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• W2051354632 abstract "The cell signaling docking protein p130 cas became tyrosine-phosphorylated in SH-SY5Y human neuroblastoma cells during induced differentiation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum or a combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The differentiating cells develop a neuronal phenotype with neurites and growth cones and sustained activation of protein kinase C (PKC) and pp60c- src . The TPA-induced p130 cas phosphorylation increased within 5 min of stimulation and persisted for at least 4 days, whereas bFGF/IGF-I-induced p130 cas phosphorylation was biphasic. However, the increase in tyrosine phosphorylation of p130 cas was not restricted to differentiation inducing stimuli. The phosphorylation was blocked by the specific PKC inhibitor GF 109203X, and transient transfection with active PKC-ε induced p130 cas tyrosine phosphorylation. pp60c- src, known to directly phosphorylate p130 cas in other cell systems, was not activated after stimulation with TPA or bFGF/IGF-I for up to 30 min, and the initial p130 cas phosphorylation was resistant to the Src family kinase inhibitor herbimycin A. However, in long term stimulated cells, herbimycin A blocked the induced phosphorylation of p130 cas . Also, overexpression of src induced phosphorylation of p130 cas . p130 cas protein and phosphorylated p130 cas were present in growth cones isolated from differentiated SH-SY5Y cells. Inhibition of PKC activity in differentiating cells with GF 109203X leads to a rapid retraction of growth cone filopodia, and p130 cas phosphorylation decreased transiently (within minutes). Growth cones isolated from these cells were virtually devoid of phosphorylated p130 cas . These data suggest a function for p130 cas as a PKC downstream target in SH-SY5Y cells and possibly also in their growth cones. The cell signaling docking protein p130 cas became tyrosine-phosphorylated in SH-SY5Y human neuroblastoma cells during induced differentiation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum or a combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The differentiating cells develop a neuronal phenotype with neurites and growth cones and sustained activation of protein kinase C (PKC) and pp60c- src . The TPA-induced p130 cas phosphorylation increased within 5 min of stimulation and persisted for at least 4 days, whereas bFGF/IGF-I-induced p130 cas phosphorylation was biphasic. However, the increase in tyrosine phosphorylation of p130 cas was not restricted to differentiation inducing stimuli. The phosphorylation was blocked by the specific PKC inhibitor GF 109203X, and transient transfection with active PKC-ε induced p130 cas tyrosine phosphorylation. pp60c- src, known to directly phosphorylate p130 cas in other cell systems, was not activated after stimulation with TPA or bFGF/IGF-I for up to 30 min, and the initial p130 cas phosphorylation was resistant to the Src family kinase inhibitor herbimycin A. However, in long term stimulated cells, herbimycin A blocked the induced phosphorylation of p130 cas . Also, overexpression of src induced phosphorylation of p130 cas . p130 cas protein and phosphorylated p130 cas were present in growth cones isolated from differentiated SH-SY5Y cells. Inhibition of PKC activity in differentiating cells with GF 109203X leads to a rapid retraction of growth cone filopodia, and p130 cas phosphorylation decreased transiently (within minutes). Growth cones isolated from these cells were virtually devoid of phosphorylated p130 cas . These data suggest a function for p130 cas as a PKC downstream target in SH-SY5Y cells and possibly also in their growth cones. p130 cas is a recently identified docking protein that contains an SH3 domain and a region with several tyrosine residues that can become tyrosine-phosphorylated and constitutes putative SH2 binding domains (1Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). The protein structure suggests a function for p130 cas in assembling signaling complexes. The only identified kinases that directly phosphorylate p130 cas is pp60c- src (2Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar) and Abl (3Mayer B.J. Hirai H. Sakai R. Curr. Biol. 1995; 5: 296-305Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, little is known about the signaling pathways that lead to induced tyrosine phosphorylation of p130 cas and thereby promote binding of SH2 domain containing proteins, and the downstream effects of this complex formation remain to be clarified. It is established that p130 cas becomes heavily tyrosine-phosphorylated after integrin stimulation (4Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 5Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 6Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and that the protein is localized to focal adhesions (4Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 7Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) and along stress fibers (4Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). p130 cas associates with focal adhesion kinase (FAK) 1The abbreviations used are: FAK, focal adhesion kinase, TPA, 12-O-tetradecanoylphorbol-13-acetate; FCS, fetal calf serum; bFGF, basic fibroblast growth factor; IGF-I, insulin-like growth factor I; PKC, protein kinase C; MARCKS, myristoylated alanine-rich protein kinase C substrate; dbcAMP, dibutyryl cyclic AMP; PAGE, polyacrylamide gel electrophoresis; PTP, protein-tyrosine phosphatase.1The abbreviations used are: FAK, focal adhesion kinase, TPA, 12-O-tetradecanoylphorbol-13-acetate; FCS, fetal calf serum; bFGF, basic fibroblast growth factor; IGF-I, insulin-like growth factor I; PKC, protein kinase C; MARCKS, myristoylated alanine-rich protein kinase C substrate; dbcAMP, dibutyryl cyclic AMP; PAGE, polyacrylamide gel electrophoresis; PTP, protein-tyrosine phosphatase.(7Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 8Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (385) Google Scholar) and pp60c- src (1Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 9Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 10Burnham M.R. Harte M.T. Richardson A. Parsons J.T. Bouton A.H. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar), both proteins involved in focal adhesion regulation. Stimulation of PC12 rat pheochromocytoma cells with nerve growth factor or epidermal growth factor also induces phosphorylation of p130 cas (11Ribon V. Saltiel A.R. J. Biol. Chem. 1996; 271: 7375-7380Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The finding that p130 cas can bind to SH2 domains of Grb2, phosphoinositide 3-kinase, Crk, Nck, and phospholipase C-γ (2Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar), for example, suggests that p130 cas is a docking protein that integrates signals from growth factor receptors and adhesion molecules.SH-SY5Y is a human neuroblastoma cell line that can be induced to differentiate into a neuronal phenotype when treated with 16 nm phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) in the presence of fetal calf serum (FCS) or a growth factor (12Påhlman S. Odelstad L. Larsson E. Grotte G. Nilsson K. Int. J. Cancer. 1981; 28: 583-589Crossref PubMed Scopus (225) Google Scholar, 13Påhlman S. Meyerson G. Lindgren E. Schalling M. Johansson I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9994-9998Crossref PubMed Scopus (121) Google Scholar) or with a combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I) in serum-free medium (14Lavenius E. Parrow V. Nånberg E. Påhlman S. Growth Factors. 1994; 10: 29-39Crossref PubMed Scopus (70) Google Scholar). The differentiated cells extended neurites with neurotransmitter containing varicosities and growth cones. The growth cones are the leading tips of the growing neurites and are mainly composed of actin filaments (reviewed in Ref.15Smith S.J. Science. 1988; 242: 708-715Crossref PubMed Scopus (265) Google Scholar), and actin reorganization is the mechanism underlying growth cone motility. In SH-SY5Y cells, the activity of pp60c- src increases during differentiation (16Bjelfman C. Meyerson G. Cartwright C.A. Mellström K. Hammerling U. Påhlman S. Mol. Cell. Biol. 1990; 10: 361-370Crossref PubMed Scopus (77) Google Scholar), and pp60c- src is enriched and activated in growth cones (17Meyerson G. Pfenninger K.H. Påhlman S. J. Cell Sci. 1992; 103: 233-243PubMed Google Scholar). pp60c- src binds to the growth cone cytoskeleton in an activity dependent manner (18Helmke S. Pfenninger K.H. Cell Motil. Cytoskeleton. 1995; 30: 194-207Crossref PubMed Scopus (53) Google Scholar).Protein kinase C (PKC) is a family of serine-threonine kinases that are subdivided into three classes based on activator and co-factor dependence. Classical PKCs (PKC-α, PKC-β, and PKC-γ) and novel PKCs (PKC-δ, PKC-ε, PKC-η, PKC-θ, and PKC-μ) are activated by TPA, but the endogenous activator is, for example, diacylglycerol that is generated after growth factor stimulation (reviewed in Refs. 19Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar and20Stabel S. Parker P.J. Pharmacol. Ther. 1991; 51: 71-95Crossref PubMed Scopus (452) Google Scholar). SH-SY5Y cells express at least PKC-α, PKC-ε, and PKC-ζ (21Parrow V. Fagerström S. Meyerson G. Nånberg E. Påhlman S. J. Neurosci. Res. 1995; 41: 782-791Crossref PubMed Scopus (31) Google Scholar). A sustained PKC activity, measured as phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS), is detected during differentiation of SH-SY5Y cells (22Parrow V. Nånberg E. Heikkilä J. Hammerling U. Påhlman S. J. Cell. Physiol. 1992; 152: 536-544Crossref PubMed Scopus (39) Google Scholar), and treatment with a high concentration of TPA (1.6 μm) that down-regulates PKC-α completely only induces poor differentiation (21Parrow V. Fagerström S. Meyerson G. Nånberg E. Påhlman S. J. Neurosci. Res. 1995; 41: 782-791Crossref PubMed Scopus (31) Google Scholar, 23Påhlman S. Ruusala A.-I. Abrahamsson L. Odelstad L. Nilsson K. Cell Differ. 1983; 12: 165-170Crossref PubMed Scopus (91) Google Scholar, 24Fagerström S. Påhlman S. Gestblom C. Nånberg E. Cell Growth Differ. 1996; 7: 775-785PubMed Google Scholar). Furthermore, inhibition of PKC activity by specific inhibitors blocks differentiation induced by 16 nm TPA in FCS or the combination of bFGF and IGF-I with respect to both morphological and transcriptional events (24Fagerström S. Påhlman S. Gestblom C. Nånberg E. Cell Growth Differ. 1996; 7: 775-785PubMed Google Scholar). PKC-α and PKC-ε are enriched in growth cones of SH-SY5Y cells (21Parrow V. Fagerström S. Meyerson G. Nånberg E. Påhlman S. J. Neurosci. Res. 1995; 41: 782-791Crossref PubMed Scopus (31) Google Scholar, 24Fagerström S. Påhlman S. Gestblom C. Nånberg E. Cell Growth Differ. 1996; 7: 775-785PubMed Google Scholar), and maintenance of growth cone structure appears to be PKC-dependent (24Fagerström S. Påhlman S. Gestblom C. Nånberg E. Cell Growth Differ. 1996; 7: 775-785PubMed Google Scholar).In this study, we have investigated tyrosine phosphorylation of p130 cas in differentiating SH-SY5Y cells and its dependence on activation and inhibition of PKC and Src family kinases. We also studied p130 cas phosphorylation in isolated growth cones. The data presented show that there are at least two signaling pathways in differentiating SH-SY5Y cells that promote tyrosine phosphorylation of p130 cas : an initial PKC-dependent but pp60c- src /Src-kinase family-independent pathway, and a second PKC and Src-kinase family-dependent pathway. A functional role for p130 cas in regulating the assembly of signals that control growth cone function is suggested.DISCUSSIONWe here report a PKC-dependent increase in tyrosine phosphorylation of p130 cas in differentiating SH-SY5Y neuroblastoma cells. Src kinase and herbimycin A data suggested an initial p130 cas tyrosine phosphorylation pathway independent of pp60c- src and a later pathway in differentiating cells involving pp60c- src or related herbimycin A-sensitive kinase(s). We also propose a role for p130 cas in assembling signals that regulate the functional growth cone in axons and dendrites.Previously, we have demonstrated that TPA- and to a large extent bFGF/IGF-I-dependent neuronal differentiation (growth cone formation, outgrowth of neurites, and gene expression) of SH-SY5Y cells is blocked by PKC inhibitors. However, the cells retain their capacity to differentiate in response to bFGF/IGF-I when classical PKCs (such as PKC-α) are down-regulated by prolonged treatment with 1.6 μm TPA (24Fagerström S. Påhlman S. Gestblom C. Nånberg E. Cell Growth Differ. 1996; 7: 775-785PubMed Google Scholar). Under these conditions, PKC-ε is still present in substantial amounts. We have therefore proposed that PKC-ε or other novel isoforms are vital for neuronal differentiation in this cell system. We have now shown that under conditions where either of TPA and growth factors induce differentiation, tyrosine phosphorylation of p130 cas is stimulated. This response is not entirely associated with the development of a differentiated phenotype, but rather with the activation of PKC, since a number of protocols inducing PKC activation but not differentiation also induced p130 cas tyrosine phosphorylation. We have previously demonstrated that both bFGF and IGF-I activates PKC in these cells, measured as in vivo phosphorylation of MARCKS, and that the combination of the factors was additive, whereas 16 nm TPA was twice as efficient (14Lavenius E. Parrow V. Nånberg E. Påhlman S. Growth Factors. 1994; 10: 29-39Crossref PubMed Scopus (70) Google Scholar). This order of magnitude was also true for p130 cas phosphorylation, speaking in favor of a PKC-dependent signaling step in growth factor- and TPA-treated cells. The use of specific inhibitors for classical and novel PKCs (GF 109203X and Go 6976) demonstrated that this phosphorylation seemed to be dependent on active novel isoforms of PKC. Also the maintenance of an elevated p130 cas phosphorylation in differentiated cells required functional PKC, as demonstrated by the rapid net dephosphorylation of p130 cas after addition of GF 109203X. The strongest indication for a PKC-driven tyrosine phosphorylation of p130 cas came from the data in Fig.1 D where TPA added to serum-free cultures gave a rapid and potent effect.Introduction of a constitutively active PKC-ε also promoted phosphorylation of p130 cas, showing that increased PKC-ε activity as such was sufficient in promoting p130 cas phosphorylation. We have not been able to distinguish any signs of differentiation in the PKC-ε+-transfected cultures. In contrast to the TPA-differentiated cells, p130 cas phosphorylation in PKC-ε+-transfected cultures was not sensitive to acute exposure to PKC inhibitors (GF 109203X in Fig. 3 and Ro 31–8425, not shown). Preliminary results from transient transfections with PKC-ε+, where GF 109203X and Ro 31–8425 were added immediately after a shorter transfection protocol (6 h) and included for another 40 h, indicated that this treatment abolished the very small induction in tyrosine phosphorylation of p130 cas seen in these cultures (not shown). One possible explanation for these somewhat contradictive results could be that selective increase of PKC-ε activity in the transfectants induced secondary effects and, consequently, at a later time point an apparent dissociation of the p130 cas phosphorylation from PKC activity under these conditions.The steady-state level of p130 cas tyrosine phosphorylation is balanced by protein-tyrosine kinase activity(ies) counteracted by protein-tyrosine phosphatase(s). The effect of the TPA- or growth factor-stimulated serine-threonine kinase PKC in SH-SY5Y cells for tyrosine phosphorylation of p130 cas could thus be due to either activation of protein-tyrosine kinases, inactivation of protein-tyrosine phosphatases, or a combination of the two (Fig.7). One of several plausible kinase candidates would be pp60c- src since p130 cas is tyrosine-phosphorylated in v-Src transformed cells (40Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar); pp60c- src has been shown to directly phosphorylate p130 cas (4Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar), and pp60c- src is activated in differentiating SH-SY5Y cells as a comparatively late event (16Bjelfman C. Meyerson G. Cartwright C.A. Mellström K. Hammerling U. Påhlman S. Mol. Cell. Biol. 1990; 10: 361-370Crossref PubMed Scopus (77) Google Scholar). A phosphatase candidate is the protein-tyrosine phosphatase (PTP)-PEST, which is inactivated by PKC, can dephosphorylate p130 cas (41Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar), and is present in SH-SY5Y cells (42Habib T. Herrera R. Decker S.J. J. Biol. Chem. 1994; 269: 25243-25246Abstract Full Text PDF PubMed Google Scholar).A direct effect of PKC on pp60c- src kinase activity has not been conclusively evaluated, but PKC can phosphorylate pp60c- src on serine 12 (36Gould K.L. Woodgett J.R. Cooper J.A. Buss J.E. Shalloway D. Hunter T. Cell. 1985; 42: 849-857Abstract Full Text PDF PubMed Scopus (126) Google Scholar,37Purchio A.F. Shoyab M. Gentry L.E. Science. 1985; 229: 1393-1395Crossref PubMed Scopus (23) Google Scholar), which also has been demonstrated in TPA-stimulated SH-SY5Y cells (16Bjelfman C. Meyerson G. Cartwright C.A. Mellström K. Hammerling U. Påhlman S. Mol. Cell. Biol. 1990; 10: 361-370Crossref PubMed Scopus (77) Google Scholar). However, we conclude that only the late but not the initial phosphorylation of p130 cas was dependent on pp60c- src or Src family members based on the following arguments. (i) The Src family inhibitor herbimycin A had no effect on the basal and initial (5–30 min) p130 cas phosphorylation in TPA- and bFGF/IGF-I-treated cells. (ii) Despite large efforts, we have never been able to detect an increased pp60c- src activity within the first 6 h of treatment of these cells with TPA (16Bjelfman C. Meyerson G. Cartwright C.A. Mellström K. Hammerling U. Påhlman S. Mol. Cell. Biol. 1990; 10: 361-370Crossref PubMed Scopus (77) Google Scholar) or bFGF/IGF-I, and neither increased amounts of pp60c- src protein (16Bjelfman C. Meyerson G. Cartwright C.A. Mellström K. Hammerling U. Påhlman S. Mol. Cell. Biol. 1990; 10: 361-370Crossref PubMed Scopus (77) Google Scholar). (iii) The small amount of p130 cas that co-immunoprecipitated with pp60c- src was unaltered after TPA treatment (not shown), indicating that stimulation of the cells did not lead to subcellular redistribution of active pp60c- src to a p130 cas containing compartment or vice versa. (iv) In differentiated cultures (4 days), i.e. when pp60c- src activity is up-regulated, p130 cas phosphorylation was abolished by herbimycin A, suggesting involvement of pp60c- src or another herbimycin A-sensitive kinase. Alternative genes, e.g. FYN and YES, are expressed in SH-SY5Y cells and are enriched and activated in growth cones of differentiating SH-SY5Y cells (43Meyerson G. Påhlman S. FEBS Lett. 1993; 332: 27-30Crossref PubMed Scopus (9) Google Scholar). p59 fyn binds p130 cas, and p130 cas phosphorylation is decreased in fibroblasts isolated from fyn knock-out mice compared with their normal counterpart (2Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar). Both p59 fyn and pp62c- yes kinases should be sensitive to herbimycin A. These kinases might therefore contribute to tyrosine phosphorylation of p130 cas at later time points but are unlikely to promote the initial signal. (v) Overexpression of functional pp60c- src caused increased tyrosine phosphorylation of p130 cas measured 40 h after transfection, and this phosphorylation was largely abolished by short term treatment with herbimycin A or GF 109203X, indicating the involvement of a PKC-dependent step also under these conditions. Another kinase, p125FAK, that also interacts directly with p130 cas (7Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 8Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (385) Google Scholar) and can be regulated by PKC (44Mogi A. Hatai M. Soga H. Takenoshita S. Nagamachi Y. Fujimoto J. Yamamoto T. Yokota J. Yaoi Y. FEBS Lett. 1995; 373: 135-140Crossref PubMed Scopus (16) Google Scholar) is not likely to significantly contribute to the phosphorylation of p130 cas in SH-SY5Y, since no active, i.e. phosphorylated, p125FAK was detected in cells stimulated with TPA. The cytosolic tyrosine kinase Abl has been shown to phosphorylate p130 cas in vitro. The phosphorylation is enhanced by binding of Crk to Abl (3Mayer B.J. Hirai H. Sakai R. Curr. Biol. 1995; 5: 296-305Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). An in vivo function for Abl in p130 cas phosphorylation remains to be investigated in SH-SY5Y and other cells.The tyrosine phosphatase PTP-PEST was recently shown to dephosphorylate specifically p130 cas in several cell lines (41Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar) and to bind directly to the SH3 domain of p130 cas (45Garton A.J. Burnham M.R. Bouton A.H. Tonks N.K. Oncogene. 1997; 15: 877-885Crossref PubMed Scopus (144) Google Scholar). PTP-PEST can be phosphorylated on serine residues in vitro by both PKC and cyclic AMP-dependent kinase, and the same residues are phosphorylated in TPA-stimulated HeLa cells. This phosphorylation negatively regulates the activity of the phosphatase by reducing the substrate affinity (46Garton A.J. Tonks N.K. EMBO J. 1994; 13: 3763-3771Crossref PubMed Scopus (106) Google Scholar). It is feasible that PKC-evoked p130 cas phosphorylation in SH-SY5Y cells could be mediated by inactivation of PTP-PEST. The finding that activation of cyclic AMP-dependent kinase with dbcAMP also promoted tyrosine phosphorylation of p130 cas is in agreement with a PTP-PEST-regulated p130 cas phosphorylation, but the possible involvement of PTP-PEST needs further studies.Little is known about the physiological function of p130 cas . Its localization to growth cones could imply that p130 cas takes part in the regulation of growth cone function. Both PKC-α and PKC-ε are enriched in intact growth cones (21Parrow V. Fagerström S. Meyerson G. Nånberg E. Påhlman S. J. Neurosci. Res. 1995; 41: 782-791Crossref PubMed Scopus (31) Google Scholar), and PKC activity (presumably PKC-ε) is necessary for maintenance of the growth cone structure (24Fagerström S. Påhlman S. Gestblom C. Nånberg E. Cell Growth Differ. 1996; 7: 775-785PubMed Google Scholar). We now suggest that p130 cas could be a downstream target of PKC in the functional growth cone based on the following: (i) tyrosine-phosphorylated p130 cas was present in growth cones of differentiated SH-SY5Y; (ii) p130 cas in the growth cone fraction as well as in whole unfractionated cells was rapidly dephosphorylated after addition of a PKC inhibitor, a treatment that induces retraction of growth cone filopodia; and (iii) the time course of the initial dephosphorylation of p130 cas measured in whole cell extracts correlated closely to filopodia retraction. Unlike treatment with GF 109203X, exposure to herbimycin A did not lead to rapid changes of growth cone morphology, but prolonged exposure over several days caused cell detachment from the culture dish. p130 cas phosphorylation decreased in these cells but not as rapid as after GF 109203X treatment. Thus, p130 cas phosphorylation in growth cones did not seem to correlate closely to growth cone structure but rather to PKC activity.An explanation for the functionally different effects of GF 109203X and herbimycin A on growth cone structure would be if PKC has a more complex role regulating and integrating a number of vital activities in the growth cone. For example, GAP-43 is a well characterized PKC substrate located in growth cones of differentiated SH-SY5Y cells (17Meyerson G. Pfenninger K.H. Påhlman S. J. Cell Sci. 1992; 103: 233-243PubMed Google Scholar) and with an important role in neuronal sprouting and growth cone migration (47Aigner L. Caroni P. J. Cell Biol. 1995; 128: 647-660Crossref PubMed Scopus (165) Google Scholar, 48Aigner L. Arber S. Kapfhammer J.P. Laux T. Schneider C. Botteri F. Brenner H.R. Caroni P. Cell. 1995; 83: 269-278Abstract Full Text PDF PubMed Scopus (551) Google Scholar). Although the results discussed above point to an important role of PKC in signaling through p130 cas in growth cones, neither p130 cas phosphorylation nor PKC activation are sufficient for neurite extension (see for instance PKC-ε transfections). Experiments with PC12 cells have previously demonstrated that introduction of v-src or mutated activeras leads to neurite outgrowth and differentiation (49Alema S. Casalbore P. Agostini E. Tato F. Nature. 1985; 316: 557-559Crossref PubMed Scopus (197) Google Scholar, 50Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (566) Google Scholar) and that the two oncogenes have a synergistic effect (for a review see Ref. 51Keegan K. Halegoua S. Curr. Opin. Neurobiol. 1993; 3: 14-19Crossref PubMed Scopus (39) Google Scholar). Introduction of active Ras (Val-12 Ras) in SH-SY5Y cells also leads to neurite formation, 2A.-K. Olsson and E. Nånberg, manuscript in preparation.clearly demonstrating signaling events leading to neurite formation which cannot solely be explained by activation of PKC or p130 cas .SH-SY5Y cells undergoing an active process of differentiation have to coordinate signals mediating neurite outgrowth and other cytoskeletal events as well as altered gene expression. Also, survival and maintenance of the differentiated cell probably demands other signaling activities or combinations of signals than unstimulated or acutely stimulated SH-SY5Y cells. It is feasible that p130 cas has different or partially non-overlapping functions and is differently regulated during early and late phases of differentiation. The rapid phosphorylation of p130 cas in cells induced to differentiate might be an early event during the dynamic phase of cytoskeleton rearrangement that precedes the formation of growth cones and neurite sprouting. The acute effects of TPA, FCS, and growth factors added separately might also lead to the same actin reorganization. Since p130 cas is the target of both growth factor and integrin stimulation (see Introduction for references) and is a signal transduction docking protein, p130 cas may sense and integrate different signals in growth cones, thus being a member of the tightly regulated machinery needed for axon growth and path finding. p130 cas is a recently identified docking protein that contains an SH3 domain and a region with several tyrosine residues that can become tyrosine-phosphorylated and constitutes putative SH2 binding domains (1Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). The protein structure suggests a function for p130 cas in assembling signaling complexes. The only identified kinases that directly phosphorylate p130 cas is pp60c- src (2Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar) and Abl (3Mayer B.J. Hirai H. Sakai R. Curr. Biol. 1995; 5: 296-305Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, little is known about the signaling pathways that lead to induced tyrosine phosphorylation of p130 cas and thereby promote binding of SH2 domain containing proteins, and the downstream effects of this complex formation remain to be clarified. It is established that p130 cas becomes heavily tyrosine-phosphorylated after integrin stimulation (4Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 5Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 6Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and that the protein is localized to focal adhesions (4Petch L.A. Bockholt S." @default.
• W2051354632 created "2016-06-24" @default.
• W2051354632 creator A5019347490 @default.
• W2051354632 creator A5051135936 @default.
• W2051354632 creator A5072303267 @default.
• W2051354632 date "1998-01-01" @default.
• W2051354632 modified "2023-10-16" @default.
• W2051354632 title "Protein Kinase C-dependent Tyrosine Phosphorylation of p130 in Differentiating Neuroblastoma Cells" @default.
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