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Department of Microbiology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908
Received 11 November 2003/ Accepted 17 December 2003
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ABSTRACT |
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INTRODUCTION |
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Propagation of the Ras/Raf/MEK/ERK signal is modulated by a variety of inputs that must be integrated to achieve a full signaling response. Cell adhesion is required to couple Ras activation to MEK and ERK activation, as stimulation of suspended cells with growth factors leads to Ras and possibly Raf activation; however, activation of MEK and ERK fails to occur (38, 47). Signal transduction through the Ras-ERK pathway can be enhanced by the Rho family GTPases Rac and Cdc42 through their effector p21-activated kinase (PAK) (12, 23, 24), and Rac and Raf can synergize to promote cellular transformation (31). Expression of constitutively active Rac or Cdc42 activates the Jun N-terminal kinases (JNKs) and p38 kinases but not the ERKs (14). However, Rac and Cdc42 are able to synergize with Raf to stimulate ERK activation through mechanisms involving PAK1 phosphorylation of the MEK1 proline-rich sequence (PRS) (12, 23, 54) and PAK3 phosphorylation of Raf-1 (11, 32). PAK1 has been reported to enhance the phosphorylation of T292 and S298 of MEK1 in cells, although only S298 is phosphorylated by PAK in vitro (12, 23). Combined mutation of both of these sites inhibits Raf binding (23) and the ability of Raf to activate MEK (12, 54).
We recently reported that Rac-PAK signaling modulates the ERK pathway, at least in part by enhancing the association of MEK1 with ERK. We also showed that mutation of MEK1 at both T292 and S298 to alanine inhibited this effect (18). In addition, we demonstrated a requirement for PAK activity in the formation of MEK1-ERK complexes and ERK activation in newly adhering cells (18). Moreover, cellular adhesion to fibronectin was shown to induce PAK phosphorylation of MEK1 on S298, an event required for MEK1 activation upon adhesion (54).
In the present study we demonstrate that PAK phosphorylation of MEK1 at S298 is required for the Rac-induced enhancement of the MEK1-ERK interaction, while T292 phosphorylation is inhibitory. The inhibitory effect of T292 phosphorylation is explained by the observation that ERK feedback phosphorylation of MEK1 on T292 inhibits the ability of PAK to phosphorylate S298. We show that ERK phosphorylation of T292, like PAK phosphorylation of S298 (54), is stimulated by adhesion. Because PAK phosphorylation of MEK1 on S298 is required for MEK1 activation upon cellular adhesion (54), preventing ERK feedback phosphorylation on T292 enhanced MEK1 S298 phosphorylation and prolonged adhesion-induced MEK1 phosphorylation on its activating sites. These data demonstrate that opposing phosphorylation events in the MEK1 PRS regulate MEK1 adhesion-dependent complex formation and activation.
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MATERIALS AND METHOD |
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TopAbstractIntroductionMaterials and MethodResultDiscussionReference |
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Coimmunoprecipitations COS-1 cells in 10-cm-diameter dishes were transfected with 0.8 µg of ERK2 plasmid along with 3.2 µg of the indicated MEK plasmid and either 0.25 µg of RacL61 or empty vector. The cells were serum starved the following day for 4 h prior to harvest in hypotonic buffer (50 mM HEPES [pH 7.4], 2 mM MgCl2, 2 mM EGTA, and aprotinin). MEK-ERK complexes were immunoprecipitated as described previously (9, 17, 18).
For MEK1 immunoprecipitation experiments on adherent cells, COS-1 cells were plated onto 6-cm-diameter dishes and were transfected with 0.3 µg of hemagglutinin (HA)-MEK, 0.25 µg of FLAG-ERK, and empty vector for a total of 2 µg of DNA. After treatment the cells were harvested in FLAG lysis buffer (17) containing sodium fluoride (50 mM), sodium pyrophosphate (5 mM), sodium orthovanadate (0.1 mM), and phenylmethylsulfonyl fluoride (2 mM). Lysates were cleared by centrifugation, and immunoprecipitations were performed with anti-HA antibody. For MEK1 immunoprecipitation experiments on suspended and replated cells, 15-cm-diameter dishes were transfected with 0.3 µg of HA-MEK1, 0.3 µg of FLAG-ERK2, and empty vector for a total of 12.5 µg of DNA. The day following transfection the cells were trypsinized by using trypsin-EDTA solution (Gibco), washed with 1 mg of soybean trypsin inhibitor (Sigma)/ml, and suspended in serum-free DMEM for 90 min at 37°C and 5% CO2. When indicated, U0126 (Calbiochem, La Jolla, Calif.) was added at a final concentration of 10 µM. Cells were either left in suspension or allowed to adhere to bovine fibronectin-coated dishes (10 µg/ml in phosphate-buffered saline, left overnight at 4°C; Sigma) for the indicated times at 37°C and 5% CO2 prior to lysis in FLAG lysis buffer. MEK1 proteins were immunoprecipitated with anti-HA antibody 12CA5.
