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Epidermal Growth Factor Modulates Tyrosine Phosphorylation of p130Cas

INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3′-KINASE AND ACTIN CYTOSKELETON*
  • Marja Ojaniemi
    Footnotes
    Affiliations
    La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037
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  • Kristiina Vuori
    Correspondence
    To whom correspondence should be addressed: La Jolla Cancer Research Center, The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3100; Fax: 619-646-3199
    Affiliations
    La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant CA 72560 (to K. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by a fellowship from the American-Scandinavian Foundation.
Open AccessPublished:October 10, 1997DOI:https://doi.org/10.1074/jbc.272.41.25993
      Epidermal growth factor (EGF) treatment of Rat-1 cells expressing human EGF receptor results in the modification of the tyrosine phosphorylation of the p130 Crk-associated substrate (Cas), a novel signaling molecule residing in focal adhesions. At low, mitogenic concentrations (<10 ng/ml), EGF treatment induced a rapid and transient tyrosine phosphorylation of Cas and promoted the formation of a Cas-adapter protein Crk complex in intact cells. The increase in tyrosine phosphorylation of Cas paralleled an increase in the cellular content of actin stress fibers and occurred via a pathway that depended on the integrity of the cytoskeleton. Further, phosphatidylinositol 3′-kinase activity was found to be required for the EGF-stimulated Cas phosphorylation and actin polymerization. At high concentrations (>30 ng/ml), EGF treatment resulted in the tyrosine dephosphorylation of Cas in a time-dependent manner with a concomitant decrease in the length and number of actin stress fibers. Thus, Cas exhibits an unusual bell-shaped dose-response curve in response to EGF stimulation. These results demonstrate a novel signaling role for EGF in inducing changes in tyrosine phosphorylation of Cas and Cas-Crk complex formation and suggest that Cas could be a signaling component in EGF-mediated signal transduction.
      The binding of growth factors to their receptors results in receptor dimerization and subsequent transphosphorylation of specific tyrosine residues in the cytoplasmic domains of the receptors. The phosphorylated residues in turn serve as attachment sites for cellular Src homology 2 (SH2)
      The abbreviations used are: SH, Src homology; Cas, p130 Crk-associated substrate; EGF, epidermal growth factor; FAK, focal adhesion kinase; PBS, phosphate-buffered saline; PI, phosphatidylinositol; PDGF, platelet-derived growth factor; TRITC, carboxytetramethylrhodamine isothiocyanate.
      1The abbreviations used are: SH, Src homology; Cas, p130 Crk-associated substrate; EGF, epidermal growth factor; FAK, focal adhesion kinase; PBS, phosphate-buffered saline; PI, phosphatidylinositol; PDGF, platelet-derived growth factor; TRITC, carboxytetramethylrhodamine isothiocyanate.
      domain-containing signaling proteins; formation of these signal transduction complexes coordinates the multiple intracellular programs that initiate various changes in cell proliferation and differentiation (
      • Schlessinger J.
      • Ullrich A.
      ). In addition to the mitogenic effects, many growth factors have been reported to induce changes in cell morphology. Upon stimulation of cells with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin, the cells exhibit rapid membrane ruffling; this is brought about by a reorganization of the actin microfilament system, which includes extensive local actin polymerization in the cell cortex (
      • Ridley A.J.
      • Hall A.
      ). A cascade of small GTP-binding proteins of the Rho family has been implicated in mitogen-stimulated changes in the actin cytoskeleton. In particular, Rac1 is responsible for membrane ruffling and lamellipodia formation, whereas activation of RhoA mediates the assembly of focal adhesions and stress fibers at a later time point following growth factor stimulation (
      • Ridley A.J.
      • Hall A.
      ,
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ,
      • Nobes C.D.
      • Hall A.
      ,
      • Hall A.
      ). Recent experiments demonstrate that activation of phosphatidylinositol 3′-kinase (PI 3-kinase) upon PDGF stimulation is required for Rac1 activation and subsequent membrane ruffling (
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.K.
      • Heldin C.H.
      • Mori S.
      • Claesson-Welsh L.
      ,
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Wymann M.
      • Arcaro A.
      ,
      • Hawkins P.T.
      • Eguinoa A.
      • Qiu R.G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • Stevens L.
      ).
      The mitogen-induced changes in actin cytoskeleton are accompanied by dramatic changes in tyrosine phosphorylation of several proteins present in focal adhesions. Focal adhesions are regions of a cell in direct contact with the extracellular matrix, providing anchorage sites for actin stress fibers and forming a link between the extracellular matrix and the actin cytoskeleton via the integrin family of cell surface receptors (
      • Burridge K.
      • Fath K.
      • Kelly T.
      • Nuckolls G.
      • Turner C.
      ,
      • Jockusch B.M.
      • Bubeck P.
      • Giehl K.
      • Kroemker M.
      • Moschner J.
      • Rothkegel M.
      • Rüdiger M.
      • Schlüter K.
      • Stanke G.
      • Winkler J.
      ). Stimulation of cells with low, mitogenic concentrations of PDGF (1–10 ng/ml) results in PI 3-kinase activation, membrane ruffle formation, and simultaneous tyrosine phosphorylation of focal adhesion proteins p125Fak (FAK), paxillin, and p130Cas (Cas) (
      • Rankin S.
      • Rozengurt E.
      ,
      • Abedi H.
      • Dawes K.E.
      • Zachary I.
      ,
      • Casamassima A.
      • Rozengurt E.
      ). Treatment of cells with wortmannin, a PI 3-kinase inhibitor, or with cytochalasin D, an agent that prevents actin polymerization, inhibits PDGF-stimulated tyrosine phosphorylation of focal adhesion proteins (
      • Casamassima A.
      • Rozengurt E.
      ,
      • Rankin S.
      • Hooshmand-Rad R.
      • Claesson-Welsh L.
      • Rozengurt E.
      ). On the other hand, stimulation of cells with insulin or with high concentrations of PDGF (30 ng/ml) results in tyrosine dephosphorylation of the focal adhesion proteins with a concomitant decrease in the length and number of actin stress fibers (
      • Rankin S.
      • Rozengurt E.
      ,
      • Casamassima A.
      • Rozengurt E.
      ,
      • Pillay T.S.
      • Sasaoka T.
      • Olefsky J.M.
      ,
      • Yamauchi K.
      • Milarski K.L.
      • Saltiel A.R.
      • Pessin J.E.
      ,
      • Knight J.B.
      • Yamauchi K.
      • Pessin J.E.
      ). These data have established a novel role for PDGF and insulin in modulating the signaling molecules residing in the focal adhesions.
      p130Cas (Cas, Crk-associatedsubstrate) is a novel, 130-kDa focal adhesion protein that was originally identified as a prominent tyrosine-phosphorylated protein in v-Src- and v-Crk-transformed cells. Molecular cloning of Cas revealed a docking protein that contains an SH3 domain, proline-rich regions, and a cluster of 15 putative SH2-binding motifs (
      • Sakai R.
      • Iwamatsu A.
      • Hirano N.
      • Ogawa S.
      • Tanaka T.
      • Mano H.
      • Yazaki Y.
      • Hirai H.
      ). The unique structure of Cas suggests a role in assembling multiprotein signaling complexes. Indeed, Cas has been reported to become tyrosine-phosphorylated in response to a number of different stimuli, many of which affect the assembly of focal adhesions and actin stress fibers. These stimuli include integrin-mediated cell adhesion (
      • Petch L.A.
      • Bockholt S.M.
      • Bouton A.
      • Parsons J.T.
      • Burridge K.
      ,
      • Vuori K.
      • Ruoslahti E.
      ,
      • Nojima Y.
      • Morino N.
      • Mimura T.
      • Hamasaki K.
      • Furuya H.
      • Sakai R.
      • Sato T.
      • Tachibana K.
      • Morimoto C.
      • Yazaki Y.
      • Hirai H.
      ), ligation of the B-cell receptor and interleukin-8 receptor (
      • Ingham R.J.
      • Krebs D.L.
      • Barbazuk S.M.
      • Turck C.W.
      • Hirai H.
      • Matsuda M.
      • Gold M.R.
      ,
      • Schraw W.
      • Richmond A.
      ), and stimulation of cells with nerve growth factor (
      • Ribon V.
      • Saltiel A.R.
      ), bombesin and other neuropeptides, phorbol esters, bioactive lipids, and PDGF (
      • Casamassima A.
      • Rozengurt E.
      ). Following tyrosine phosphorylation, Cas interacts with a number of SH2-containing signaling molecules, such as the adapter proteins Crk and Nck, possibly recruiting these molecules to focal adhesions (
      • Vuori K.
      • Hirai H.
      • Aizawa S.
      • Ruoslahti E.
      ,
      • Schlaepfer D.D.
      • Broome M.A.
      • Hunter T.
