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Department of Cell Biology,1 Hematology Division, Brigham & Women's Hospital, Department of Medicine,2 Department of Pathology, Division of Cancer Biology and Angiogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 021153
Received 26 November 2003/ Accepted 6 January 2004
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ABSTRACT |
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INTRODUCTION |
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Activation of RSK requires multiple phosphorylation events. Inputs from the upstream activators, including ERK1/2 and PDK1, as well as autophosphorylation, are necessary for RSK activation (8, 17, 23, 24, 29, 38, 40). Like phosphorylation, subcellular localization of RSK may play a role in its activation. We have recently shown that after epidermal growth factor (EGF) stimulation, RSK transiently localizes to the plasma membrane prior to nuclear translocation (6, 23). Constitutive membrane targeting of RSK leads to the phosphorylation of key regulatory sites and bypasses the requirement for ERK, indicating that membrane localization is important for activation (23, 26). Changes in subcellular localization thereby contribute to its activation and generate the potential for RSK to regulate a variety of proteins throughout the cell.
We have previously shown that filamin A (FLNa), a membrane-associated, cytoskeletal protein, is a RSK substrate (21). FLNa (also known as actin-binding protein 280, filamin 1, and nonmuscle filamin) is the most widely distributed isoform of a family of high-molecular-weight dimeric proteins that cross-link actin filaments (reviewed by Stossel et al. [28]). FLNa is a very efficient actin gelation factor, capable of inducing a liquid-to-gel transition of actin filaments at a ratio of one FLNa dimer per actin filament (15). In addition to actin, FLNa has more than 20 other binding partners, including membrane receptors, small GTPases, and stress-activated protein kinase (reviewed by Stossel et al. [28]).
FLNa is essential for mammalian cell locomotion. Human melanoma cell lines that do not express detectable levels of FLNa do not migrate (2), and restoring expression of wild-type levels of FLNa in these cells rescues their locomotion (7, 12). Mutations of the human filamin A gene on the X chromosome leading to failure of FLNa protein expression are responsible for a disease called periventricular heterotopia (PH). Males or females homozygous for PH die in utero, presumably due to severe defects in embryonic cell migration. Female PH heterozygotes present with epileptic seizures caused by the periventricular heterotopias, collections of embryonic neurons that fail to migrate from the lateral ventricles to the cerebral cortex. Heterozygous PH females often exhibit other neuronal and extraneuronal anomalies as well as premature vascular problems (13).
FLNa is known to be a phosphoprotein but how phosphorylation regulates its function is not understood. In addition to RSK (21), many kinases, including cyclic AMP (cAMP)-dependent protein kinase (PKA) (5, 16, 18, 39), CaM kinase II (20), Pak1 (36), and PKC (34), can phosphorylate FLNa in vitro. Purified human RSK2 phosphorylates the C-terminal half of FLNa in vitro at a serine residue(s) that is phosphorylated in vivo in response to lysophosphatidic acid (21). However, the RSK phosphorylation site(s) on FLNa has not been identified. Pak1 can also phosphorylate FLNa, and Ser2152 was identified as the phosphorylation site. Interestingly, mutation of Ser2152 to Ala prevents Pak1-mediated membrane ruffle formation (36). These data suggest that FLNa Ser2152 phosphorylation is required for FLNa to effect cell shape changes.