In vitro MEK1 phosphorylation
Wild-type or mutant GST-MEK1 protein (1 µg), purified as described previously (17), was incubated with or without purified, activated ERK2 (specific activity, 1.7 µmol/min/mg with myelin basic protein as substrate) in kinase buffer (25 mM HEPES-NaOH [pH 7.5], 10 mM MgCl2, 0.5 mM ATP, 1 mM dithiothreitol) for 40 min at 30°C. Control experiments indicated that MEK1 was phosphorylated to a stoichiometry of 
1 mol/mol under these conditions. Purified recombinant PAK3 (0.675 or 6.75 ng per reaction; a gift from Mark Marshall) or buffer control was added and reactions continued at 30°C for an additional 10 min. Reactions were terminated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and were resolved by electrophoresis on a 10% polyacrylamide gel. After transfer to Immobilon P membrane, phosphorylation of T292 and S298 was assessed by blotting with appropriate phospho-MEK1-specific antisera. Coomassie staining confirmed equivalent loading of all glutathione S-transferase (GST)-MEK1 proteins.
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RESULT |
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ERK phosphorylation of T292 inhibits S298 phosphorylation by PAK PAK1 phosphorylates MEK1 at S298 (12, 23, 54). Transfection of constitutively active PAK into cells increased not only S298 phosphorylation but also T292 phosphorylation (12), although only S298 was phosphorylated by PAK in vitro (12, 23). Investigation of the PAK phosphorylation of Raf-1 on S338 revealed a consensus sequence containing a tyrosine at the +2 position and arginine residues at the -2 and -5 positions (33). The sequence surrounding S298 of MEK1 loosely conforms to this consensus, with a tyrosine at the +2 position and arginines at the -3 and -7 positions. T292, a reported ERK phosphorylation site (5, 40), lies directly C terminal to the -7 arginine (Fig. 2A). We hypothesized that the negative charge generated by phosphorylation of T292 might neutralize the positive charge at arginine 291, disrupting the PAK consensus and inhibiting the ability of PAK to phosphorylate MEK1 at S298.
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We then examined whether active ERK could affect phosphorylation of MEK1 on S298. COS-1 cells were transfected with expression plasmids for MEK1 and either wild-type ERK2 or ERK2 T183A, a phosphorylation site mutant that cannot be activated by MEK. In addition, mutationally activated Ras (Ras V12) or Raf (Raf-CAAX) was cotransfected to stimulate ERK2 activity. Transfection of Ras V12 alone, which would stimulate the ERK pathway as well as potentially activate the Rac-PAK pathway via activation of phosphatidylinositol 3 kinase (48), had little effect on S298 phosphorylation of MEK1 (Fig. 3A). However, cotransfection of Ras V12 with ERK2, but not ERK2-T183A, greatly inhibited phosphorylation of MEK1 at S298. Cotransfection of Raf-CAAX, which would primarily stimulate only the ERK pathway, inhibited S298 phosphorylation somewhat more than the empty vector. However, cotransfection of Raf-CAAX with ERK2, but not ERK2 T183A, strongly inhibited MEK1 S298 phosphorylation. The greater inhibition of S298 phosphorylation by Raf-CAAX and ERK2 compared to that of Ras-V12 and ERK2 correlated with a greater activation of ERK2 by Raf-CAAX (Fig. 3A). A similar inhibition of S298 phosphorylation was observed following cotransfection of ERK1 or ERK2 and a mutationally activated form of MEK1, MEK1 S218/222D (designated MEK1 Act.), in which the activating phosphorylation sites (62) were mutated to aspartate to mimic phosphorylation (Fig. 3B). These data demonstrate that both activated ERK1 and ERK2 were able to inhibit the phosphorylation of MEK1 at S298.
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In vitro experiments were performed to further test the hypothesis that ERK phosphorylation of MEK1 at T292 inhibited PAK phosphorylation of S298. Recombinant wild-type MEK1, MEK1 T292A, or MEK1 T292D were incubated in kinase buffer containing ATP in the presence or absence of recombinant, active ERK for 40 min. Recombinant PAK3 (1 or 10 U; 1 U = 6.75 ng) or buffer control was then added, and reactions were continued for 10 min. MEK1 proteins were then blotted to assess phosphorylation of S298 by PAK3 and phosphorylation of T292 by ERK (Fig. 5A). Wild-type MEK1 and MEK1 T292A were robustly phosphorylated on S298 in a dose-dependent manner by PAK3 (Fig. 5A, lanes 1 to 3). In contrast, MEK1 T292D was phosphorylated poorly on S298 (lanes 7 to 9), suggesting that a negative charge at position 292 inhibits PAK phosphorylation at S298. Importantly, prior phosphorylation of MEK1 on T292 by ERK greatly inhibited subsequent PAK3 phosphorylation of MEK1 S298 (lanes 10 to 12). These data suggest that prior T292 phosphorylation or substitution with a phosphomimetic residue inhibits subsequent PAK phosphorylation of MEK1.
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Our results suggest that ERK phosphorylation of T292 is necessary to inhibit PAK3 phosphorylation of MEK1 S298. They further suggest that any other MEK1 sites phosphorylated by ERK in these assays (e.g., T386) alone cannot account for ERK-mediated inhibition of PAK phosphorylation. To further test this contention, we asked whether S298 phosphorylation of MEK1 T292D could be further inhibited by prior incubation with ERK. Our data (lanes 7 to 9 and 16 to 18) indicate that prior incubation of MEK1 T292D with ERK does not change its sensitivity to subsequent PAK3 phosphorylation. Together these data indicate that phosphorylation of T292 is necessary and sufficient for ERK to inhibit PAK3 phosphorylation of MEK1 S298 in vitro. These data are in good agreement with the experiments performed in cultured cells. Note that the slight MEK1 S298 phosphorylation stimulated by ERK alone (lanes 10, 13, and 16) has been reported previously as an in vitro artifact (12).
We hypothesized that excess PAK might overcome the inhibition of S298 phosphorylation generated by ERK phosphorylation of T292. We therefore determined if excess PAK activity in cells could overcome the inhibition of S298 phosphorylation caused by a negative charge at T292. COS-1 cells were transfected with expression plasmids for MEK1, MEK1 T292D, or MEK1 S298A, along with increasing amounts of mutationally activated PAK1 (PAK1 T423E). Cells were placed in suspension to reduce endogenous PAK activity, and phosphorylation of exogenous MEK1 proteins was assessed following immunoprecipitation. PAK1 T423E increased the level of S298 phosphorylation of MEK1 in suspended cells in a dose-dependent manner (Fig. 5B). MEK1 T292D was resistant to S298 phosphorylation with low levels of activated PAK1. However, S298 phosphorylation of this mutant was observed to levels similar to those in wild-type MEK1 upon cotransfection of the highest concentrations of activated PAK1. Similarly, excess activated PAK3 resulted in efficient phosphorylation of ERK-phosphorylated MEK1 in vitro (data not shown). Collectively, these data clearly demonstrate that a negative charge at T292 of MEK1 inhibits the ability of PAK to phosphorylate S298, but that this inhibition can be overcome, in vitro and in vivo, with high concentrations of active PAK. Importantly, these data also demonstrate that the phospho-MEK1 S298 antibody recognizes MEK1 that was either phosphorylated or had an acidic residue at T292. This is further verified by our finding that the phospho-S298 MEK1 antibody efficiently immunoprecipitates MEK1 doubly phosphorylated on both S298 and T292 (data not shown).
To directly test whether interference with the positive charge at MEK1 R291 affects the ability of PAK1 to phosphorylate S298, we generated mutants of MEK1 at position 291 to either neutralize (alanine mutation) or mimic (lysine mutation) the positive charge of the arginine. While mutation to lysine resulted in S298 phosphorylation equal to that of wild-type MEK1, mutation to alanine inhibited the phosphorylation of MEK1 on S298 (Fig. 6A). These results demonstrate that the positive charge at R291 contributes to PAK phosphorylation of S298 and that neutralization of this charge by ERK phosphorylation of T292 may be one mechanism whereby ERK feedback phosphorylation inhibits the ability of PAK to phosphorylate MEK. Interestingly, cotransfection of ERK2 further inhibited S298 phosphorylation of the MEK1 R291A mutant, suggesting that there may be an additional mechanism by which ERK inhibits S298 phosphorylation (see Discussion).
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Regulation of MEK1 T292 phosphorylation by cellular adhesion It was previously reported that MEK1 S298 and T292 were unaffected by growth factor treatment of adherent CCL39 cells (10). Recent results show that cell adhesion stimulates PAK phosphorylation of MEK1 on S298 and subsequent MEK activation (54). Therefore, we examined whether phosphorylation of T292 is regulated by cellular adhesion. In addition, we determined if phosphorylation of T292 could affect MEK1 S298 phosphorylation induced by adhesion to fibronectin. Transfected COS-1 cells were placed in suspension (S) in serum-free media with either the MEK inhibitor U0126 or dimethylsulfoxide (DMSO) (Fig. 7A). The cells were then either harvested or replated onto fibronectin-coated dishes for the indicated time. Immunoblotting revealed that T292 of MEK1 was phosphorylated at a low level in cells in suspension, and phosphorylation of this site gradually increased after cellular adhesion to a fibronectin matrix, not becoming maximal until 30 min after replating. U0126 treatment prevented the induction of T292 phosphorylation upon cellular adhesion. Cotransfection of ERK2 slightly enhanced T292 phosphorylation in suspended cells but greatly stimulated this phosphorylation upon cellular adhesion. As demonstrated previously (54), immunoblotting these immunoprecipitates with the phosphospecific MEK1 S298 antibody showed that phosphorylation of this site was also low in suspended cells but became maximally induced within 10 min of adhesion to fibronectin and remained constant throughout the time course. Treatment of the cells with U0126 did not affect S298 phosphorylation, while cotransfection of ERK2 slightly delayed the kinetics of S298 phosphorylation.
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We next determined if MEK1 prephosphorylated on T292 would prevent phosphorylation of S298 by PAK upon cellular adhesion. MEK1 Act. transfected alone displayed no T292 or S298 phosphorylation in suspended cells, but phosphorylation of both sites was induced upon cell adhesion (Fig. 8A). Cotransfection of ERK2 enhanced T292 phosphorylation in suspended and newly adherent cells, correlating with an inhibition of S298 phosphorylation. Treatment of the cells with U0126 when the cells were suspended and replated prevented T292 phosphorylation and slightly enhanced S298 phosphorylation upon adhesion. In the presence of U0126, cotransfection with ERK2 did not enhance T292 phosphorylation or inhibit S298 phosphorylation. These data demonstrate that ERK activation in newly adherent cells controls T292 phosphorylation of MEK1, and this in turn regulates the phosphorylation of MEK1 S298 by PAK.
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PAK phosphorylation of MEK1 on S298 is required for MEK1 activation during cellular adhesion to fibronectin (54). Therefore, we determined whether T292 phosphorylation also affected adhesion-stimulated MEK1 activation. Transfected cells were trypsinized and suspended in serum-free media for 90 min and either harvested or replated onto fibronectin-coated dishes for 20 min. Immunoblotting HA immunoprecipitates with a phosphospecific antibody that recognizes the activating phosphorylation sites on MEK (phospho-S218/222) demonstrated MEK1 activation induced by cellular adhesion to fibronectin (Fig. 9A and B). Mutation of T292 to alanine did not inhibit MEK1 activation after 20 min of adhesion (Fig. 9A). Consistent with previous observations, mutation of S298 to alanine inhibited MEK1 activation under these conditions (Fig. 9B) (54), demonstrating that PAK phosphorylation of S298 was required for adhesion-induced MEK1 activation by Raf.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodResultDiscussionReference |
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Each level of the MAP kinase pathway can be regulated by feedback phosphorylation. MEK-dependent phosphorylation of SOS downregulates Ras activation in mammalian cells in response to growth factor stimulation (28). Degradation of the Saccharomyces cerevisiae MEK activator Ste11 is induced through a MAP kinase feedback mechanism (20), and mammalian Raf-1 is also a substrate for ERK, although the importance of this phosphorylation is unknown (3). In addition, ERKs phosphorylate MEK1 on T292 and T386 (5, 40). These phosphorylations were reported to have no effect on MEK1 activity in vitro (40), although mutation of these sites to alanine enhanced the amplitude of MEK1 activity after serum stimulation of cells (5). We propose that T292 phosphorylation by ERK is a feedback mechanism to limit the extent to which integrin signaling enhances MAP kinase signaling.
