Crk adaptor proteins regulate CD3 zeta chain phosphorylation and TCR/CD3 down-modulation in activated T cells

摘要

T cell receptor (TCR) recognition of a peptide antigen in the context of MHC molecules initiates positive and negative cascades that regulate T cell activation, proliferation and differentiation, and culminate in the acquisition of effector T cell functions. These processes are a prerequisite for the induction of specific T cell mediated adaptive immune responses. A key event in the activation of TCR-coupled signaling pathways is the phosphorylation of tyrosine residues within the cytoplasmic tails of the CD3 subunits, predominantly CD3 zeta. These transiently formed phosphotyrosyl epitopes serve as docking sites for SH2-domain containing effector molecules, predominantly the ZAP70 protein tyrosine kinase, which is critical for signal propagation. We found that CrkI and CrkII adaptor proteins also interact with CD3 zeta in TCR activated-, but not in resting-, T cells. Crk binding to CD3 zeta was independent of ZAP70 and also occurred in ZAP70-deficient T cells. Binding was mediated by Crk-SH2 domain interaction with phosphotyrosine-containing motifs on CD3 zeta, via a direct physical interaction, as demonstrated by Far-Western blot. CrkII binding to CD3 zeta could also be demonstrated in a heterologous system, where coexpression of a catalytically active Lck was used to phosphorylate the CD3 zeta chain. TCR activation-induced Crk binding to CD3 zeta resulted in increased and prolonged phosphorylation of CD3 zeta, as well as ZAP70 and LAT, suggesting a positive role for CrkI/II binding to CD3 zeta in regulation of TCR-coupled signaling pathways. Furthermore, Crk-dependent increased phosphorylation of CD3 zeta coincided with inhibition of TCR downmodulation, supporting a positive role for Crk adaptor proteins in TCR-mediated signal amplification.