In this report, we provide clear evidence that FLNa is a membrane-associated RSK substrate. We show that RSK phosphorylates FLNa on Ser2152 in vitro and that FLNa Ser2152 phosphorylation is stimulated by a variety of agonists in cultured cells. In addition, we demonstrate that FLNa Ser2152 phosphorylation is dramatically reduced by pharmacological inhibition of ERK signaling using the MEK inhibitor UO126. In contrast, FLNa phosphorylation is not diminished by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or stimulated by insulin, which potently activates PI3K and Akt. Moreover, expression of wild-type RSK potentiates EGF-stimulated FLNa phosphorylation, whereas expression of kinase-inactive RSK or RSK RNA interference (RNAi) antagonizes this phosphorylation. Supporting our hypothesis of RSK-dependent regulation of FLNa function, we show that FLNa-dependent, EGF-induced migration of human melanoma cells is significantly reduced by UO126 treatment. Since FLNa Ser2152 phosphorylation is necessary for membrane ruffling, our data suggest that RSK plays a role in control of actin cytoskeletal dynamics.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20N) was previously referred to as pCMV-myc-filamin 1 (22). Monoclonal antibodies, phospho-ERK1/2 (Sigma), and FLNa MAb1678 (Chemicon) were purchased from commercial suppliers. Rabbit polyclonal phospho-specific RSK antibody that specifically recognizes Ser380 was obtained from R&D Systems. myc antibody was produced from 9E10 hybridoma (American Type Culture Collection). HA antibody (affinity purified from 12CA5 hybridoma) was a gift from M. Chou. C-terminal peptide-specific rabbit polyclonal RSK-C3 antibody (11) and rabbit polyclonal ERK1/2 antibody (6) have been previously described. RSK1-specific antibody was generated by injecting rabbits with PIESSILAQRRVKKLPSTTL peptide, derived from amino acid residues 733 to 752 in avian RSK and corresponding to residues 716 to 735 in human RSK1 (Invitrogen). RSK2-specific antibody was made by injecting rabbits with the peptide RNQSPVLEPVGRSTL, derived from amino acids 712 to 726 in human RSK2 (Invitrogen). To make phospho-FLNa antibody, rabbits were injected with RRAPpSVANVGS peptide (containing phosphorylated Ser2152 [pS]), derived from amino acids 2148 to 2158 in human FLNa (Invitrogen).
Site-directed mutagenesis.
pGEX-4T-1-FLNa-Ala2152 was made using the QuikChange site-directed mutagenesis kit (Stratagene). pGEX-4T-1-FLNa-C was amplified with Pfu DNA polymerase, 5' FLNa-Ala2152 primer (GCAGGCGTCGGGCTCCTGCAGTGGCCAACGTTG) and 3' FLNa-Ala2152 primer (CAACGTTGGCCACTGCAGGAGCCCGACGCCTGC). The PCR was performed as suggested by the manufacturer's manual except that extension of template was performed at 66°C. pBluescript II SK+-FLNa (ClaI) was made by excision of a ClaI-ClaI fragment from human filamin A in pCMV5-FLNa (20N) and ligation into ClaI-cut pBluescript II SK+ (Stratagene). pBluescript II SK+-FLNa-Ala2152 (ClaI) was created by amplification of pBluescript II SK+-FLNa (ClaI) with Pfu DNA polymerase and the above primers. The Ser2152-to-Ala mutation was confirmed by sequencing.
Construction of pcDNA3-myc-FLNa (human filamin A).
pcDNA3-myc-FLNa was created using the following strategy. (i) pREP4-FLNa was amplified with Ex Taq polymerase (Takara), 5' FLNa (EcoRI) primer containing an EcoRI site (TTAGAATTCAGTAGCTCCCACTCTCGG), and 3' FLNa (SalI/XbaI) primer containing SalI and XbaI sites (TGTTCTAGAGTCCCGCGTGTCGACG). The 70-bp PCR product was gel purified, digested with EcoRI and XbaI (New England Biolabs), and ligated into EcoRI-XbaI-digested pCMV5 vector to make pCMV5-5'FLNa. (ii) pCMV5-full-length FLNa was made by excision of an 8.6-kb SalI-SalI filamin A fragment from pCMV5-FLNa (20N) and ligation into SalI-cut pCMV5-5'FLNa. (iii) pcDNA3-myc-FLNa (EcoRI-XbaI) was made by excision of a 1.9-kb EcoRI-XbaI fragment of FLNa from pCMV5-FLNa (20N) and ligation into EcoRI-XbaI-digested pcDNA3 vector containing an N-terminal myc tag (27). (iv) pcDNA3-myc-FLNa was made by excision of a 6.7-kb EcoRI-EcoRI fragment from pCMV5-full-length FLNa and ligation into EcoRI-cut pcDNA3-myc-FLNa (EcoRI-XbaI).
pcDNA3-myc-FLNa-Ala2152 was created by replacement of the 2.4-kb ClaI-ClaI fragment of FLNa in pcDNA3-myc-FLNa with a ClaI-ClaI fragment containing the Ala mutation that was excised from pBluescript II SK+-FLNa-Ala2152 (ClaI).
