title: 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.
abstract: D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase catalyzes the production of D-myo-inositol 1,3,4,5-tetrakisphosphate from the second messenger Ins (1,4,5)P3. Transient and okadaic acid-sensitive activation of Ins(1,4,5)P3 3-kinase by 8-10-fold is observed in homogenates prepared from rat cortical astrocytes after incubation with either carbachol or UTP. 12-O-Tetradecanoylphorbol-13-acetate provokes the activation of Ins(1,4,5)P3 3-kinase by 2-fold in both cell systems. The kinase was purified by calmodulin-Sepharose from the two cell systems. Enzyme activity corresponding to the silver-stained 88-kDa protein could be regenerated after SDS-polyacrylamide gel electrophoresis. Antibodies to two distinct peptides chosen in the primary structure of human Ins(1,4,5)P3 3-kinase B recognized the astrocytic native isoform. In [32P]orthophosphate-preincubated cells, a major phosphorylated 88-kDa enzyme could be purified and identified in cells in response to receptor activation or 12-O-tetradecanoylphorbol-13-acetate treatment. Calmodulin kinase II inhibitors (i.e. KN-93 and KN-62) and a protein kinase C inhibitor (i.e. calphostin C) prevented the phosphorylation of the 88-kDa isoenzyme. In addition to enzyme activation, a redistribution of Ins(1,4,5)P3 3-kinase from soluble to particulate fraction of astrocytes was observed. In vitro phosphorylation of the purified enzyme by calmodulin kinase II and protein kinase C added together resulted in a maximal 60-70-fold activation.
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats