Calcium-calmodulin-dependent protein kinase II and protein kinase C-mediated phosphorylation and activation of D-myo-inositol 1,4,5-trisphosphate 3-kinase B in astrocytes

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) 3-kinase catalyzes the p roduction of D-myo-inositol 1,3,4,5-tetrakisphosphate from the second messe nger Ins (1,4,5)P-3. Transient and okadaic acid-sensitive activation of Ins (1,4,5)P-3 3-kinase by 8-10-fold is observed in homogenates prepared from r at cortical astrocytes after incubation with either carbachol or UTP. 12-O- Tetradecanoylphorbol-13-acetate provokes the activation of Ins(1,4,5)P-3 3- kinase by a-fold in both cell systems. The kinase was purified by calmoduli n-Sepharose from the two cell systems. Enzyme activity corresponding to the silver-stained 88-kDa protein could be regenerated after SDS-polyacrylamid e gel electrophoresis. Antibodies to two distinct peptides chosen in the pr imary structure of human Ins(1,4,5)P-3 3-kinase B recognized the astrocytic native isoform. In [P-32]orthophosphate-preincubated cells, a major phosph orylated 88-kDa enzyme could be purified and identified in cells in respons e to receptor activation or 12-O-tetradecanoylphorbol-13-acetate treatment. Calmodulin kinase II inhibitors (i.e. KN-93 and KN-62) and a protein kinas e C inhibitor (i.e. calphostin C) prevented the phosphorylation of the 88-k Da isoenzyme. In addition to enzyme activation, a redistribution of Ins(1,4 ,5)P-3 3-kinase from soluble to particulate fraction of astrocytes was obse rved. In vitro phosphorylation of the purified enzyme by calmodulin kinase II and protein kinase C added together resulted in a maximal 60-70-fold act ivation.