Phosphorylation of the Linker for Activation of T-Cells by Itk Promotes Recruitment of Vav
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- 作者：Juan J. Perez-Villar ; Gena S. Whitney ; Mitchell T. Sitnick ; Robert J. Dunn ; Srividhya Venkatesan ; Kathleen O'Day ; Gary L. Schieven ; Tai-An Lin ; and Steven B. Kanner
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：August 27, 2002
- 年：2002
- 卷：41
- 期：34
- 页码：10732 - 10740
- 全文大小：179K
- 年卷期：v.41,no.34(August 27, 2002)
- ISSN：1520-4995
文摘
The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor proteinthat resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites,several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptorsGads and Grb2, as well as to phospholipase C
1 (PLC1), thereby facilitating the recruitment of keysignal transduction components to drive T-cell activation. The LAT tyrosine residues Y132, Y171, Y191,and Y226 have been shown previously to be critical for binding to Gads, Grb2, and PLC1. In this report,we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studiesusing purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines inLAT to phenylalanine residues, we demonstrate that Y171 and potentially Y226 are docking sites for theVav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk inT-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav toLAT complexes. These data indicate that kinases from distinct PTK families are likely responsible forLAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment ofVav to LAT.
1 (PLC1), thereby facilitating the recruitment of keysignal transduction components to drive T-cell activation. The LAT tyrosine residues Y132, Y171, Y191,and Y226 have been shown previously to be critical for binding to Gads, Grb2, and PLC1. In this report,we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studiesusing purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines inLAT to phenylalanine residues, we demonstrate that Y171 and potentially Y226 are docking sites for theVav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk inT-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav toLAT complexes. These data indicate that kinases from distinct PTK families are likely responsible forLAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment ofVav to LAT.