Cell culture conditions, transfection, and lysate preparation. Human embryonic kidney (HEK) 293E, rat 3Y1, and HeLa cells were cultured at 37°C in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. HEK 293E cells were transfected by calcium phosphate precipitation. For lysate preparation, HEK 293E or rat 3Y1 cells were washed once in 1x phosphate-buffered saline (PBS) and then harvested on ice in cold lysis buffer (10 mM KPO4, 1 mM EDTA [pH 7.05], 10 mM MgCl2, 5 mM EGTA [pH 7.2], 50 mM ß-glycerophosphate [pH 7.2], 0.5% Nonidet P-40 [NP-40], 0.1% Brij-35 plus 1 mM Na3VO4, 5 µg of pepstatin ml-1, 10 µg of leupeptin ml-1, 1 mM phenylmethylsulfonyl fluoride). Lysates were cleared by centrifugation at 16,000 x g for 10 min at 4°C.
In vitro kinase assay. Lysates were made from HEK 293E cells that had been starved for 24 h or cells that were starved and then stimulated for 10 min with 10% FBS and incubated with RSK-C3 antibody or anti-HA antibody (for overexpressed RSK or Akt) for 1 h at 4°C with rocking. Complexes were precipitated by incubation with protein A Sepharose beads for 1 h. Beads were collected by centrifugation and washed once with each of the following buffers: Hermann's buffer (pH 7.2) (10 mM Tris [pH 7.2], 100 mM NaCl, 1 mM EDTA [pH 8.0], 1% NP-40, 0.5% deoxycholate), buffer B (pH 7.2) (10 mM Tris [pH 7.2], 1 M NaCl, 0.1% NP-40), ST buffer (pH 7.2) (50 mM Tris-HCl, 5 mM Tris base, 150 mM NaCl), and kinase buffer (20 mM HEPES [pH 7.2], 10 mM MgCl2, 3 mM ß-mercaptoethanol, 100 µg of bovine serum albumin ml-1). In vitro kinase assay was performed as previously described (26) using glutathione S-transferase (GST) fusion proteins purified from bacteria or proteins expressed by a baculovirus (19) as substrates. Kinase reactions were stopped by the addition of 2x sample buffer (100 mM Tris [pH 6.8], 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 20% glycerol, 286 mM ß-mercaptoethanol). Kinase assays were performed at least two times with comparable results.
In vivo labeling. HEK 293E cells were transfected with plasmid DNA and then starved for about 20 h. Cells were subsequently labeled for 3 h with 32Pi (1 to 2 mCi per 10-cm-diameter dish of cells). Cells were pretreated with 5 or 10 µM UO126 (BIOMOL Research Laboratories, Inc.) for 30 min and then stimulated with 10% FBS for 10 min or with 50 ng of EGF ml-1 for 15 min. Lysates were prepared. Overexpressed FLNa was immunoprecipitated with anti-myc antibody, and endogenous RSK was immunoprecipitated from the supernatant of the myc immunoprecipitates with RSK-C3 antibody. Immunoprecipitates were washed twice with each of the following buffers: Hermann's buffer (pH 7.2), buffer B (pH 7.2), and ST buffer (pH 7.2). Samples were denatured in 2x sample buffer, boiled, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Phosphate incorporation was quantitated using a phosphorimager and reported as fold activation normalized to the serum-starved samples. Each in vivo labeling experiment was performed a minimum of two times, and representative data are shown.
RNAi. High-performance liquid chromatography-purified (>97% pure) short interfering RNA (siRNA) oligonucleotides were purchased from Qiagen. The sequences of the sense strands of the siRNA oligonucleotides follow: RSK1, 5'-GGCAACGCUGAAAGUACGUdTdT-3', RSK2, 5'-GGCCACACUGAAAGUUCGAdTdT-3', and nonsilencing control, 5'-UUCUCCGAACGUGUCACGUdTdT-3'. HeLa cells (2 x 105) were seeded in 35-mm-diameter wells. On the following day, cells were transfected with 200 pmol of RSK1, RSK2, or nonsilencing control siRNA oligonucleotides using Oligofectamine as described by the manufacturer (Invitrogen). Approximately 24 h later, cells were trypsinized and seeded in 60-mm-diameter plates. Cells were serum starved for 1 day and/or stimulated with EGF (with and without UO126 treatment). Cells were lysed 72 h posttransfection, since optimal silencing of RSK1 and RSK2 was observed at this time point.
