Article |
Address correspondence to T. Hunter, Molecular and Cell Biology Laboratory, The Salk Institute for Biological Sciences, 10010 North Torrey Pines Rd., La Jolla, CA 92037-1099. Tel.: (858) 453-4100, ext. 1385. Fax: (858) 457-4765. email: hunter{at}salk.edu
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Abstract |
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Key Words: Abl/Arg/ fibroblasts; fibronectin adhesion; F-actin microspikes; cytoskeleton; Nck
Abbreviations used in this paper: FN, fibronectin; KD, kinase-deficient; MEF, mouse embryo fibroblast; PH, pleckstrin homology; pTyr, phosphotyrosine; SH2, Src homology 2 domain; STI, signal transduction inhibitor; TLC, thin layer cellulose; WT, wild-type.
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Introduction |
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Actin polymerization provides the force to drive filopodial membrane protrusions (Mallavarapu and Mitchison, 1999). Current data suggest that filopodia arise from the elongation, convergence, and bundling of preexisting actin filaments in the lamellipod (Svitkina et al., 2003). WASP family proteins regulate filopodia by recruiting the Arp2/3 complex, which nucleates actin polymerization (Pollard and Borisy, 2003). Although activators of WASP have been identified (e.g., Rho family GTPases, PIP2, and SH3 adaptor proteins), the signaling pathways controlling the formation, maintenance, and termination of filopodia during specific cellular processes remain largely undefined.
Recent observations suggest that c-Abl tyrosine kinase can regulate filopodia formation. c-Abl extends the duration of membrane protrusive activity during the early stages of cell spreading on fibronectin (FN), resulting in increased numbers of filopodia and F-actin microspikes (Woodring et al., 2002), which are precursors to filopodia (Kozma et al., 1995; Svitkina et al., 2003). Active Abl also increases the length of neurites and the number of F-actinrich branches on embryonic cortical neurons grown on laminin (Zukerberg et al., 2000; Woodring et al., 2002). The effect of c-Abl on neurite branching and persistence of filopodia during cell spreading is dependent on c-Abl tyrosine kinase activity, implicating c-Abl substrates in these processes. Indeed, cell adhesion to ECM components, such as FN, collagen, vitronectin, and laminin, increases c-Abl activity (Lewis et al., 1996; Frasca et al., 2001; Woodring et al., 2001). Several collaborators that synergize with c-Abl in F-actinmediated processes have been suggested (for review see Lanier and Gertler, 2000; Woodring et al., 2003; Hernandez et al., 2004); however, essential components downstream of c-Abl remain elusive.
Here, we investigated the c-Abl substrates involved in the persistence of F-actin microspikes and filopodia during fibroblast spreading. Using an unbiased biochemical strategy to detect substrates combined with a genetic strategy using fibroblasts from knockout mouse embryos, we found that the p62 docking protein (Dok1) is an essential substrate for c-Abl in the induction of filopodia during FN-stimulated cell spreading. c-Abl and Dok1 are both present in filopodia, supporting a role for both proteins in filopodia function. The effects of c-Abl on filopodia appear to be mediated by the phosphorylation of Y361 of Dok1, which promotes association of Dok1 and Nck, a Src homology 2 domain (SH2)/SH3 adaptor protein that can trigger localized actin polymerization (Campellone et al., 2004; Rivera et al., 2004).
