Regulation of Actin Dynamics by Tyrosine Phosphorylation: Identification of Tyrosine Phosphorylation Sites within the Actin-Severing Domain of Villin

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• 作者：Liwei Zhai ; Narendra Kumar ; Alfredo Panebra ; Peilin Zhao ; Abby L. Parrill ; and Seema Khurana
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：October 1, 2002
• 年：2002
• 卷：41
• 期：39
• 页码：11750 - 11760
• 全文大小：354K
• 年卷期：v.41,no.39(October 1, 2002)
• ISSN：1520-4995

文摘

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosinephosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated byphosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, wesought to identify the major phosphorylation site(s) in human villin and study its role in actin filamentassembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in theprokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed inTKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a regionin the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261).Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine(F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villinmutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitrokinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosineresidues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one ofthe identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between theamino-terminal end of villin and other actin-severing proteins, the results provide a structural basis forthe actin-severing mechanism and help understand the relationship of phosphorylation with this function.