Regulation of phospholipase C-gamma(1) by the actin-regulatory protein villin.
Regulation of phospholipase C-gamma(1) by the actin-regulatory protein villin.
复制标题
肌动蛋白调节蛋白绒毛蛋白对磷脂酶 C-gamma(1) 的调节。
DOI：
10.1152/ajpcell.2001.281.3.c1046
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Khurana,S
中科院分区：
文献类型：
--
作者：
Panebra,A;Ma,SX;Zhai,LW;Wang,XT;Rhee,SG;Khurana,S
The actin-regulatory protein villin is tyrosine phosphorylated and associates with phospholipase C-γ1(PLC-γ1) in the brush border of intestinal epithelial cells. To study the mechanism of villin-associated PLC-γ1activation, we reconstituted in vitro the tyrosine phosphorylation of villin and its association with PLC-γ1. Recombinant villin was phosphorylated in vitro by the nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using in vitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain of PLC-γ1. The catalytic activity of PLC-γ1was inhibited by villin in a dose-dependent manner with half-maximal inhibition at a concentration of 12.4 μM. Villin inhibited PLC-γ1activity by sequestering the substrate phosphatidylinositol 4,5-bisphosphate (PIP2), since increasing concentrations of PIP2reversed the inhibitory effects of villin on PLC activity. The inhibition of PLC-γ1activity by villin was reversed by the tyrosine phosphorylation of villin. Further, we demonstrated that tyrosine phosphorylation of villin abolished villin's ability to associate with PIP2. In conclusion, tyrosine-phosphorylated villin associates with the COOH-terminal SH2 domain of PLC-γ1and activates PLC-γ1catalytic activity. Villin regulates PLC-γ1activity by modifying its own ability to bind PIP2. This study provides biochemical proof of the functional relevance of tyrosine phosphorylation of villin and identifies the molecular mechanisms involved in the activation of PLC-γ1by villin.