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Results |
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The p62 docking protein Dok1 binds Abl-SH2 when c-Abl is active
Because Dok1 (downstream of tyrosine kinases) is a 62-kD protein that is tyrosine phosphorylated in Bcr-Abltransformed cells (Wisniewski et al., 1994; Carpino et al., 1997; Yamanashi and Baltimore, 1997), we checked if Dok1 was a component of the 60-kD pTyr-proteins isolated with Abl-SH2. Using antibodies specific for Dok1 and lysates from latrunculin-treated suspended wild-type (WT; Dok1+/+) or Dok1/ MEFs, we found that Abl-SH2 did isolate Dok1 (Fig. 2 A). The level of
60-kD pTyr-proteins isolated with Abl-SH2 was significantly decreased using lysates from Dok1/ MEFs compared with Dok1+/+ MEFs (unpublished data), indicating that Dok1 is likely a component of pTyr-p60. To explore this possibility further, we performed side-by-side analysis of Abl-SH2 pull downs and Dok1 immunoprecipitates (Fig. 2 B). The levels of pTyr on Dok1 correlated with the amount of pTyr-p60 isolated with Abl-SH2. Direct evidence that Dok1 was a component of the
60-kD pTyr-proteins isolated with Abl-SH2 was obtained using Dok1 antibodies to immunoprecipitate Dok1 from proteins that were eluted from Abl-SH2 (Fig. 2 C). We also found that the pTyr on Dok1 was increased during latrunculin treatment and fibroblast spreading, and this increase was largely blocked by STI571 (Fig. 2 D). These data indicate that Dok1 is a component of the
60-kD pTyr-proteins isolated with Abl-SH2 and support the conclusion that Dok1 is a substrate for c-Abl in spreading MEFs.
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Results obtained with the Y361F Dok1 mutant suggested that Y361 is phosphorylated by c-Abl in MEFs spreading on FN (Fig. 3, A and D). Using the pY361 Dok1 antibody, we found that FN stimulation increased the pY361 content of Dok1 relative to that of Dok1 from suspended cells (Fig. 4 C, middle). STI571 largely blocked the stimulatory effect of FN, and pY361 was not detected on Y361F Dok1. We also detected pY361 on endogenous Dok1 from spreading Abl[Abl/Arg/] MEFs, whereas very little pY361 was detected on Dok1 from pMSCV[Abl/Arg/] MEFs, and no signal was detected in an immunoprecipitate from Dok1/ cells, as expected (Fig. 4 C, right). Based on the results shown in Figs. 3 and 4, we conclude that c-Abl induces phosphorylation of Dok1 at Y361 in MEFs spreading on FN.
Tyrosine phosphorylation of Dok1 at Y361 coincides with F-actin microspike formation
c-Abl activity prolongs the persistence of exploratory filopodia and F-actin microspikes during the early stages of cell spreading (Woodring et al., 2002). In addition, deletion of four amino acids at the extreme COOH terminus generates a form of Abl (c-Abl*) that remains active in suspended cells (Woodring et al., 2001). Consistent with these two observations, suspended Abl/ cells expressing WT c-Abl contained few to no microspikes (Fig. 5 A, top) and basal c-Abl activity, whereas suspended cells expressing c-Abl* contained numerous F-actin microspikes (Fig. 5 A, bottom). To explore Abl substrates involved in c-Ablinduced microspike formation in suspended cells, we used Abl-SH2 to isolate Abl substrates from each of these cell lines (Fig. 5 B). After FN stimulation, Abl-SH2 isolated similar amounts of pTyr-containing proteins from the lysates of c-Abl and c-Abl*expressing cells (Fig. 5 B, lanes 1 and 3). In suspended cells, Abl-SH2 isolated more pTyr-p62 from lysates of cells expressing c-Abl* than from lysates of cells expressing WT c-Abl (Fig. 5 B, lanes 2 and 4). Thus, there was an increase in F-actin microspikes in detached cells expressing active Abl and a corresponding increase in the quantity of pTyr-p62 (Dok1) isolated using Abl-SH2.
