EARLY ACTIVATION OF ENDOGENOUS PP60SRC KINASE-ACTIVITY DURING NEURONAL DIFFERENTIATION OF CULTURED HUMAN NEUROBLASTOMA-CELLS
EARLY ACTIVATION OF ENDOGENOUS PP60SRC KINASE-ACTIVITY DURING NEURONAL DIFFERENTIATION OF CULTURED HUMAN NEUROBLASTOMA-CELLS
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DOI：
10.1128/mcb.10.1.361
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发表时间：
1990-01-01
影响因子：
5.3
通讯作者：
PAHLMAN, S
中科院分区：
文献类型：
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作者：
BJELFMAN, C;MEYERSON, G;PAHLMAN, S
原癌基因产物 pp60c-src 是一种酪氨酸特异性激酶，其细胞功能尚未解决。神经元中高水平的 pp60c-src 以及神经元 pp60c-src 变体 pp60c-srcN 的存在表明参与神经元分化表型的进展或维持。我们之前曾报道过，佛波酯，例如 12-O-十四烷酰佛波醇-13-乙酸酯 (TPA)，可刺激人 SH-SY5Y 神经母细胞瘤细胞进行神经元分化，根据形态学、生化和功能分化标记物的监测。在本报告中，我们描述了用 TPA 诱导 SH-SY5Y 细胞后 6 小时观察到的 pp60src (pp60c-src pp60c-srcN) 激酶活性的激活。这种现象与神经突生长、生长锥样结构的形成以及 GAP43 mRNA 表达的增加同时发生，这些是这些细胞中神经元分化的最早迹象。在用 16 nM TPA 处理的细胞中观察到最高的特异性 src 激酶活性（诱导 4 天后增加三到四倍）；该浓度对于 TPA 诱导的神经元表型的发育是最佳的。在分化过程中，未处理的 SH-SY5Y 细胞中 pp60c-src 与 pp60c-srcN 的 1:1 比例没有变化。来自体内 32P 标记细胞的 pp60src 的 V8 蛋白酶和胰蛋白酶磷酸肽图谱表明，分化细胞中 pp60src 的总体磷酸化高于未处理细胞，这主要是因为强烈的丝氨酸 12 磷酸化。在这两种细胞类型中均未检测到酪氨酸 416 磷酸化，并且在分化过程中未观察到酪氨酸 527 磷酸化发生变化。
The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration to the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tryosine 527 phosphorylation was observed.