Isolation of stable human melanoma cell lines. Human melanoma cells were cultured in minimum essential medium Eagle (MEM) containing Earle's salts and L-glutamine (Mediatech, Inc.), 10% iron-enriched calf serum, 10 mM HEPES (pH 7.2), 500 µg of G418 (Invitrogen) ml-1, and antibiotics. pcDNA3 vector or pcDNA3-myc-FLNa (FLNa-WT) plasmids were each transfected into FLNa-deficient M2 human melanoma cells (7) by calcium phosphate coprecipitation. After 3 days, the transfected cells were replated, and the medium was replaced with medium containing G418 (500 µg ml-1). Cells were cultured for 17 additional days under G418 selection, and the medium was changed every few days. Colonies were picked, and resistant clonal cell lines were cultured in medium containing G418 (500 µg ml-1).
Single-cell tracking migration assay. Human melanoma cells (5 x 104 cells) were seeded in 25-mm-diameter flasks. On the following day, cells were washed once with PBS and starved for 16 h in MEM containing 20 mM HEPES. After the starvation period, the medium was changed to medium containing 10% serum for 1 h. EGF (5 ng ml-1) is added to the medium to induce migration. Cells were viewed using a 4x objective on a Nikon Diaphot 300 inverted microscope equipped with a heated stage. Images were captured with a charge-coupled device camera (Dage-MTI) every 15 min for 1 h beginning 15 min after EGF stimulation. Centroid displacement was determined using IP Lab Spectrum software.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP. BAD was included as a positive control, because it is a known RSK substrate (1, 26, 32). Activated RSK from serum-stimulated cells phosphorylated FLNa proteins A and C, both of which contain Ser2152 of FLNa (Fig. 1A). In addition, the sequence N-terminal to this Ser residue (R-R-R-A-P-S) fits the known consensus phosphorylation sequence for the RSK N-terminal (D1) kinase domain (R-X-R-X-X-S). To prove that Ser2152 is phosphorylated by RSK, this residue was mutated to Ala in FLNa-C (FLNa-Ala2152) and used as a substrate in a kinase reaction. Mutation of Ser2152 to Ala abolished RSK-mediated, serum-stimulated phosphorylation of FLNa, confirming that Ser2152 is the major RSK phosphorylation site (Fig. 1B, compare lanes 8 and 6). Furthermore, whereas wild-type RSK and RSK-K464R (C-terminal kinase domain mutant) phosphorylated FLNa, RSK-K112R (N-terminal kinase domain mutant) did not (Fig. 1B, compare lanes 6 and 14 to lane 10). These results demonstrate that kinase activity of the D1 kinase domain is required for FLNa phosphorylation but that activity of the D2 domain is dispensable. As expected, kinase-inactive RSK-K112/464R, which contains ATP-binding site mutations in both the N- and C-terminal kinase domains (11), did not phosphorylate FLNa, indicating that phosphorylation was not due to an associated kinase in the immunoprecipitate (Fig. 1B, lane 18). Our data suggest that the N-terminal kinase domain of RSK is responsible for FLNa phosphorylation on Ser2152.
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RSK does not phosphorylate FLNb in vitro. We wanted to determine whether RSK phosphorylates the other members of the filamin family, FLNb and FLNc. The Ser2152 residue of FLNa is conserved in human FLNb (Ser2107) and FLNc (Ser2126). However, FLNb and FLNc do not have arginine residues at both positions -3 and -5 of the RSK consensus sequence (Fig. 2A), suggesting that they are not likely to be good RSK substrates. FLNc is expressed only in muscle cells and has been shown to interact with sarcoglycans (33) and other muscle-specific proteins, such as myotilin (37), FATZ (10), and myozenin (30). Since a clone containing FLNc Ser2126 is not available, we were unable to test the ability of RSK to phosphorylate FLNc. To our knowledge, phosphorylation of FLNc has not been reported. Excluding a divergent hinge I region, FLNb is about 70% identical to FLNa on the amino acid level. Similar to FLNa, FLNb is expressed in many human tissues (31). In addition, FLNb has been shown to interact with FLNa in human cells (25). We tested FLNb as a substrate for RSK in an in vitro kinase assay. Overexpressed RSK was immunoprecipitated from HEK 293E cells that had been serum starved or serum starved and then stimulated with serum. Kinase reactions were performed using full-length, baculovirus-expressed human FLNa and FLNb proteins as substrates. In response to serum stimulation, RSK phosphorylated FLNa but not FLNb (Fig. 2B, compare lanes 5 and 10). These data suggest that FLNa, not FLNb, is a substrate of RSK.
