D-myo-inositol 1,4,5-trisphosphate 3-kinase A is activated by receptor activation through a calcium:calmodulin-dependent protein kinase II phosphorylation mechanism.
D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase, the enzyme responsible for production of D-myo-inositol 1,3,4,5-tetrakisphosphate, was activated 3- to 5-fold in homogenates of rat brain cortical slices after incubation with carbachol. The effect was reproduced in response to UTP in Chinese hamster ovary (CHO) cells overexpressing Ins(1,4,5)P3 3-kinase A, the major isoform present in rat and human neuronal cells. In ortho-32P-labelled cells, the phosphorylated 53 kDa enzyme could be identified after receptor activation by immunoprecipitation. The time course of phosphorylation was very similar to that observed for carbachol (or UTP)-induced enzyme activation. Enzyme phosphorylation was prevented in the presence of okadaic acid. Calmodulin (CaM) kinase II inhibitors (i.e. KN-93 and KN-62) prevented phosphorylation of Ins(1,4,5)P3 3-kinase. Identification of the phosphorylation site in transfected CHO cells indicated that the phosphorylated residue was Thr311. This residue of the human brain sequence lies in an active site peptide segment corresponding to a CaM kinase II-mediated phosphorylation consensus site, i.e. Arg-Ala-Val-Thr. The same residue in Ins(1,4,5)P3 3-kinase A was also phosphorylated in vitro by CaM kinase II. Phosphorylation resulted in 8- to 10-fold enzyme activation and a 25-fold increase in sensitivity to the Ca2+:CaM complex. In this study, direct evidence is provided for a novel regulation mechanism for Ins(1,4,5)P3 3-kinase (isoform A) in vitro and in intact cells.
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Institute of Interdisciplinary Research, Free University of Brussels, Belgium. dcommuni@ulb.ac.be
Vanweyenberg
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Erneux
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D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002540:Cerebral Cortex / Q000201:enzymology*; D006224:Cricetinae; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058473:Receptors, Purinergic P2Y2; D013912:Threonine / Q000378:metabolism; D014544:Uridine Triphosphate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D018721:Muscarinic Agonists; C546385:P2RY2 protein, human; C546387:P2ry2 protein, rat; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D019319:Okadaic Acid; D013912:Threonine; D002217:Carbachol; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium; D014544:Uridine Triphosphate