Phosphorylation of the N-Terminal and C-Terminal CD3-ϵ–ITAM Tyrosines Is Differentially Regulated in T Cells

Abstract

Tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within CD3 chains is crucial for the recruitment of protein tyrosine kinases and effector molecules into the T cell receptor. Thus, phenylalanine substitution at the N-terminal tyrosine residue of the CD3-ϵ–ITAM abolished signal transduction functions of this ITAM, including phosphorylation at the C-terminal ITAM tyrosine, and its association with ZAP-70. In contrast, mutation at the C-terminal tyrosine of CD3-ϵ–ITAM did not prevent phosphorylation at the N-terminal tyrosine, nor its association with Lck, or p85 PI 3-K regulatory subunit. In contrast to the ZAP-70/diphosphorylated CD8-ϵ–ITAM interaction, the Lck/monophosphorylated CD8-ϵ–ITAM interaction was sensitive to octylglucoside, an agent that disrupts Lck interaction with membrane rafts. Therefore, association of Lck with membrane rafts seems to be essential for stabilization of Lck/CD3-ϵ protein–protein interactions. Overall, the data suggest that the sequential and coordinated phosphorylation of CD3-ϵ–ITAM tyrosines provides to CD3-ϵ the potential to interact with multiple downstream effectors and signaling pathways.