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We generated a polyclonal phospho-specific FLNa (phospho-FLNa) antibody that recognizes FLNa protein phosphorylated on Ser2152 to confirm the in vivo labeling results. We tested our phospho-FLNa antibody using cell extracts prepared from HEK 293E and rat 3Y1 cells that have been serum starved or serum starved and stimulated with EGF. Phospho-FLNa antibody strongly recognized endogenous FLNa in stimulated cells, while FLNa antibody detected FLNa protein from unstimulated and stimulated cells to a similar extent (Fig. 4A). A small pool of FLNa may be basally phosphorylated, since our phospho-FLNa antibody weakly detected FLNa in unstimulated cells. We used peptide competition to validate the specificity of our phospho-specific antibody. Prior to incubation with the Western blot membrane, phospho-FLNa antibody was incubated with phosphorylated FLNa peptide or unphosphorylated FLNa peptide. Although blocking the antibody with unphosphorylated FLNa peptide had no effect (Fig. 4B, top gel, right), blocking with phosphorylated FLNa peptide abolished the signal (Fig. 4B, top gel, left). Furthermore, phospho-FLNa antibody detected overexpressed FLNa-WT protein but not FLNa-Ala2152 protein (Fig. 4C). These results suggest that our phospho-FLNa antibody specifically recognizes FLNa when it is phosphorylated on Ser2152.
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Wild-type RSK potentiates EGF-stimulated FLNa phosphorylation, while kinase-dead RSK blocks phosphorylation. In order to provide further evidence that RSK mediates FLNa Ser2152 phosphorylation in vivo, we overexpressed RSK in HEK 293E cells and monitored FLNa phosphorylation by 32P labeling and Western blotting. In vivo labeling was performed as previously described on HEK 293E cells cotransfected with myc-tagged FLNa and HA-tagged RSK-WT, kinase-inactive RSK-K112/464R, or vector. Labeled FLNa protein was immunoprecipitated from extracts using myc antibody. Overexpression of RSK-WT led to a significant increase in phosphate incorporation by FLNa (Fig. 6A, compare lanes 5 and 2). In contrast, overexpression of RSK-K112/464R reduced EGF-induced FLNa phosphorylation to basal level (Fig. 6A, compare lanes 7 and 4). The effect of RSK on FLNa Ser2152 phosphorylation was also investigated using the phospho-FLNa antibody. HEK 293E cells were transfected with FLNa and RSK constructs, serum starved, and stimulated with EGF (with and without UO126 pretreatment). myc immunoprecipitates were analyzed by Western blotting using phospho-FLNa antibody. Similar to the results of the in vivo labeling experiment, overexpression of RSK-WT greatly enhanced EGF-stimulated FLNa Ser2152 phosphorylation (Fig. 6B, compare lanes 5 and 2). In addition, phosphorylation was completely UO126 sensitive (Fig. 6B, lane 6). Overexpression of RSK-K112/464R, on the other hand, antagonized EGF-stimulated FLNa Ser2152 phosphorylation (Fig. 6B, lane 8). These results strengthen our hypothesis that RSK is a physiological FLNa Ser2152 kinase.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Other kinases, such as PKA and Pak1, have also been shown to phosphorylate FLNa protein. Several groups have demonstrated that treatment of cells with cAMP or agonists that increase intracellular cAMP levels activates PKA and stimulates FLNa phosphorylation in platelets, smooth muscle cells, and fibroblasts (4, 5, 9, 39, 41). The in vivo PKA phosphorylation site(s) was not identified in these studies. The effect of PKA-mediated FLNa phosphorylation on FLNa function is somewhat controversial. One group reported that PKA phosphorylation of FLNa in vitro confers resistance to cleavage by calpain (5). Another study concluded that phosphorylation of FLNa is required for the ability of FLNa to cross-link F-actin in vitro (42). Ser2152 on FLNa has been identified as a PKA phosphorylation site in vitro (16); however, we present the first evidence that Ser2152 phosphorylation is stimulated in vivo after PKA activation (Fig. 5A, lane 5).
