SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: Identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C

Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of ~100 amino acids called the src homology 2 (SH2) domain, and a region of ~50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-γ (PLCγ). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLCγ. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLCγ were expressed in Eschenchia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLCγ are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLCγ-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 μM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLCγ. Higher concentrations of PLC γ-SH2 also led to the protection from dephosphorylation of Tyr1068 (≥ 1 μM), Tyr1173 (≥ 6 μM) and Tyr1086 (≥ 100 μM). These results provide further support for the central regulatory role of SH2 domains in signal transduction pathways, and reveal a hierarchy in the interactions between tyrosine phosphorylation sites of the EGF receptor and PLCγ-SH2.