Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C.
Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4). cDNAs encoding two isoenzymes of Ins(1,4,5)P(3) 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052 bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P(3) 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75. 207 kDa and a K(m) for Ins(1,4,5)P(3) of 6 microM. Northern-blot analysis showed the presence of a transcript of approx. 3.9 kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5alphaF' bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P(3) 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin-Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P(3) 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin-Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.
Dewaste
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Interdisciplinary Research Institute (IRIBHN), Université Libre de Bruxelles, Campus Erasme, Bldg. C, 808 Route de Lennik, 1070 Brussels, Belgium.
Pouillon
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V
Moreau
C
C
Shears
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S
Takazawa
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K
Erneux
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C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium