Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases (original) (raw)
Related papers
Nerve growth factor induces protein-tyrosine phosphorylation
Proceedings of the National Academy of Sciences, 1988
When the sympathetic nerve-like cell line PC12 is exposed to nerve growth factor (NGF), there is a rapid and transient phosphorylation of tyrosine residues in cellular proteins, as demonstrated by immunoblotting of cell extracts with high-affmity polyclonal antibodies specific for phosphotyrosine residues. Epidermal growth factor (EGF), which does not cause the morphological differentiation of PC12 cells that is produced by NGF, also induces protein-tyrosine phosphorylation. The methyltransferase inhibitor, 5'-methylthioadenosine, which is known to block the NGF-mediated morphological differentiation of PC12 cells, also inhibits the induction of protein-tyrosine phosphorylation by NGF. 5'-Methylthioadenosine has no effect, however, on EGF-stimulated phosphorylation of tyrosine residues in cellular proteins. In addition, low temperature markedly slows the rate of protein-tyrosine phosphorylation stimulated by NGF, but it has no effect on the time course of protein-tyrosine phosphorylation induced by EGF.
Cellular Signalling, 1998
We investigated the ability of bryostatin 1 to block nerve growth factor (NGF)-induced differentiation of pheochromocytoma PC12 cells and to effect expression of protein kinase C (PKC) isoforms. Compared with phorbol myristate acetate (PMA), a likewise potent activator of PKC, high doses of bryostatin (Ͼ 200 nM) failed to down-regulate ␦-PKC, as with-PKC, whereas, ␣-PKC was completely down-regulated. Two forms of ␦-PKC were expressed in PC12 cells, a phosphorylated 78.000 M r species and a de-phosphorylated 76.000 M r form. High-dose bryostatin treatment resulted in a 4.5-fold increase in phosphorylated ␦-PKC and a 2.5-fold increase in phosphotyrosine. Inhibition of tyrosine kinase activity, with either herbimycin or genistein, prior to addition of bryostatin abrogated protection from down-regulation and led to simultaneous increases in ubiquitinated 110.000 M r-␦-PKC. Similarly, pre-treatment of cells with N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal, an inhibitor of the proteasome pathway, prior to low-dose treatment with bryostatin resulted in a dose-dependent accumulation of ␦-PKC and inhibition of down-regulation. Protection of ␦-PKC from down-regulation by highdose bryostatin requires a counterbalance between protein tyrosine kinase and phosphatase systems. High doses of bryostatin blocked NGF-induced neurite outgrowth without altering Y-490 TrK A phosphorylation or an alteration in pp44/42 mitogen-activated protein kinase. Our findings suggest that the phosphorylation state of ␦-PKC may regulate its ability to participate in signal coupling and modulation of cell growth and differentiation pathways. Moreover, these data reveal the existence of a signalling pathway independent of MAP kinase that affects NGF differentiation in a negative fashion.
Molecular and cellular biology, 1993
A myriad of gene induction events underlie nerve growth factor (NGF)-induced differentiation of PC12 cells. To dissect the signal transduction pathways which lead to NGF actions, we have assessed the relative roles of NGF receptor, Src, Ras, and Raf activities in mediating specific gene inductions. We have used the PC12 cell line as well as sublines which inducibly express activated forms of either Src, Ras, or Raf or a dominant inhibitory form of Ras (p21N17 Ras) to study the expression of multiple NGF-inducible mRNAs. The NGF induction of NGFI-A, transin, and VGF mRNAs was mimicked by activated forms of Src, Ras, or Raf and was blocked by p21N17 Ras. The NGF induction of SCG10 mRNA was mimicked only by activated Src and Ras and was blocked by p21N17 Ras, while the induction of Thy-1 mRNA was mimicked only by activated Src and was not blocked by p21N17 Ras. The NGF induction of mRNAs for two sodium channel types was neither mimicked by any activated oncoprotein nor blocked by p21N1...
Journal of Biological Chemistry, 1998
Induction of neuronal differentiation of the rat pheochromocytoma cell line, PC12 cells, by nerve growth factor (NGF) requires activation of the mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK). cAMP-dependent protein kinase (protein kinase A (PKA)) also can induce differentiation of these cells. Like NGF, the ability of PKA to differentiate PC12 cells is associated with a sustained activation of ERKs. Here we show that maximal sustained activation of ERK1 by NGF requires PKA. Inhibitors of PKA partially blocked activation of ERK1 by NGF but had no effect on activation of ERK1 by EGF. Inhibition of PKA also reduced the ability of NGF and cAMP, but not EGF, to activate the transcription factor Elk-1, reduced the induction of both immediate early and late genes after NGF treatment, and blocked the nuclear translocation of ERK1 induced by NGF. We propose that PKA is an important contributor to the activation of ERK1 by NGF and is required for maximal induction of gene expression by NGF.
European Journal of Biochemistry, 1998
The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (A, β and γ) are activated in response to NGF. Gprotein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.
Molecular and Cellular Biology, 2000
Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit...
Molecular and Cellular Biology, 2001
Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-. Recruitment of PKC-into the complex was dependent on the tyrosine phosphorylation state of PKC-. The association of src and PKC-was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-(amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-, whereas the Y325F mutation significantly reduced src-induced activation of PKC-. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-B, with significant impairment by the Y325F PKC-mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.
Nerve growth factor activates kinases that phosphorylate atypical protein kinase C
Cellular Signalling, 2002
Activation of atypical protein kinase C by nerve growth factor (NGF) involves phosphorylation. In order to identify kinases that regulate atypical PKC (aPKC), we surveyed PC12 cell lysates for protein kinases that are activated by NGF and which could phosphorylate aPKC. Employing an in-gel kinase assay where aPKC-z was copolymerized within the gel matrix as a substrate, three kinases, pp175, pp87 and pp60, were identified as enzymes that phosphorylated aPKC. Phosphorylation of aPKC by these three kinases coincided with NGF-induced activation of the enzyme. Each kinase possessed a unique subcellular distribution pattern and could be activated by either ceramide or H 2 0 2 , second messengers that mimic NGF signaling events. Upstream, pp175 and pp60 lie in a ras pathway, whereas pp87 lies in a pathway dependent upon src. Altogether, these findings reveal that the aPKCs are subject to regulation by a novel group of kinases.