Heasley L E, Storey B, Fanger G R, Butterfield L, Zamarripa J, Blumberg D, Maue R A
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262, USA.
Mol Cell Biol. 1996 Feb;16(2):648-56. doi: 10.1128/MCB.16.2.648.
Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.
持续刺激特定的蛋白激酶途径被认为是受体酪氨酸激酶和细胞内癌蛋白的一个关键特征，这些受体酪氨酸激酶和细胞内癌蛋白可介导大鼠嗜铬细胞瘤（PC12）细胞的神经元分化信号。在迄今已鉴定出的蛋白丝氨酸/苏氨酸激酶中，p42/44丝裂原活化蛋白（MAP）激酶因其在介导PC12细胞分化信号中的潜在作用而受到关注。我们在此报告，逆转录病毒介导的异源三聚体Gq家族成员GαqQ209L和Gα16Q212L的GTP酶缺陷型、组成型活性形式在PC12细胞中的表达，可诱导神经元分化，表现为神经突生长和电压依赖性钠通道表达增加。在细胞表达GTP酶缺陷型的αi2或α0后未观察到分化，这表明对G蛋白的Gq家族具有选择性。如预期的那样，αqQ209L和α16Q212L的过表达使PC12细胞中的基础磷脂酶C活性组成型升高约10倍。重要的是，在用α16Q212L或αqQ209L分化的PC12细胞中几乎检测不到p42/44 MAP激酶活性，尽管这些蛋白在组成型活性cRaf-1表达后被强烈激活。相反，在表达αqQ209L和α16Q212L的PC12细胞中观察到cJun NH2末端激酶（JNKs）持续三倍的激活。这种JNK激活水平与神经生长因子（一种PC12细胞分化的强诱导剂）所达到的水平相似。支持JNK激活在PC12细胞分化中起作用的是，逆转录病毒介导的JNK靶标cJun在PC12细胞中的过表达诱导了神经突生长。这些结果确定了一种PC12细胞分化的不依赖p42/44 MAP激酶的机制，并表明GTP酶缺陷型Gαq和Gα16亚基对脯氨酸定向蛋白激酶家族的JNK成员的持续激活足以诱导PC12细胞分化。
