Identification of Factors Involved in Neuronal Differentiation

PC12大鼠嗜铬细胞瘤细胞对神经生长因子有反应并发生神经元分化。微量注射抗ras癌基因产物p21的抗体可抑制ngf依赖性分化，表明ras功能对分化至关重要。为了研究NGF诱导信号转导的分子机制，我们分离了几个对NGF表现出极低敏感性的突变细胞。我们现在正试图分离出挽救突变表型的基因。我们分析了各种因素对PC12细胞分化的影响。其中只有NGF和表皮生长因子（EGF）能激活ras p21。酪氨酸激酶抑制剂阻断了NGF诱导的ras p21的激活，这表明酪氨酸激酶活性可能对NGF引起的信号转导至关重要。为了研究ras基因产物p21的生化和酶学特性，我们分析了刺激ras p21内在GTPase活性的因素。通过这种方法，我们确定了I型神经纤维瘤病（NF1）基因的产物，其功能丧失可能参与神经皮肤病的发病，I型神经纤维瘤病NF1基因产物主要位于颗粒部分。而GTPase激活蛋白（GAP）主要存在于可溶性部分。此外，还发现了另一种具有GTPase刺激活性的因子。

PC12 rat pheochromocytoma cells respond to NGF and undergo neuronal differentiation. Microinjection of antibody against ras oncogene product p21 inhibits NGF-dependent differentiation which indicates that ras function is essential for the differentiation. To study the molecular mechanism for the transduction of NGFinduced signals, we isolated several mutant cells which exhibit extremely low sensitivity to NGF. We are now trying to isolate gene (s) which rescue the mutated phenotype.We analyzed the effect of various factors which make PC12 cells undergo the differentiation. Among them only NGF and epidermal growth factor (EGF) were shown to activate ras p21. Tyrosine kinase inhibitors blocked NGF-induced activation of ras p21, which suggested that tyrosine kinase activity may be essential for transduction of the signals evoked by NGF.To characterize factors which modulate biochemical and enzymatic properties of ras gene product p21, we analyzed factors which stimulate intrinsic GTPase activity of ras p21. By this approach we identified the product of neurofibromatosis type I(NF1)gene whose loss of function may be involved in the onset of neurocutaneous disease, neurofibromatosis type I. NF1 gene product was located mainly in the particulate fraction. In contrast GTPase activating protein (GAP) mainly exists in the soluble fraction. Furthermore there found to be another factor with GTPase stimulating activity.