Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells

Abstract

T-cell-receptor (TCR)-mediated LAT (linker for activation of T cells) phosphorylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT is required for recruitment and activation of signalling proteins, the molecular mechanism associated with this event is unclear. In the present study we reconstituted the LAT signalling pathway by demonstrating that a direct tyrosine phosphorylation of LAT with activated protein-tyrosine kinase Zap70 is necessary and sufficient for the association and activation of signalling proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127. By substituting these tyrosine residues in LAT with phenylalanine and by utilizing phosphorylated peptides derived from these sites, we mapped the tyrosine residues in LAT required for the direct interaction and activation of Vav, p85/p110alpha and phospholipase Cgamma1 (PLCgamma1). Our results indicate that Tyr(226) and Tyr(191) are required for Vav binding, whereas Tyr(171) and Tyr(132) are necessary for association and activation of phosphoinositide 3-kinase activity and PLCgamma1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr(191) and Tyr(171) are required for T-cell activation and Tyr(132) is required for the activation of PLCgamma1 and Ras signalling pathways.

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 T cells, PI3K regulates the recruitment of proteins that facilitate TCR signal transduction such as PLC-g1, Sos1, RasGAP, Itk, Tec, and Vav. Early studies showed that PI3K bound directly to LAT (Fukazawa et al 1995a;Lahesmaa et al 1995;Paz et al 2001); however more recent studies showed binding of p85 to tyrosine-phosphorylated SLP-76 (Shim et al 2004). Inhibition of PI3K in T cells resulted in reduced TCR-induced calcium influx, Rac1 activation, and Erk1/2 phosphorylation.…”

“…Interaction with LAT was shown to be required for both PLC-g1 activation and localization near its substrate PIP 2 at the plasma membrane (Zhang et al 1999a;Zhang et al 2000;Lin and Weiss 2001). Subsequently, the amino-terminal SH2 domain of PLC-g1 was shown to bind phosphorylated LAT Y132 with high affinity (Paz et al 2001;Houtman et al 2004). Furthermore, a single Y132F mutation abolished the association between LAT and PLC-g1 in Jurkat T cells (Zhang et al 2000).…”

HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76–Signaling Microclusters following TCR Stimulation

“…We next addressed the effect of Nef on the behavior of SLP-76, a signaling adaptor that is recruited into MCs following TCR engagement in a ZAP70-dependent manner (12). J14SLP-76.YFP cells (Jurkat T lymphocytes stably expressing SLP-76 fused to YFP) were used for these analyses as established model of SLP-76 MC formation (43,44).…”

“…LAT facilitates the formation of several multiprotein complexes and acts as nucleation center for T cell signal transduction (11). Upon phosphorylation at Tyr 191 by ZAP70 (12), LAT recruits SLP-76 into MCs via an additional adaptor protein, GADS (13). Together, the LAT-SLP-76 complex nucleates multiple important downstream signaling events such as calcium flux, MAPK activation, integrin activation, and cytoskeletal organization (14).…”