Characterization of the interaction between Syk kinase and SH2 domains important in B cell signaling

Regulation of B cell growth and differentiation occurs via signaling cascades that originate at membrane bound receptors. SH2 domains, one component of this regulation, play an important part in controlling protein-protein interactions through tyrosine phosphorylation. A recently identified mode of interaction between Syk kinase and SH2 domains important in B cell signaling is mediated by two phosphotyrosines, Y342 and Y346, within the Interdomain B region of Syk, and binding studies have revealed that both tyrosine residues are important for complex formation. In order to characterize this unusual interaction, we have identified the binding site of a doubly phosphorylated peptide derived from Syk kinase (designated pYpY) on the C-terminal SH2 domain of phospholipase Cγ-1 (PLCC) and solved the structure of the pYpY-PLCC complex using NMR techniques. These experiments revealed that the second tyrosine of pYpY binds on the opposite side of the central β-sheet from the primary pTyr binding pocket. The second pTyr binding pocket is made from several aromatic residues from the BG loop of PLCC as well as two lysine residues (K54 and K56). The primary pTyr residue interacts with the conserved arginine residue of the central β-sheet but not with the arginine residues surrounding the primary pTyr pocket. Instead, these residues interact with two glutamates from the N-terminal half of pYpY. Surprisingly, there are large conformational changes within PLCC upon binding of pYpY when compared to binding of PLCC to a singly phosphorylated peptide from the platelet derived growth factor receptor. The large changes to the binding surface of PLCC were unexpected because previous studies of SH2 domains have shown that phosphopeptide binding was not accompanied by a conformational change of the protein. Surface plasmon resonance was used to determine the binding kinetics of the interaction between Syk and wild type (WT) PLCC and two mutants of PLCC. Binding studies were also attempted with the SH2 domain of Lck, which has been found to bind to the same region of Syk, but were unsuccessful. The characteristics of the pYpY-PLCC complex will be used to elucidate the mechanism of control of different pathways of antigen-activated signaling in B cells