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Phosphorylation of Y361 can promote Dok1 binding to the SH2 domains of p120RasGAP (p120) and the Nck adaptor protein (Tang et al., 1997; Murakami et al., 2002; Shah and Shokat, 2002). Whereas association with Nck involves the phosphorylation of Y361 of Dok1, association with p120 appears to involve phosphorylation of five additional tyrosines (Murakami et al., 2002). We did not detect an association between p120 and Dok1 in lysates of spreading MEFs, nor did we detect any change in the pTyr content of p190RhoGAP, which associates with p120 (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200312171/DC1). In contrast, we did detect endogenous Nck in immunoprecipitates of Dok1 using lysates from spreading Abl[Abl/Arg/] MEFs (Fig. 9 A, right). WT Dok1 was present in Nck immunoprecipitates, whereas Y361F Dok1 was not (Fig. 9 A, left). Correspondingly, the Dok1 that coimmunoprecipitated with Nck was phosphorylated at Y361 (Fig. 9 A). Coimmunoprecipitation of endogenous Dok1 and Nck was detected in spreading MEFs (Fig. 9 B, lanes 6 and 7), and this association was enhanced in cell lines stably expressing elevated levels of Dok1 (Fig. 9 B, lanes 4 and 5; note that lanes 7 and 8 were exposed to film longer than lanes 46). In addition, the interaction between the Dok1 and Nck was reduced in STI571-treated spreading MEFs (Fig. 9 B, lane 8) and in suspended MEFs (Fig. 9 B, lane 9). Together these data strongly imply that the association between Dok1 and Nck is stimulated by c-Abl.
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Results from cotransfection experiments suggest that Nck1, Dok1, and Abl may form a ternary complex in cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200312171/DC1). The association of all three proteins was significantly decreased when Y361F Dok1 was used instead of WT Dok1, when KD Abl was used instead of WT Abl, or when cells were treated with STI571. Thus, a signaling complex containing Abl, Dok1, and Nck may form in cells, and its formation is dependent on Abl activity and phosphorylation of Y361 of Dok1. In summary, our data suggest that c-Abl transduces signals to actin at the cell periphery by phosphorylating Y361 of Dok1 and recruiting Nck.
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Discussion |
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Several publications support a role for Abl kinases in regulation of F-actin polymerization in cells (for review see Lanier and Gertler, 2000; Woodring et al., 2003; Hernandez et al., 2004). Overall, it appears that c-Abl stimulates filopodia formation, membrane ruffling, and neurite extension, but inhibits chemotaxis (Frasca et al., 2001; Kain and Klemke, 2001). At first glance, this finding may appear as a paradox: how can c-Abl increase actin polymerization yet inhibit cell movement? This enigma might be resolved by considering the function of filopodia. Filopodia (and membrane ruffles) are present on cells that have paused to explore their environment (Machesky 2000). If a cell remains in the exploratory phase for an extended time, directed migration may progress slower. We (and others) have found that primary cultured Abl-null cells spread faster than the Abl-reconstituted or WT cells (Sheetz, M., personal communication; unpublished data). Thus, c-Abl may slow migration or spreading by promoting formation of actin structures that permit a cell to pause and explore the surroundings before moving toward a target.
Based on overexpression studies, it has been suggested that Dok1 and Dok2 regulate cell motility (Noguchi et al., 1999; Hosooka et al., 2001; Master et al., 2001). Dok1 may also affect plasma membrane ruffling because GFP-Dok1 undergoes a PH domaindependent translocation to the plasma membrane in response to PDGF (Zhao et al., 2001) and is found in the cytoskeletal and membrane fractions of cells (Noguchi et al., 1999; Hosooka et al., 2001; Zhao et al., 2001). The PH domain of Dok1 is required for tyrosine phosphorylation of Dok1 (Zhao et al., 2001), suggesting that the tyrosine kinases that phosphorylate Dok1 localize at or near the plasma membrane. These observations are consistent with Dok1 regulating localized F-actinmediated processes.
