Inhibitory effect of src homology (SH) 2/SH3 fragments of phospholipase C-gamma on the catalytic activity of phospholipase C isoforms. Identification of a novel phospholipase C inhibitor region.
In order to study the regulatory mechanisms of phospholipase C-gamma (PLC-gamma) via the intrinsic SH2/SH3 region (Z region), two recombinant Z proteins, rP45Z and rP38Z, derived from rat PLC-gamma 1 and PLC-gamma 2, respectively, were purified from the inclusion bodies of Escherichia coli. We examined their direct effects on phosphoinositide hydrolysis induced by four different PLC isoforms purified from bovine brain and thymus, and found that both of these Z proteins suppress the enzyme activity of all four PLC isoforms in a dose-dependent manner. This suppressive effect is very potent and stoichiometric. The kinetics studies indicate that the suppression is non-competitive. This suppression is eliminated by treatment with proteases but is not affected by heat treatment at 95 degrees C for 15 min, indicating that the primary structure might be important for the action of Z proteins. Comparative studies suggested that two Z proteins but not Src and phosphatidylinositol 3-kinase possess, adjacent to their SH2 and SH3 motifs, a phospholipase C inhibitor (PCI) region that strongly suppresses their phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity. A series of synthetic peptides identical with the sequence of the proposed PCI region, including an octamer, YRKMRLRY, inhibited PIP2 hydrolysis induced by four different phospholipase C isoforms. These results demonstrate that both types of phospholipase C-gamma contain the PCI sequence which is responsible for the inhibition of PIP2 hydrolysis, indicating that phospholipase C-gamma is a self-regulating enzyme.
Homma
Y
Y
Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Japan.
Takenawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D002417:Cattle; D004795:Enzyme Stability; D006358:Hot Temperature; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src); D010446:Peptide Fragments / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013950:Thymus Gland / Q000201:enzymology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D015688:Oncogene Protein pp60(v-src); D010738:Type C Phospholipases