SRC-HOMOLOGY-2 (SH2) DOMAIN LIGATION AS AN ALLOSTERIC REGULATOR - MODULATION OF PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE-C-GAMMA-1 STRUCTUREAND ACTIVITY

Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) cat alyses the hydrolysis of PtdIns(4,5)P-2 to generate the second messeng ers diacylglycerol and Ins(1,4,5)P-3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to gr owth-factor-induced tyrosine phosphorylation, and, in vivo, is translo cated from the cytosol to the particulate cell fraction. Here we repor t the bacterial expression of rat brain PI-PLC gamma 1 under the contr ol of the T7 promoter. Production of the active enzyme in amounts suit able for structure-function analysis depended on coupling the translat ion of PLC gamma 1 to the expression of the phage-phi 10 coat protein. Purification of the enzyme was facilitated by the presence of a three -amino-acid C-terminal antibody epitope tag (Glu-Glu-Phe) engineered i nto the cloned PLC gamma 1. Examination of the specific activity, pH-r ate profile, [Ca2+]-dependence and substrate specificity of bacteriall y expressed PLC gamma indicated that it had kinetic properties similar to those of PLC gamma isolated from bovine brain. The substrate speci ficity was dependent on [Ca2+]: at low [Ca2+] (1-10 mu M) PtdIns(4,5)P -2 was a better substrate than PtdIns. Addition of phosphotyrosine-con taining peptides (12-mers) with the cognate sequence of the high-affin ity binding site for PLC gamma 1 on the activated epidermal-growth-fac tor (EGF) receptor (Tyr-992) increased enzyme activity (up to 85%) in vitro. Cognate non-phosphorylated peptides had no effect on activity. When c.d. spectroscopy was used to monitor the effect of added phospho tyrosine-containing peptide on the structure of recombinant PLC gamma 1, significant spectral shifts, indicative of a conformational change, were observed upon complexation with the EGF-receptor phosphotyrosine -containing 12-residue peptide (Tyr-992). How SH2 domains from PLC ga mma 1 can mediate structural rearrangements and modulate enzymic activ ity on their ligation by growth-factor receptors is discussed.