There are five Dok family members (Dok1Dok5). We detected expression of Dok1 and Dok2 in the MEFs used in this paper (unpublished data). The COOH-terminal YXXP Abl consensus phosphorylation sites are not conserved in Dok3, Dok4, or Dok5, and these proteins appear to have functions distinct from Dok1 and Dok2 (Cong et al., 1999; Grimm et al., 2001). Recently, it was reported that pTyr-Dok2 can increase Abl kinase activity when coexpressed with c-Abl in HEK293T cells, presumably by binding to and displacing both the SH2 and SH3 domains of c-Abl (Master et al., 2003). This finding raises the interesting possibility that phosphorylation of Dok2 might amplify the effect of c-Abl; however, we did not detect pTyr on Dok2 immunoprecipitated from MEFs plated on FN (unpublished data), so it is unlikely that Dok2 activates c-Abl or mediates the effects of c-Abl in FN-stimulated MEFs. In our analysis, we found that, similar to the overexpression of other adaptor or docking proteins (e.g., Nck, Crk, Abi, and Dok2), Dok1 also increased the pTyr content of c-Abl when coexpressed in HEK293T cells (Woodring et al., 2003; unpublished data). This effect was not dependent on Y361 phosphorylation because the Y361F and Y295F/Y361F Dok1 mutants also increased the pTyr content of c-Abl to a similar level as WT Dok1. This finding suggests that Dok1 may activate c-Abl through an intermediate protein or by a binding mechanism that does not require Y361 of Dok1 in HEK293T cells. Although these overexpression studies are interesting, the results obtained may not be applicable to all systems. For example, the endogenous c-Abl was still activated during FN cell spreading in Dok1/ MEFs (unpublished data). For these reasons, we believe that neither Dok2 nor activation of c-Abl by Dok1 is responsible for the effect of c-Abl on filopodia in spreading MEFs.
Dok1 has several I/LYXXP consensus sites, including LY295AEP, LY314SDP, IY361DEP, and LY376DLP. Although c-Abl can phosphorylate several tyrosine residues of Dok1 in vitro (Fig. 3 B), it appears that c-Abl predominantly phosphorylates Y361 in spreading MEFs. Y361 of Dok1 is also a substrate for Src in v-Src transformed cells (Shah and Shokat, 2002) and in CHO cells overexpressing the insulin receptor (Noguchi et al., 1999). Src may contribute to the pTyr of Dok1 in MEFs because some Dok1 pTyr remains when cells are treated with STI571 (Fig. 2 D). Src and Abl may even act cooperatively to stimulate pTyr-Dok1, as has been suggested previously in MEFs stimulated with PDGF (Plattner et al., 1999).
The pTyr-Dok1 isolated from spreading cells associated with purified Abl-SH2 domain (Fig. 1 B). The binding of Abl substrates to the Abl-SH2 domain can promote processive phosphorylation of Abl substrates (Duyster et al., 1995; Mayer et al., 1995). It is possible that the tyrosine phosphorylation of Dok1 may allow Dok1 to stably associate with the Abl-SH2 domain to promote further tyrosine phosphorylation of Dok1. Because Dok1 can oligomerize through its pTyr binding domain (Songyang et al., 2001), c-Abl could also promote phosphorylation of Y361 of other Dok1 molecules in an oligomer. However, although Abl-SH2 did bind Dok1 from spreading MEFs, we were unable to detect coimmunoprecipitation of the endogenous proteins, suggesting that their association is transient and/or that minor populations of Dok1 and c-Abl associate, perhaps those molecules localized in filopodia.
Dok1 appears to be a multifunctional signaling protein with reported roles in cell growth, transformation, axonal guidance, and immune response (Holland et al., 1997; Yamanashi et al., 2000; Di Cristofano et al., 2001; Songyang et al., 2001; Zhao et al., 2001; Murakami et al., 2002). We have uncovered a new function of Dok1 in regulating F-actin microspikes and filopodia during fibroblast spreading. Notably, instead of using overexpression and dominant-negative approaches, which most other investigators have used to study Abl or Dok, we have used a genetic strategy with cells from knockout mice reexpressing levels of protein that are comparable to endogenous levels. We have defined a novel signaling pathway downstream of c-Abl leading to F-actin assembly and filopodia formation. Precisely localized c-Abl signaling at the tips of filopodia may be important for regulating the localized dynamics of filopodia extension, retraction, or attachment to the ECM to mediate the exploratory process. Further studies are required to determine if c-Abl, pY361Dok1, and Nck can fine-tune cell guidance by modulating cell exploration in vivo during embryonic development, axon path finding, or wound healing.