PAK phosphorylation of S298 is regulated by cellular adhesion and is required for MEK1 activation upon adhesion (54). ERK feedback phosphorylation of MEK1 on T292 inhibited the ability of PAK to phosphorylate S298 in vitro and in vivo and negatively regulated adhesion-stimulated MEK1 activation. Therefore, the MEK1 PRS serves as an integration point for positive and negative phosphorylation signals that regulate the formation of MEK1 complexes and activation during cellular adhesion to the extracellular matrix.
Opposing phosphorylation events regulate MEK1-ERK complex formation Frost et al. (23) observed that mutation of MEK1 at both T292 and S298 inhibited the basal association of MEK1 and Raf-1, but they were unable to detect an induction of MEK1-Raf-1 complexes upon cotransfection of activated Rac. We recently demonstrated that activated Rac could stimulate the formation of stable MEK1-ERK2 complexes in serum-deprived cells and that this stimulation of complexes was inhibited by alanine mutations of MEK1 at both T292 and S298 (18). These observations are consistent with previous reports that the MEK1 PRS is important for functional MEK1-ERK signaling (10, 15). Neither phosphorylation site on MEK1 was required for basal association with ERK2 in adherent cells; however, phosphorylation of S298, but not T292, was required for the enhancement of MEK1-ERK association by Rac. MEK1 S298 phosphorylation could act to stimulate the MEK1-ERK association in several ways. First, S298 phosphorylation might stimulate a conformational change in MEK1 that promotes its interaction with ERK. Our attempts to test this possibility in vitro have been inconclusive (data not shown). Second, the S298 phosphorylation may allow MEK1 to interact with an ERK-binding scaffold protein. We previously demonstrated that the MEK1-ERK association is enhanced by the small scaffold protein MP1, which binds to the MEK1 PRS (51). However, MP1 is an unlikely candidate scaffold, because the MEK1-MP1 interaction does not require S298 phosphorylation (data not shown). Third, S298 phosphorylation may allow the formation of MEK1-ERK complexes upon cytoskeletal structures induced by active Rac or Cdc42. Interestingly, mutation of S298 to aspartate did not detectably enhance binding to ERK2 (data not shown), suggesting that it is either the actual phosphoserine 298 form of MEK1 that is required for the enhanced interaction or a combination of the phosphoserine and Rac downstream signals.
In contrast to MEK1 S298, T292 phosphorylation negatively regulates Rac-enhanced MEK1-ERK association. Mutation of T292 to alanine enhanced both basal and Rac-inducible ERK association, while mutation to aspartate prevented the ability of Rac to enhance ERK association over the basal level. Together, these data suggest that phosphorylation of T292 might inhibit PAK phosphorylation of MEK1 on S298.
ERK regulates PAK phosphorylation of MEK1 One potential mechanism for ERK regulation of PAK phosphorylation of MEK1 is revealed by an analysis of the putative PAK consensus sequence. PAK phosphorylates Raf-1 on S338 (32). Marshall and coworkers (33) observed that arginines N terminal to S338 in Raf-1 are important for PAK phosphorylation of this site. MEK1 contains arginines at similar positions relative to the S298 phosphorylation site. Indeed, we demonstrate that a positive charge at R291 is necessary for efficient S298 phosphorylation by PAK. Phosphorylation of T292 may act to neutralize the positive charge at R291, interfering with PAK recognition of S298. Support for this charge neutralization hypothesis comes from the observation that aspartate mutation at T292 completely inhibited MEK1 S298 phosphorylation in cells, except in the presence of excess activated PAK. Additionally, prevention of T292 phosphorylation by alanine mutation enhanced S298 phosphorylation. MAP kinase has been shown to phosphorylate MEK1 on both T292 and T386, although the functional consequences of these phosphorylations have been unclear (5, 40). Both our in vivo and in vitro data support a major role for ERK phosphorylation of T292, but little or no role for T386, in the regulation of PAK phosphorylation of MEK1. Either expression of active ERK in cells or prior phosphorylation of MEK1 by ERK in vitro inhibited PAK phosphorylation of MEK1. Interestingly, cotransfection of ERK in cells further inhibited PAK phosphorylation of both MEK1 R291A and MEK1 T292A, suggesting that there is an additional mechanism by which ERK inhibits PAK phosphorylation of S298. This other mechanism could involve inhibition of PAK activity, sequestration of MEK1, or steric interference with the PAK-MEK1 interaction.
