Nerve growth factor activates kinases that phosphorylate atypical protein kinase C (original) (raw)
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Molecular and Cellular Biology, 2000
The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-ι was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-ι with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-ι by NGF. At the level of Raf-1, neither PKC-ι nor PI3 kinase was required for activation; however, PKC-ι could weakly activate MEK. Inhibitors of PKC-ι activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-ι were likewise required for NGF-induced NF-κB activation...
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.
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.
Journal of Neurophysiology, 2012
Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK1/2 (PD-98059), PLC (U-73122, neomycin), or conventional/novel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKMζ, PKCζ, and PKCλ/ι) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKCζ. Both Western blots and quantitative real-time PCR established that...
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.
1999
In this study, we examined the role of specific protein kinase C (PKC) isoforms in the differentiation of PC12 cells in response to nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells express PKC- a ,- b ,- g ,- d ,- e ,- m, and -z. For PKC-d ,- e, and -z, NGF and EGF exerted differential effects on translocation. Unlike overexpression of PKC-a and -d, overexpression of PKC- e caused enhanced neurite outgrowth in response to NGF. In the PKC-e-overexpressing cells, EGF also dramatically induced neurite outgrowth, arrested cell proliferation, and induced a sustained phosphorylation of mitogen-activated protein kinase (MAPK), in contrast to its mitogenic effects on control cells or cells overexpressing PKC-a and -d. The induction of neurite outgrowth by EGF was inhibited by the MAPK kinase inhibitor PD95098. In cells overexpressing a PKC-e dominant negative mutant, NGF induced reduced neurite outgrowth and a more transient phosphorylation of MAPK than in controls. O...
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.
Neuroscience, 2017
Nerve growth factor plays a key role in the initiation as well as the prolonged heightened pain sensitivity of the inflammatory response. Previously, we showed that NGF rapidly augmented both the excitability of isolated rat sensory neurons and the mechanical sensitivity of the rat's hind paw. The increase in excitability and sensitivity were blocked by the myristolated pseudosubstrate inhibitor of atypical PKCs (mPSI), suggesting that an atypical PKC may play a key regulatory role in generating this heightened sensitivity. Our findings raised the question as to whether NGF directs changes in translational control, as suggested for long-lasting LTP, or whether NGF leads to the activation of an atypical PKC by other mechanisms. The current studies demonstrate that enhanced action potential firing produced by NGF was blocked by inhibitors of translation, but not transcription. In parallel, in vitro studies showed that NGF elevated the protein levels of PKMζ, which was also prevent...