title: src-homology 2 (SH2) domain ligation as an allosteric regulator: modulation of phosphoinositide-specific phospholipase C gamma 1 structure and activity.
abstract: Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) catalyses the hydrolysis of PtdIns(4,5)P2 to generate the second messengers diacylglycerol and Ins(1,4,5)P3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to growth-factor-induced tyrosine phosphorylation, and, in vivo, is translocated from the cytosol to the particulate cell fraction. Here we report the bacterial expression of rat brain PI-PLC gamma 1 under the control of the T7 promoter. Production of the active enzyme in amounts suitable for structure-function analysis depended on coupling the translation 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 into the cloned PLC gamma 1. Examination of the specific activity, pH-rate profile, [Ca2+]-dependence and substrate specificity of bacterially expressed PLC gamma indicated that it had kinetic properties similar to those of PLC gamma isolated from bovine brain. The substrate specificity was dependent on [Ca2+]: at low [Ca2+] (1-10 microM) PtdIns(4,5)P2 was a better substrate than PtdIns. Addition of phosphotyrosine-containing peptides (12-mers) with the cognate sequence of the high-affinity binding site for PLC gamma 1 on the activated epidermal-growth-factor (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 phosphotyrosine-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 gamma 1 can mediate structural rearrangements and modulate enzymic activity on their ligation by growth-factor receptors is discussed.
authors.affiliation: Department of Cancer Research, Merck Research Laboratories, West Point, PA 19486.
authors.forename: K S
authors.initials: KS
authors.lastname: Koblan
authors.forename: M D
authors.initials: MD
authors.lastname: Schaber
authors.forename: G
authors.initials: G
authors.lastname: Edwards
authors.forename: J B
authors.initials: JB
authors.lastname: Gibbs
authors.forename: D L
authors.initials: DL
authors.lastname: Pompliano
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019000:Phosphotyrosine; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
chemical_list: D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase