Akiyama T, Matsuda S, Namba Y, Saito T, Toyoshima K, Yamamoto T
Department of Oncogene Research, Osaka University, Japan.
Mol Cell Biol. 1991 Feb;11(2):833-42. doi: 10.1128/mcb.11.2.833-842.1991.
The mutant c-erbB-2 protein with Glu instead of Val-659 exhibited transforming activity in NIH 3T3 cells. This protein showed enhanced tyrosine kinase activity in vitro and enhanced autophosphorylation at Tyr-1248 located proximal to the carboxyl terminus. Enhanced tyrosine phosphorylation of several cellular proteins was detected in cells expressing the Glu-659 c-erbB-2 protein. Introduction of an additional mutation at the ATP-binding site (Lys-753 to Met) of this protein resulted in abolition of its transforming ability. These data indicate that the transforming potential of c-erbB-2 is closely correlated with elevated tyrosine kinase activity of the gene product. To investigate the role of autophosphorylation in cell transformation, we introduced an additional mutation at the autophosphorylation site of the Glu-659 c-erbB-2 protein (Tyr-1248 to Phe). This mutant protein exhibited lower tyrosine kinase activity and lower transforming activity. On the other hand, when the carboxyl-terminal 230 amino acid residues were deleted from the c-erbB-2 protein, the tyrosine kinase activity and cell-transforming activity of the protein were enhanced. Thus, the carboxyl-terminal domain, which contains the major autophosphorylation site, Tyr-1248, may regulate cellular transformation negatively and autophosphorylation may eliminate this negative regulation.
具有谷氨酸而非缬氨酸-659的突变型c-erbB-2蛋白在NIH 3T3细胞中表现出转化活性。该蛋白在体外显示出增强的酪氨酸激酶活性，并且在靠近羧基末端的Tyr-1248处的自身磷酸化增强。在表达谷氨酸-659 c-erbB-2蛋白的细胞中检测到几种细胞蛋白的酪氨酸磷酸化增强。在该蛋白的ATP结合位点（赖氨酸-753突变为甲硫氨酸）引入额外突变导致其转化能力丧失。这些数据表明，c-erbB-2的转化潜力与该基因产物升高的酪氨酸激酶活性密切相关。为了研究自身磷酸化在细胞转化中的作用，我们在谷氨酸-659 c-erbB-2蛋白的自身磷酸化位点（Tyr-1248突变为苯丙氨酸）引入了额外突变。这种突变蛋白表现出较低的酪氨酸激酶活性和较低的转化活性。另一方面，当从c-erbB-2蛋白中缺失羧基末端230个氨基酸残基时，该蛋白的酪氨酸激酶活性和细胞转化活性增强。因此，包含主要自身磷酸化位点Tyr-1248的羧基末端结构域可能对细胞转化起负调节作用，而自身磷酸化可能消除这种负调节。
