Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav

Juan J. Perez-Villar, Gena S. Whitney, Mitchell T. Sitnick, Robert J. Dunn, Srividhya Venkatesan, Kathleen O'Day, Gary L. Schieven, Tai An Lin, Steven B. Kanner

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Abstract

The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that 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 adaptors Gads and Grb2, as well as to phospholipase Cγ1 (PLCγ1), thereby facilitating the recruitment of key signal 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 PLCγ1. 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 studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y171 and potentially Y226 are docking sites for the 8Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.

Original languageEnglish
Pages (from-to)10732-10740
Number of pages9
JournalBiochemistry
Volume41
Issue number34
DOIs
Publication statusPublished - 27 Aug 2002

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Perez-Villar, J. J., Whitney, G. S., Sitnick, M. T., Dunn, R. J., Venkatesan, S., O'Day, K., ... Kanner, S. B. (2002). Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav. Biochemistry, 41(34), 10732-10740. https://doi.org/10.1021/bi025554o