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)P-3] 3-kinase, the enzym e responsible for production of D-myoinositol 1,3,4,5-tetrakisphosphat e, was activated 3- to 5-fold in homogenates of rat brain cortical sli ces after incubation with carbachol. The effect was reproduced in resp onse to UTP in Chinese hamster ovary (CHO) cells overexpressing Ins(1, 4,5)P-3 3-kinase A, the major isoform present in rat and human neurona l cells, In ortho-P-32-labelled cells, the phosphorylated 53 kDa enzym e 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 phosphorylat ion was prevented in the presence of okadaic acid. Calmodulin (CaM) ki nase II inhibitors (i.e. KN-93 and KN-62) prevented phosphorylation of Zns(1,4,5)P-3 3-kinase. Identification of the phosphorylation site in transfected CHO cells indicated that the phosphorylated residue was T hr311. This residue of the human brain sequence lies in an active site peptide segment corresponding to a CaM kinase II-mediated phosphoryla tion consensus site, i.e. Arg-Ala-Val-Thr. The same residue in Ins(1,4 ,5)P-3 3-kinase A was also phosphorylated in vitro by CaM kinase II, P hosphorylation resulted in 8- to 10-fold enzyme activation and a 25-fo ld increase in sensitivity to the Ca2+:CaM complex. In this study, dir ect evidence is provided for a novel regulation mechanism for Ins(1,4, 5)P-3 S-kinase (isoform A) in vitro and in intact cells.