Determination of Cytoplasmic Tyrosine Residues of Tim-3 Involved in Regulation of TCR signaling (35.30)

Abstract

T-cell immunoglobulin and mucin 3 protein (Tim-3) is a type-I transmembrane protein expressed on murine T-helper type 1 (Th1) cells which regulates Th1 cell proliferation and development of tolerance. Tim-3 has six tyrosine residues in the cytosplasmic tail, two and three tyrosines residues are clustered in proximal and distal region of the cytoplasmic tail respectively. Using a motif scan program, we have predicted that two tyrosine residues from each cluster may be phosphorylated by Src family kinases, and three of the five residues may interact with SH2 domains. We generated serial deletion and point mutation constructs of Tim-3 and demonstrated that deletion of the cytoplasmic tail region containing the distal tyrosine residues (Y250, Y251, Y253) slightly increased NFAT-Luciferase reporter activity, while deletion of the region containing distal and proximal (Y235, Y242) tyrosine residues abolished the reporter activity to basal level in Jurkat cells. Point mutation of both Y235 and Y242 decreased NFAT-Luc reporter activity close to basal level while single point mutation of Y235 or Y242 did not result in significant changes in reporter activity. Therefore we conclude that phosphorylation of Y235 and Y242 residues of Tim-3 are responsible for potentiation of signal transduction initiated by activation of TCR, upstream of NFAT/AP1 pathways, in a redundant manner.