Phosphorylation of the Linker for Activation of T-Cells by Itk Promotes Recruitment of Vav

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 Cgamma1 (PLCgamma1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y(132), Y(171), Y(191), and Y(226) have been shown previously to be critical for binding to Gads, Grb2, and PLCgamma1. 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 Y(171) and potentially Y(226) are docking sites for the Vav 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.

Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton

“…Itk has been shown to phosphorylate tyrosine 171 of linker for activation of T cells (LAT), which can bind Vav (38). However, our results argue that proper Vav localization does not require Itk kinase activity.…”

Mitogenic CD28 Signals Require the Exchange Factor Vav1 to Enhance TCR Signaling at the SLP-76-Vav-Itk Signalosome

“…The most likely candidates fall into the Tec-kinase family, whose activation is not only dependent on Syk-family kinases, but which typically have substrate specificity overlapping with that of Syk-family kinases (42). For example, Itk has recently been shown to phosphorylate LAT, and another Tec-family kinase Rlk/Txk phosphorylates SLP-76 in overexpression systems (43,44). Both of these substrates were until recently thought to be specific Zap-70 substrates.…”

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

“…Though a full understanding of how the kinases phosphorylate the individual tyrosines on LAT in vivo remains unresolved, overexpression and in vitro studies have implicated primarily ZAP-70, but also Lck and Itk in LAT phosphorylation (Zhang et al 1998a;Paz et al 2001;Perez-Villar et al 2002;Jiang and Cheng 2007). Even less is known about the phosphatases that dephosphorylate LAT, however CD148 has been implicated (Baker et al 2001).…”

“…In Vav-deficient T cells, the interaction between SLP-76 and PLC-g1 was substantially reduced and Itk was not phosphorylated (Reynolds et al 2002;Braiman et al 2006). The Tec family kinase Itk phosphorylates PLC-g1 (Readinger et al 2009) and LAT (Perez-Villar et al 2002). Itk is recruited to the LAT complex by binding of its SH2 domain to tyrosine phosphorylated SLP-76 (Bunnell et al 2000).…”