Adhesion-regulated phosphorylation and activation of MEK1 Feedback phosphorylations by MAP kinases on upstream members of their activation pathway generally act to downregulate pathway activation. Mansour et al. (40) reported that while ERK phosphorylated MEK1 on T292 and T386, these phosphorylations had no effect on MEK1 kinase activity in vitro. However, Brunet et al. (5) reported that mutation of these phosphorylation sites enhanced the activity, but not the duration, of MEK1 activity in response to serum stimulation through an unknown mechanism. Our results demonstrate that MEK1 T292 phosphorylation, like S298 phosphorylation (this work and reference 54), is regulated by cellular adhesion. The requirement for MEK1 S298 phosphorylation in MEK1 activation (54) suggests that this phosphorylation precedes MEK1 and ERK activation as well as T292 phosphorylation by ERK. Indeed, T292 phosphorylation upon cellular adhesion was delayed relative to S298 phosphorylation. This suggests a sequential phosphorylation sequence upon cellular adhesion whereby PAK phosphorylates MEK1, MEK1 becomes phosphorylated on its activating sites, MEK1 activates ERK, and ERK phosphorylates MEK1 on T292, preventing further phosphorylation of MEK1 by PAK. In agreement with this, prephosphorylation of T292 in suspended cells with activated ERK inhibited S298 phosphorylation by PAK following adhesion to fibronectin.
We demonstrate that one level of regulation of MAP kinase pathway activation during adhesion is through ERK feedback phosphorylation. The importance of T292 phosphorylation in limiting MEK1 activation was demonstrated by the prolonged activation of the MEK1 T292A mutant compared to wild-type MEK1. One explanation for this observation is that the elevated level of S298 phosphorylation observed for the MEK1 T292 mutant enhances coupling to Raf. Thus, under normal conditions of cellular adhesion, ERK feedback phosphorylation of T292 prevents increasing levels of S298 phosphorylation by PAK, potentially inhibiting the duration of MEK1 activation by Raf. Our observations suggest a plausible mechanism for the transient activation of MEK1 and ERK during adhesion to the extracellular matrix and provide a biological context for previous observations regarding the integration of Rho family GTPases into the MAP kinase pathway. The data presented here and elsewhere (12, 23, 54) suggest that adhesion-regulated assembly and disassembly of signaling complexes around MEK1 and subsequent activation of MEK1 and ERK are controlled by the integration of phosphorylation signals within the MEK1 PRS.
In addition to being regulated by cellular adhesion and spreading, both PAK and ERK have been shown to regulate cell spreading and migration (1, 34, 35, 52). While the mechanisms and pathways that regulate these processes are poorly understood, both PAK and ERK regulate cell spreading, possibly through the regulation of actin/myosin cytoskeletal dynamics and focal adhesion turnover (22, 50, 53, 57). Preliminary results suggest that transfection of MEK1 T292A into cells does not affect cell adhesion or spreading (data not shown). However, feedback inhibition of MEK1 activation by ERK may act to regulate the localized activation of ERK during adhesion and spreading. Active ERK localizes to the periphery of newly adherent cells (22). In addition, ERK phosphorylates paxillin (36, 39), a mutlifunctional focal adhesion docking protein, and phosphorylates and activates the protease M-calpain (25, 26), which cleaves several focal adhesion proteins, such as FAK, ezrin, talin, and integrin ß1 and ß3 cytoplasmic tails (8, 13, 42, 45, 46, 55). Thus, the coordinated regulation of ERK activity may help control focal adhesion dynamics and turnover. PAK has been reported to regulate actin dynamics through phosphorylation of LIM kinase and its subsequent effects on cofilin (19, 61) as well as through regulation of myosin light-chain kinase (50). The ability of ERK and PAK to regulate cell adhesion dynamics has implications not only for cell migration but also for tumorigenesis and progression. Activated PAK has been reported to enhance tumor growth, invasion, and metastasis in model systems (2, 4, 41, 56, 59). Furthermore, its elevated expression and activity in tumors (6, 49, 56) and its ability to stimulate adhesion-independent activation of ERK (29) suggest that activated PAK may be able to enhance MEK1 activation either directly or to overcome the negative regulation of MEK1 activation by ERK, potentially promoting cell proliferation, migration, and oncogenesis.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grants CA39076 and CA40042 (M.J.W.) and also GM68111 (A.D.C.).
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FOOTNOTES |
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Present address: Department of Pharmacology and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA 70115.
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