Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25Mm upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1
Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25Mm upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1
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DOI：
10.1074/jbc.m001378200
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发表时间：
2000-09-22
影响因子：
4.8
通讯作者：
Satoh, T
中科院分区：
文献类型：
--
作者：
Kiyono, M;Kato, J;Satoh, T
Ras-GRF 1是Ras的脑特异性鸟嘌呤核苷酸交换因子（GEF），其活性响应于Ca 2+内流和G蛋白偶联受体信号而调节。此外，当G蛋白偶联受体刺激后酪氨酸磷酸化时，Ras-GRF 1充当Rac的GEF。然而，Ras-GRF 1功能的调节机制仍不完全清楚。我们在这里表明，激活的ACK 1，一种非受体酪氨酸激酶，属于黏着斑激酶家族，导致Ras-GRF 1的酪氨酸磷酸化。另一方面，激酶缺陷型ACK 1没有发挥作用。通过体外GDP结合和释放测定，Ras-GRF 1对Ha-Has的GEF活性在ACK 1酪氨酸磷酸化后增强。相反，GEF对RAD的活性仍然是潜伏的，这意味着ACK 1并不代表酪氨酸激酶，作用于G蛋白偶联受体的下游。与增强的Ras-GEF活性一致，通过使用has结合结构域下拉测定显示细胞内GTP结合形式的Ras的积累。此外，Ras依赖性激活ERK 2的Ras-GRF 1增强后，激活的ACK 1的共表达。这些结果暗示ACK 1作为Ras-GRF 1的上游调节剂，并表明在神经元细胞中由Cdc 42、ACK 1、Ras-GRF 1和Has组成的信号级联。
Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca2+ influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Has, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rad remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of has-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Has in neuronal cells.