Identification of a Phospholipase C-γ1 (PLC-γ1) SH3 Domain-Binding Site in SLP-76 Required for T-Cell Receptor-Mediated Activation of PLC-γ1 and NFAT

Abstract

SLP-76 is an adapter protein required for T-cell receptor (TCR) signaling. In particular, TCR-induced tyrosine phosphorylation and activation of phospholipase C-γ1 (PLC-γ1), and the resultant TCR-inducible gene expression, depend on SLP-76. Nonetheless, the mechanisms by which SLP-76 mediates PLC-γ1 activation are not well understood. We now demonstrate that SLP-76 directly interacts with the Src homology 3 (SH3) domain of PLC-γ1. Structure-function analysis of SLP-76 revealed that each of the previously defined protein-protein interaction domains can be individually deleted without completely disrupting SLP-76 function. Additional deletion mutations revealed a new, 67-amino-acid functional domain within the proline-rich region of SLP-76, which we have termed the P-1 domain. The P-1 domain mediates a constitutive interaction of SLP-76 with the SH3 domain of PLC-γ1 and is required for TCR-mediated activation of Erk, PLC-γ1, and NFAT (nuclear factor of activated T cells). The adjacent Gads-binding domain of SLP-76, also within the proline-rich region, mediates inducible recruitment of SLP-76 to a PLC-γ1-containing complex via the recruitment of both PLC-γ1 and Gads to another cell-type-specific adapter, LAT. Thus, TCR-induced activation of PLC-γ1 entails the binding of PLC-γ1 to both LAT and SLP-76, a finding that may underlie the requirement for both LAT and SLP-76 to mediate the optimal activation of PLC-γ1.