Cloning, sequencing, expression, and Gq-independent activation of phospholipase C-beta 2.
cDNAs corresponding to a previously uncharacterized phospholipase C were isolated from an HL-60 cell cDNA library. The cDNAs encodes a putative polypeptide of 1181 amino acids with a calculated molecular mass of 133,700 daltons. Comparison of the amino acid sequence of the predicted protein with those of five mammalian phospholipase C isoforms (PLC-beta 1, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) revealed that the new enzyme is most closely related to PLC-beta 1 with an overall amino acid sequence identity of 48%. Thus, the new phospholipase C was named PLC-beta 2. The least similarity between PLC-beta 1 and PLC-beta 2 is apparent in the carboxyl-terminal 450 amino acids. Both PLC-beta 1 and PLC-beta 2 were purified from extracts of HeLa cells that had been transfected with vaccinia virus containing the corresponding cDNAs. Like other mammalian PLC isoforms, including PLC-beta 1, the catalytic activity of PLC-beta 2 was entirely dependent on Ca2+, and PLC-beta 2 preferred phosphatidyl-inositol 4,5-bisphosphate to phosphatidylinositol as substrate. Recently, the alpha subunit of the pertussis toxin-insensitive G-protein alpha q has been shown to activate PLC-beta 1 but not PLC-gamma 1 and PLC-delta 1. When alpha q purified from bovine brain was reconstituted with PLC-beta 1 or PLC-beta 2, no stimulation of PLC-beta 2 was observed in the presence of either AlF4- or guanosine 5-O-(3-thiotriphosphate) (GTP gamma S), whereas PLC-beta 1 activity was enhanced markedly in the presence of AlF4- and less markedly but significantly in the presence of GTP gamma S. These results suggest that the receptor-dependent stimulation of PLC-beta 1 and that of PLC-beta 2 may require different G-protein alpha subunits. (see also accompanying article (Lee, C. H., Park, D., Wu, D., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 16044-16047).
Park
D
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Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Jhon
D Y
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Kriz
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R
Knopf
J
J
Rhee
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SG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D010738:Type C Phospholipases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium