Specific Activation of a c-Jun NH2-terminal Kinase Isoform and Induction of Neurite Outgrowth in PC-12 Cells by Staurosporine

Abstract

Staurosporine, a protein kinase inhibitor, is known to mimic the effect of nerve growth factor (NGF) in promoting neurite outgrowth. To elucidate the mechanism by which staurosporine induces neurite outgrowth in PC-12 cells, we performed an in-gel kinase assay using myelin basic protein as a substrate, and found that staurosporine induced the activation of a kinase with an apparent molecular mass of 57 kDa. The dose of staurosporine required to activate this kinase was consistent with that required to induce neurite outgrowth. Interestingly, the staurosporine-activated kinase was immunoprecipitated by anti-c-Jun NH 2 -terminal kinase (JNK) isoforms antibody, but not by anti-JNK1-specific antibody or anti-ERK1 antibody, raising the possibility that this kinase is a novel JNK isoform. The substrate specificity of the kinase was distinct from those of osmotic shock-activated JNKs and NGF-activated ERK1. The kinase phosphorylates transcription factors including cJun, Elk-1, and ATF2, as well as myelin basic protein, suggesting that it plays a role in gene induction. Furthermore, staurosporine induced immediate-early genes including Nur77 and fos, but not jun. The activation of the staurosporine-activated kinase, as well as the induction of neurite outgrowth, did not require Ras function, while Ras was required for the activation of ERKs and neurite outgrowth induced by NGF. Taken together, these results indicate staurosporine specifically activates a JNK isoform, which may contribute to biological activities including neurite outgrowth.Neurotrophic factors play a key role in the normal development of the nervous system by regulating both differentiation and apoptosis of neurons (1). Nerve growth factor (NGF) 1 is the prototype of this family of neurotrophins and its intracellular signaling pathways have been intensely studied using rat pheochromocytoma PC-12 cells, which undergo neuronal differentiation in response to NGF stimulation (1, 2). NGF binds and activates its receptor, Trk, which leads to the activation of a guanine nucleotide-binding protein, Ras (3-5). Expression of oncogenic Ras in PC-12 cells induces neuronal differentiation (6), while introduction of anti-Ras antibody or dominant inhibitory Ras mutant into PC-12 cells blocks NGF-induced differentiation (7,8), suggesting that Ras is necessary and sufficient for neuronal differentiation in PC-12 cells. The formation of GTP-Ras is followed first by the activation of Raf and then by activation of MEK (9, 10). MEK in turn activates the ERK family of MAP kinases, which then phosphorylate a variety of proteins including Elk-1 transcription factor (11-13). The expression of a dominant inhibitory mutant of Ras inhibits activation of the Raf/MEK/ERK kinase cascade (14 -16). Furthermore, recent studies revealed that MEK and ERK play an important role in NGF-induced differentiation (17, 18). These results indicate the Ras/Raf/MEK/ERK signaling pathway plays a key role in NGF-induced differentiation in PC-12 cells.The molecular cloning of c-Jun NH 2 -terminal ki...

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