      ).
      Previous studies have demonstrated that stimulation of cells with EGF results in dramatic changes in cell morphology (
      • Peppelenbosch M.P.
      • Tertoolen L.G.
      • Hage W.J.
      • de Laat S.W.
      ); in the present study, we report that stimulation of cells with EGF induces changes in actin cytoskeleton with a concomitant modulation of the tyrosine phosphorylation of Cas. Similar to the PDGF response (
      • Casamassima A.
      • Rozengurt E.
      ), we found that the phosphorylation of Cas exhibits an unusual bell-shaped dose-response curve upon EGF stimulation and that the tyrosine phosphorylation of Cas by low concentrations of EGF requires the integrity of the actin cytoskeleton and PI 3-kinase activity. Furthermore, low concentrations of EGF promote the formation of a Cas-Crk complex in intact cells. These results identify a novel signaling role for EGF and support the notion that Cas could play a role in mitogen-mediated signal transduction.

      DISCUSSION

      Although the exact role of p130Cas (Cas) in growth factor signaling has not been elucidated to date, it appears to be an important integration point for multiple signaling pathways. In the present report, we demonstrate that in contrast to many growth factors, EGF stimulation of fibroblasts results in an unusual bell-shaped dose-response curve of tyrosine phosphorylation of Cas. The changes in tyrosine phosphorylation of Cas paralleled EGF-induced changes in the polymerization content of actin and were found to result in changes in the complex formation between Cas and the adapter protein Crk. These results suggest a novel function for Cas as a signaling component in EGF-mediated signal transduction.
      One of the most striking findings reported here is that the tyrosine phosphorylation of Cas in response to low concentrations of EGF is dramatically different from that induced by high concentrations of the growth factor; low concentrations (0.5–5 ng/ml) of EGF resulted in increased tyrosine phosphorylation of Cas, whereas higher concentrations either had no effect or induced a marked dephosphorylation of Cas (concentrations >30 ng/ml EGF). This bell-shaped dose-response curve was observed in three different cell lines (RatER, A431, and Rat-1 cells) expressing vastly different amounts of EGF receptor on the cell surface. To our knowledge, none of the previous studies that examined EGF-stimulated tyrosine phosphorylation identified substrates exhibiting a bell-shaped response curve as a function of EGF concentration. As a comparison, we tested the phosphorylation of p120 Ras-GAP in response to EGF and found an increase in the tyrosine phosphorylation in a dose-dependent manner; similar results regarding p120 Ras-GAP have been reported earlier by others (
      • Ellis C.
      • Moran M.
      • McCormick F.
      • Pawson T.
      ). Recent results by Ribon and Saltiel (
      • Ribon V.
      • Saltiel A.R.
      ) differ from the results reported here; they found that stimulation of cells with a high concentration (100 ng/ml) of EGF results in tyrosine phosphorylation of Cas. The reason for this difference is unknown at present, but it may indicate cell type-specific differences in the response; Ribon and Saltiel (
      • Ribon V.
      • Saltiel A.R.
      ) used PC-12 rat pheochromocytoma cells in their experiments.
      Our results demonstrate that EGF increases Cas tyrosine phosphorylation at concentrations (0.5–5 ng/ml) significantly lower than the concentrations required to elicit a detectable increase in the tyrosine phosphorylation of several substrates of the EGF receptor, including p120 Ras-GAP (see Fig. 1; Refs.
      • Margolis B.
      • Rhee S.G.
      • Felder S.
      • Mervic M.
      • Lyall R.
      • Levitzki A.
      • Ullrich A.
      • Zilberstein A.
      • Schlessinger J.
      and
      • Ellis C.
      • Moran M.
      • McCormick F.
      • Pawson T.
      ). In contrast, the concentrations that were found to stimulate a robust tyrosine phosphorylation of p120 Ras-GAP (>30 ng/ml) demonstrated a dramatic tyrosine dephosphorylation of Cas. These results suggest that EGF may trigger tyrosine phosphorylation of Cas and possibly other associated cellular components via a signal transduction pathway fundamentally distinct from that involving other known EGF receptor substrates. FAK, a focal adhesion protein whose tyrosine phosphorylation status closely parallels that of Cas, may exhibit a similar dose-response for EGF stimulation; studies by Rankin et al. have demonstrated that stimulation of Swiss 3T3 cells with low concentrations of EGF induces tyrosine phosphorylation of FAK (
      • Rankin S.
      • Hooshmand-Rad R.
      • Claesson-Welsh L.
      • Rozengurt E.
      ). PDGF receptor may connect to similar intracellular signaling pathways as the EGF receptor; Casamassima and Rozengurt (
      • Casamassima A.
      • Rozengurt E.
      ) have shown a similar bell-shaped dose-response curve for Cas tyrosine phosphorylation in response to PDGF stimulation. Low concentrations of EGF (present study) and PDGF (
      • Casamassima A.
      • Rozengurt E.
      ) that stimulate tyrosine phosphorylation of focal adhesion proteins are mitogenic, raising the interesting possibility that focal adhesion proteins might play a role in amplifying and disseminating the mitogenic signal delivered by low, physiological concentrations of EGF and PDGF.
      The intracellular signaling events leading to the modulation of the tyrosine phosphorylation status of Cas are not well characterized. Many of the stimuli leading to the enhancement of tyrosine phosphorylation of Cas have been shown to induce a rapid increase in stress fibers and in focal adhesions; these stimuli include integrin-mediated cell adhesion, as well as neuropeptides, lysophosphatidic acid, and sphingosylphosphorylcholine (
      • Burridge K.
      • Fath K.
      • Kelly T.
      • Nuckolls G.
      • Turner C.
      ,
      • Seufferlein T.
      • Rozengurt E.
      ,
      • Rankin S.
      • Morii N.
      • Narumiya S.
      • Rozengurt E.
      ). We found here that induction of Cas tyrosine phosphorylation by low concentrations of EGF requires the presence of intact cytoskeleton since the tyrosine phosphorylation can be prevented by cytochalasin D treatment. A similar situation has been observed when Cas phosphorylation has been induced by bombesin, low concentrations of PDGF, or integrin-mediated cell adhesion (
      • Casamassima A.
      • Rozengurt E.
      ,
      • Vuori K.
      • Ruoslahti E.
      ,
      • Nojima Y.
      • Morino N.
      • Mimura T.
      • Hamasaki K.
      • Furuya H.
      • Sakai R.
      • Sato T.
      • Tachibana K.
      • Morimoto C.
      • Yazaki Y.
      • Hirai H.
      ). It thus appears that tyrosine kinase(s) capable of phosphorylating Cas associate with focal adhesions upon EGF treatment and other stimuli, and an intact functional cytoskeleton may be required to bring together the various components of this signaling complex. One of the candidate kinases for phosphorylating Cas is FAK, which has been shown to directly interact with and mediate the tyrosine phosphorylation of Cas (
      • Vuori K.
      • Hirai H.
      • Aizawa S.
      • Ruoslahti E.
      ,
      • Polte T.R.
      • Hanks S.K.
      ) and which becomes activated by the same stimuli that induce tyrosine phosphorylation of Cas (
      • Hanks S.K.
      • Polte T.R.
      ). Other candidates include Src family tyrosine kinases (
      • Vuori K.
      • Hirai H.
      • Aizawa S.
      • Ruoslahti E.
      ) and the FAK-homologue Pyk2/CAK-β/RAFTK/CADTK (
      • Astier A.
      • Avraham H.
      • Manie S.N.
      • Groopman J.
      • Canty T.
      • Avraham S.
      • Freedman A.S.
      ,
      • Li X.
      • Earp H.S.
      ), which also directly interact with Cas.
      The effects on focal adhesions and stress fibers by extracellular stimuli are likely to be mediated by Rac and Rho, which belong to the Ras-related small G protein superfamily (see the Introduction and Refs.
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      and
      • Hall A.
      ). Previous studies have demonstrated for the existence of a linear signal transduction pathway in the action of PDGF involving PI 3-kinase and Rac that leads to the tyrosine phosphorylation of Cas (
      • Casamassima A.
      • Rozengurt E.
      ). Our studies show that stimulation of cells with low concentrations of EGF may initiate a similar intracellular signaling cascade, since the EGF-induced tyrosine phosphorylation of Cas was readily inhibitable by PI 3-kinase inhibitors. In contrast, neuropeptide-, lysophosphatidic acid, and sphingosylphosphorylcholine-induced tyrosine phosphorylation of Cas occurs in a PI 3-kinase independent fashion, suggesting multiple distinct signaling pathways leading to Cas phosphorylation (
      • Casamassima A.
      • Rozengurt E.
      ).
      Not much is known about signaling pathways that induce tyrosine dephosphorylation of Cas. When cells are stimulated with insulin (
      • Knight J.B.
      • Yamauchi K.
      • Pessin J.E.
      ) or with high concentrations of EGF (present study), the dephosphorylation of focal adhesion proteins correlates with actin stress fiber breakdown and focal adhesion disassembly; the exact cause-effect relationship between the dephosphorylation events and focal adhesion disassembly is not known. It is possible that dephosphorylation of Cas takes place upon activation of tyrosine phosphatases in response to the extracellular stimuli. Candidate phosphatases include PTP1B and PTP-PEST, which have been shown to directly interact with and dephosphorylate Cas (
      • Liu F.
      • Hill D.E.
      • Chernoff J.
      ,
      • Garton A.J.
      • Flint A.J.
      • Tonks N.K.
      ). Interestingly, PTP-PEST was recently demonstrated to become recruited to activated EGF receptor complexes (
      • Charest A.
      • Wagner J.
      • Kwan M.
      • Tremblay M.L.
      ). In one study, insulin-induced tyrosine dephosphorylation of FAK and paxillin was found to be mediated by protein phosphatase SHP2 (Ref.
      • Ouwens D.M.
      • Mikkers H.M.
      • van der Zon G.C.
      • Stein-Gerlach M.
      • Ullrich A.
      • Maassen J.A.
      ; but see also Ref.
      • Yamauchi K.
      • Milarski K.L.
      • Saltiel A.R.
      • Pessin J.E.
      ); SHP2 is also a known component of the EGF receptor signaling pathway (
      • Fantl W.J.
      • Johnson D.E.
      • Williams L.T.
      ). Dephosphorylation may also take place through inactivation of tyrosine kinases; Tobe and co-workers (
      • Tobe K.
      • Sabe H.
      • Yamamoto T.
      • Yamauchi T.
      • Asai S.
      • Kaburagi Y.
      • Tamemoto H.
      • Ueki K.
      • Kimura H.
      • Akanuma Y.
      • Yazaki Y.
      • Hanafusa H.
      • Kadowaki T.
      ) found that C-terminal Src kinase Csk is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of Src family kinases.
      Recent reports have shown that integrin-dependent cell adhesion and stimulation of cells with neuropeptides, bioactive lipids, and low concentrations of PDGF can induce an SH2-mediated association of Crk with Cas (
      • Casamassima A.
      • Rozengurt E.
      ,
      • Vuori K.
      • Hirai H.
      • Aizawa S.
      • Ruoslahti E.
      ). Tyrosine-phosphorylated Cas also binds to Src and Nck in an SH2-dependent manner (
      • Schlaepfer D.D.
      • Broome M.A.
      • Hunter T.
      ,
      • Nakamoto T.
      • Sakai R.
      • Ozawa K.
      • Yazaki Y.
      • Hirai H.
      ). Our results demonstrate that low concentrations of EGF induce the formation of a Cas-Crk complex in intact RatER cells that is dependent on the integrity of the actin cytoskeleton; concentrations of EGF that do not stimulate tyrosine phosphorylation of Cas fail to induce Cas-Crk complex formation. Crk binds to a number of signaling proteins through its SH3 domain including C3G (
      • Matsuda M.
      • Hashimoto Y.
      • Muroya K.
      • Hasegawa H.
      • Kurata T.
      • Tanaka S.
      • Nakamura S.
      • Hattori S.
      ,
      • Tanaka S.
      • Morishita T.
      • Hashimoto Y.
      • Hattori S.
      • Nakamura S.
      • Shibuya M.
      • Matuoka K.
      • Takenawa T.
      • Kurata T.
      • Nagashima K.
      • Matsuda M.
      ), which is a guanine nucleotide exchange factor for the small GTP-binding protein Rap-1 (
      • Gotoh T.
      • Hattori S.
      • Nakamura S.
      • Kitayama H.
      • Noda M.
      • Takai Y.
      • Kaibuchi K.
      • Matsui H.
      • Hatase O.
      • Takahashi H.
      • Kurata T.
      • Matsuda M.
      ). Interestingly, recent work by Okayama and co-workers suggests that the oncogenic growth signal from EGF receptor to Ras is predominantly mediated by Crk in rat fibroblasts (
      • Kizaka-Kondoh S.
      • Matsuda M.
      • Okayama H.
      ). The interaction between Cas and Crk may be important in regulating the subcellular localization of Crk or the activity of the downstream effectors in EGF signal transduction pathways. The possibility that tyrosine phosphorylation of Cas plays a role in mitogenic signaling warrants further experimental work.

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