Lck-mediated tyrosine phosphorylation of Discs Large Homolog 1 at position Y222 is required to coordinate alternative p38 activation in T cells (109.33)

Abstract Stimulation through the T cell receptor (TCR) leads to rapid activation of proximal tyrosine kinases, Lck and ZAP70, which couple to distinct downstream signaling cascades and functional outcomes. We have shown that MAGUK family scaffold protein Discs Large Homolog 1 (Dlgh1) juxtaposes activated Lck and ZAP70 with p38 MAP kinase to selectively activate the alternative p38 pathway and downstream NFAT; but not NFκB. Lck, ZAP70 and p38 are known to complex with Dlgh1 in response TCR engagement. However, Lck and ZAP70 binding sites within Dlgh1 and the mechanism by which Dlgh1 positions and activates Lck and ZAP70 allowing p38 activation remain incompletely understood. Knowing YxxL/I is a preferred motif for Lck tyrosine phosphorylation and that SH2 domains of ZAP70 and Lck prefer to bind Lck-phosphorylated substrates; we identified Dlgh1 Y222 as a potential target of phosphorylation and/or binding. We subsequently assessed the potential role of Dlgh1 Y222 phosphorylation in coordinating the alternative p38 pathway. Preliminary studies using rLck, rZAP70 and GST-Dlgh1 Y222F fusion proteins demonstrate that Y222 is a primary site of Lck phosphorylation required for optimal alternative p38 activation. Experiments are underway to assess the role of Dlgh1 Y222 in Lck and ZAP70 binding and alternative p38 activation in primary T cells. These experiments will elucidate molecular mechanisms behind Dlgh1 coordination of the alternative p38 pathway and TCR signal specificity.