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Materials and methods |
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Antibodies
pTyr (4G10) and Myc (9E10) mAbs were purchased from Upstate Biotechnology; Dok1 mAb (A3) was purchased from Santa Cruz Biotechnology, Inc.; HA mAb (12CA5) was purchased from Babco; Flag mAb was obtained from Sigma-Aldrich; and Abl mAb (Ab3) was obtained from Oncogene Sciences. Abl mAb (8E9) was a gift from J.Y.J. Wang. Nck (5547) and Dok1 (6043) rabbit antibodies were generated by J. Meisenhelder and N. Carter (The Salk Institute, La Jolla, CA) using GST-Nck1 and GST-Dok1 as immunogens. 5547 immunoprecipitates Nck1/2 but recognizes only Nck1 on immunoblots. Affinity-purified pY361Dok1 rabbit polyclonal antibodies were a gift from M. Lakkis (Biosource International, Hopkinton, MA), and pY412-Abl rabbit polyclonal antibodies were purchased from Biosource International. Secondary HRP-conjugated antibodies used for ECL were obtained from Amersham Biosciences. Secondary antibodies (Texas red, FITC, or Alexa Fluor 488 conjugates) used for immunofluorescence were obtained from Southern Biotechnology Associates, Inc. or Molecular Probes.
Cell culture
The immortal Abl/Arg-deficient fibroblast cell line Abl/Arg/ was generated from E9 mouse embryos by A. Koleske (Yale University, New Haven, CT). The Dok1/ primary MEFs (Di Cristofano et al., 2001) and the Nck1/ Nck2/ MEFs (Gruenheid et al., 2001) were gifts from P.P. Pandolfi and T. Pawson, respectively. Cells were maintained in high glucose DME supplemented with 10% FBS (Gemini Bioproducts), L-glutamine, and antibiotics. Early passage MEFs were critical in obtaining the results reported here because MEFs were altered when grown in culture for longer than 48 wk. Latrunculin A (Molecular Probes) was added to serum-free media at a concentration of 1 µM for a 2-h pretreatment before cell detachment. Cells were held in suspension in the same media for an additional 4060 min. The Abl inhibitor STI571 (GleevecTM; Novartis) was added to growth media at a concentration of 15 µM, and the Src inhibitor SU6656 (Sugen) was used at a concentration of 0.51 µM for 18 h. The latrunculin and kinase inhibitors were also present in the media throughout the suspension and reattachment period. Control cells were incubated with DMSO, the vehicle for inhibitors.
Biochemistry
Fibroblast experiments involving cell detachment, FN stimulation, and immunoprecipitation were performed as described previously (Woodring et al., 2001) using 1% Triton X-100 lysis buffer and brief sonication to prepare cellular lysates. GST-Abl-SH2 experiments were performed in the presence of 0.5 M NaCl to reduce nonspecific binding. Purified GST or GST-Abl-SH2 protein was cross-linked to glutathione-Sepharose using dimethyl-pimelimidate (50 µg of protein coupled to 10 µl of beads per pull down). 200 µg of cell lysate protein were used for pull downs or immunoprecipitation, while 23 mg of protein were used for coimmunoprecipitation experiments.
PAK1 kinase assays were performed as described previously (Zhou et al., 2003). For in vitro Abl kinase assays, c-Abl was purified via FLAG antibody chromatography (Woodring et al., 2001) and used to phosphorylate GST-Dok1 protein in vitro. GST-Dok1 was isolated with glutathione-Sepharose before SDS-PAGE, and the 32P-labeled Dok1 was processed for phosphotryptic analysis (Shah and Shokat, 2002).
