Yao R, Yoshihara M, Osada H
Laboratory of Antibiotics, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-01, Japan.
J Biol Chem. 1997 Jul 18;272(29):18261-6. doi: 10.1074/jbc.272.29.18261.
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 NH2-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 c-Jun, 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.
星形孢菌素是一种蛋白激酶抑制剂，已知它能模拟神经生长因子（NGF）促进神经突生长的作用。为了阐明星形孢菌素诱导PC - 12细胞神经突生长的机制，我们以髓鞘碱性蛋白为底物进行了凝胶内激酶分析，发现星形孢菌素诱导了一种表观分子量为57 kDa的激酶的激活。激活该激酶所需的星形孢菌素剂量与诱导神经突生长所需的剂量一致。有趣的是，星形孢菌素激活的激酶能被抗c - Jun NH2末端激酶（JNK）亚型抗体免疫沉淀，但不能被抗JNK1特异性抗体或抗ERK1抗体免疫沉淀，这增加了这种激酶是一种新型JNK亚型的可能性。该激酶的底物特异性与渗透压休克激活的JNK和NGF激活的ERK1不同。该激酶能磷酸化包括c - Jun、Elk - 1和ATF2在内的转录因子以及髓鞘碱性蛋白，表明它在基因诱导中起作用。此外，星形孢菌素诱导了包括Nur77和fos在内的即早基因，但未诱导jun基因。星形孢菌素激活的激酶的激活以及神经突生长的诱导不需要Ras功能，而Ras是NGF诱导的ERK激活和神经突生长所必需的。综上所述，这些结果表明星形孢菌素特异性激活一种JNK亚型，这可能有助于包括神经突生长在内的生物学活性。
