RACK1, a receptor for activated C kinase and a homolog of the β subunit of G proteins, inhibits activity of Src tyrosine kinases and growth of NIH 3T3 cells
RACK1, a receptor for activated C kinase and a homolog of the β subunit of G proteins, inhibits activity of Src tyrosine kinases and growth of NIH 3T3 cells
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DOI：
10.1128/mcb.18.6.3245
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发表时间：
1998-06-01
影响因子：
5.3
通讯作者：
Cartwright, CA
中科院分区：
文献类型：
--
作者：
Chang, BY;Conroy, KB;Cartwright, CA
为了分离和表征与Src的独特结构域和SH 3和SH 2结构域相互作用并可能调节Src活性的蛋白质，我们使用酵母双杂交测定来筛选人肺成纤维细胞cDNA文库。我们确定RACK 1，活化C激酶的受体和G蛋白的β亚基的同源物，作为Src结合蛋白。使用GST-Src融合蛋白，我们确定RACK 1结合Src的SH 2结构域。用NIH 3 T3细胞证明Src和RACK 1的免疫共沉淀。纯化的GST-RACK 1以浓度依赖的方式抑制Src的体外激酶活性。GST-RACK 1（2 μ M）抑制纯化的Src和Lck酪氨酸激酶的活性达40 - 50%，但不抑制我们测试的三种丝氨酸/苏氨酸激酶的活性。在瞬时过表达RACK 1的293 T细胞中，许多细胞蛋白上的酪氨酸磷酸化降低。在稳定过表达RACK 1的NIH 3 T3细胞中，Src活性和细胞生长速率降低了40 - 50%。流式细胞术分析显示RACK 1过表达细胞的坏死或凋亡率并没有增加，但在G（0）/G（1）中的时间明显多于野生型细胞。G（0）/G（1）比值的延长可能是RACK 1过表达细胞倍增时间延长的原因。我们认为，RACK 1发挥其对NIH 3 T3细胞周期的影响，部分通过抑制Src活性。
To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 mu M) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested, Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1 overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G(0)/G(1) than do wild-type cells. Prolongation of G(0)/G(1) could account for the increased doubling time of RACK1 overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.