Pak1 has also been shown to phosphorylate FLNa on Ser2152 in vitro (36). Treatment of MCF-7 cells with heregulin increased FLNa phosphorylation, and overexpression of kinase-inactive Pak1 or the Pak1 autoinhibitory domain antagonized this phosphorylation. Activated Pak1 (Thr423-to-Glu mutant) stimulated ruffling in FLNa-expressing melanoma cells but not in FLNa-deficient cells, suggesting that FLNa is required for Pak1-mediated ruffle formation. In addition, ruffling was induced in FLNa-deficient melanoma cells expressing activated Pak1 and FLNa, but not in cells expressing activated Pak1 and FLNa-Ala2152 mutant (36). These data provide convincing evidence that phosphorylation of FLNa on Ser2152 regulates actin cytoskeletal changes.
Our data strongly support the conclusion that RSK phosphorylates FLNa on Ser2152 in vivo. Although Pak1 phosphorylates FLNa on the same site, we believe that RSK and Pak1 are likely activated under different conditions. First, we demonstrated that EGF-stimulated FLNa phosphorylation is sensitive to the MEK inhibitor UO126. UO126 specifically inhibits MEK1/2 and has not been reported to affect Pak1 activation. Second, whereas expression of wild-type RSK greatly enhanced EGF-stimulated FLNa Ser2152 phosphorylation, kinase-inactive RSK inhibited phosphorylation. Third, FLNa Ser2152 phosphorylation was greatly diminished by specific inhibition of endogenous RSK1 and/or RSK2 expression using siRNA oligonucleotides. Finally, inhibition of MAP kinase signaling to RSK significantly reduced FLNa-dependent migration of human melanoma cells in response to EGF. Taken together, these results strengthen our hypothesis that RSK is a physiological FLNa kinase.
What are the downstream events controlled by phosphorylation of FLNa on Ser2152? FLNa Ser2152 phosphorylation may directly stimulate binding or cross-linking of actin filaments. Preliminary data suggest, however, that these functions are not affected by phosphorylation. FLNa-Ala2152 proteins retained the ability to bind actin in sedimentation assays and to cross-link actin filaments into three-dimensional gels in vitro (F. Nakamura, personal communication).
Alternatively, FLNa Ser2152 phosphorylation may regulate binding to and/or activity of one of its many binding partners. To date, more than 20 proteins have been reported to interact with FLNa (reviewed by Stossel et al. [28]). Two FLNa binding proteins are the Rho family GTPases and ß integrins. Given that Rho family GTPases play a major role in the regulation of actin cytoskeletal dynamics, their connection to FLNa is intriguing. FLNa interacted with the small GTPase RalA in a GTP-dependent manner, and induction of RalA-induced filopodia did not occur in FLNa-deficient cells, indicating that FLNa protein is necessary for the formation of membrane protrusions (22). ß integrins have also been shown to interact with FLNa. Interestingly, valine-to-isoleucine substitutions in the cytoplasmic tail of ß1A integrin caused ß1A integrin to bind FLNa with greater affinity. Furthermore, increased binding of ß integrin to FLNa inhibited cell migration (3). Whether FLNa Ser2152 phosphorylation influences the binding of FLNa to GTPases and ß integrins has yet to be determined.
In conclusion, we provide the first evidence that RSK mediates phosphorylation of FLNa on Ser2152, a site that regulates the biological function of FLNa. Exploring this connection between RSK and FLNa will give us a better understanding of how activation of the Ras-MAP kinase pathway elicits actin cytoskeletal changes.
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ACKNOWLEDGMENTS |
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This work was supported in part by grant CA09595 from NCI/NIH (M.S.W.), grants RO1 CA46595 and RO1 GM51405 from the NIH (J.B.), USPHS grant HL19429, and the American Cancer Society (T.P.S.).
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FOOTNOTES |
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REFERENCES |
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