For in vivo phosphotryptic peptide mapping, Dok1 was coexpressed with c-Abl in HEK293T cells. After transfection, cells were labeled with 1 mCi/ml 32P-orthophosphate overnight, and then lysed in RIPA buffer. Dok1 was immunoprecipitated (HA) and processed for phosphotryptic peptide mapping and phosphoamino acid analysis as described previously (Meisenhelder et al., 1999). Tryptic phosphopeptides were separated on thin layer cellulose (TLC) plates (EM Science) by electrophoresis (pH 1.9, 1.5 kV, 35 min) and ascending chromatography (phosphochromo buffer, 18 h). Synthetic peptides corresponding to the tryptic peptides containing mouse Dok1 pTyr361 were synthesized on an ABI 432A Synergy peptide synthesizer: (LTDSKEDPIpYDEPEGLAPAPPRGLY (peptide 1 + GLY) and EDPIpYDEPEGLAPAPPRGLY (peptide 2 + GLY). When digested with 110 µg TPCK trypsin these peptides yielded peptides 1 and 2 (underlined). Very little peptide 2 was generated from in vitro digestion of synthetic peptide 1. When mixed together and added to the 32P-Dok1 phosphotryptic samples, the purified synthetic peptides were found to comigrate with the two pTyr361 spots in maps of Dok1 isolated from 32P-labeled cells.
Immunofluorescence staining
Cells were prepared as indicated, fixed in suspension, or plated onto coverslips coated with 10 µg/ml purified human FN (Calbiochem) for 2035 min at 37°C as described previously (Woodring et al., 2002). For Abl/Dok costaining experiments, Abl (mouse mAb 8E9) was directly labeled with Alexa Fluor 546 according to kit instructions (Molecular Probes). Fluorescent reagents and antibodies are indicated in the figure legends. Wide-field microscopy was performed using the 60x objective (Olympus). Applied Precision software (DeltaVision) was used to deconvolve z-section series of images. For quantification of filopodia and peripheral actin microspikes during cell spreading, we counted the number of filopodia and microspikes present on cells visualized with phalloidin, including all visible protruding microspikes >1 µm. Random fields of cells (n = 2001,000) were selected on coverslips using Applied Precision software. Statistical analysis of the data was performed using Microsoft Excel software.
Online supplemental material
Three supplemental figures address the mechanism by which pY361-Dok1 induces filopodia during cell spreading. Data in Figs. S1 and S3 suggest that p120RasGAP, p190RhoGAP, and PAK1 do not lie downstream of pY361-Dok1, whereas data in Fig. S2 suggest that pY361-Dok1 may form a ternary complex with both c-Abl and Nck1 to promote filopodia in spreading MEFs. Fig. S1 shows that Dok1 did not coimmunoprecipitate with p120RasGAP in spreading MEFs (A) and that tyrosine phosphorylation of p190RhoGAP was similar among Abl/Arg/, Abl-, and KD-reconstituted MEFs spreading on FN (B). Fig. S2 shows that pY361-Dok1 coimmunoprecipitated with Nck1 and c-Abl when all three proteins were transiently expressed in 293T cells. Nck1 (A) and c-Abl (B) immunoprecipitates are shown. Fig. S3 shows PAK1 kinase activity was similar among WT, Abl/Arg/, and Abl-reconstituted MEFs spreading on FN. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200312171.DC1.
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Acknowledgments |
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This work was supported by a postdoctoral fellowship from the National Cancer Institute, National Institutes of Health (NIH) grant CA76710 (to P.J. Woodring), and NIH grants HL57900 (to J.Y.J. Wang) and CA14195 and CA82863 (to T. Hunter). J.Y.J. Wang is the Herbert Stern Endowed Chair of Biology, University of California, San Diego, and T. Hunter is a Frank and Else Schilling American Cancer Society Research Professor.
Submitted: 26 December 2003
Accepted: 16 April 2004
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