min. To test the effects of growth factors and cell adhesion on MEK and MAPK activation, REF52 cells were serum-starved for 16 h in DME containing 0.2% FBS, suspended in serum-free DME for 90 min, and either kept in suspension, stimulated in suspension with the indicated concentrations of EGF or with 2 ng/ml EGF and 100 ng/ml IGF-1, or allowed to adhere to 10 µg/ml FN in the presence or absence of growth factors for 30 min. The role of MEK1 S298 phosphorylation in MEK1 activation was determined by transfecting REF52 cells with HA-tagged wild-type MEK1 or HA-tagged MEK1 S298A. After 32 h, the cells were serum-starved in DME containing 0.2% FBS for 16 h. Cells were suspended in serum-free DME for 90 min and either treated with 3 ng/ml EGF in suspension, allowed to adhere to 10 µg/ml FN, or allowed to adhere to FN in the presence of EGF for the indicated times. For all transfection experiments, MEK1 phosphorylation was determined after immunoprecipitation using HA antibodies as described in the next section.
Immunoprecipitation and Western analysis
Suspended cells or those that were allowed to reattach to 10 µg/ml FN were lysed in supplemented RIPA buffer (50 mM Hepes, 0.15 M NaCl, 2 mM EDTA, 0.1% Nonidet P-40, and 0.05% sodium deoxycholate, pH 7.2) containing 1 mM PMSF, 100 mM leupeptin, and 0.05 TIU/ml aprotinin (1 mM Na3VO4, 40 mM NaF, and 10 mM Na4P2O7). Attached cells were lysed directly on the plate in supplemented RIPA buffer. Lysates were incubated for 1 h at 4°C with protein ASepharose beads (Amersham Biosciences) preconjugated with specific IgG. The immunoprecipitated proteins were resolved on 10% SDS-PAGE gels, transferred to nitrocellulose, and blotted with p-S218/S222 MEK1, 12CA5 (mAb to the HA tag), MEK1, or MEK2 followed by HRP-conjugated secondary antibody. The immunoblots were stripped (100 mM ß-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7) and blotted with p-S298 MEK1 followed by incubation with HRP-conjugated goat antirabbit secondary antibodies and visualization by ECL (Amersham Biosciences). Western analysis of whole cell lysates was performed using 2550 µg protein.
Kinase assays
REF52 cells were suspended in the presence of PP2 (50 µM) or DMSO for 90 min and allowed to adhere to 10 µg/ml FN in the continued presence of PP2 for the indicated times. Endogenous PAK1 or matched antibody-specific GAL4 control immunoprecipitates were formed. Kinase reactions were initiated by adding a mix containing 1 µg of recombinant, kinase-defective MEK1 (MEK1 K97A; a gift from T. Vomastek (University of Virginia, Charlottesville, VA), 100 µM ATP, 10 mM MgCl2, 1 mM DTT, and 25 mM Hepes-NaOH, pH 7.4, for 20 min at 30°C. Reactions were terminated with SDS-PAGE sample buffer and resolved on 10% gels. C-Raf kinase assays were performed as described previously (Slack et al., 1999).
Immunofluorescence
REF52 cells were suspended in serum-free DME for 1 h at 37°C and plated on coverslips coated with 10 µg/ml FN for 10 min or 1 h. The cells were rinsed three times with PBS before fixing and staining. P-MAPK staining was performed as described previously (Zecevic et al., 1998). P-S298 MEK1 staining was performed after fixing the cells with 4% PFA in PBS, permeabilizing with 0.5% Triton X-100, and blocking in 20% NGS and 2% BSA. Cells were incubated with antip-S298 MEK1 and antipaxillin antibodies diluted in 10% NGS; 1% BSA followed by FITC-conjugated goat antimouse IgG, and Texas redconjugated goat antirabbit IgG in 0.5% BSA at a final concentration of 1.52 µg/ml each.
Online supplemental material
Online supplemental figures are available at http://www.jcb.org/cgi/content/full/jcb.200212141/DC1. The figure legends associated with each figure provide descriptions as to how the experiments were performed.
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Acknowledgments |
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This work was supported by the National Institutes of Health grants CA 40042 (to M.J. Weber and J.T. Parsons), CA 29243 (to J.T. Parsons), and CA 80606 (to J.T. Parsons).
Submitted: 23 December 2002
Revised: 23 May 2003
Accepted: 11 June 2003
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