title: Expression, purification, and regulation of two isoforms of the inositol 1,4,5-trisphosphate 3-kinase.
abstract: The level of inositol 1,4,5-trisphosphate in the cytoplasm is tightly regulated by two enzymes, the inositol 1,4,5,5-phosphatase and the inositol 1,4,5-trisphosphate 3-kinase. Two isoforms of the inositol 1,4,5-trisphosphate 3-kinase have been identified, the A form and the B form. The regulatory properties of the two isoforms were compared following overexpression and purification of the proteins from a v-src transformed mammalian cell line. The highly purified, recombinant inositol 1,4,5-trisphosphate 3-kinases were differentially regulated by calcium/calmodulin and via phosphorylation by protein kinase C or the cyclic AMP-dependent protein kinase. Both enzymes had similar affinities for inositol 1,4, 5-trisphosphate (Km 2-5 mu M). Calcium/calmodulin stimulated the activity of isoform A about 2.5-fold, whereas the activity of isoform B was increased 20-fold. The cyclic AMP-dependent protein kinase phosphorylated the inositol 1,4,5-trisphosphate 3-kinase A to the extent of 0.9 mol/mol and isoform B to 1 mol/mol. Protein kinase C phosphorylated isoform A to the extent of 2 mol/mol and isoform B to 2.7 mol/mol. Phosphorylation of isoform A by the cyclic AMP-dependent protein kinase caused a 2.5-fold increase in its activity when assayed in the absence of calcium/calmodulin, whereas phosphorylation by protein kinase C decreased activity by 72%. The activity of isoform B in the absence of calcium/calmodulin was not affected by phosphorylation using either kinase. When assayed in the presence of calcium/calmodulin, phosphorylation of isoform A by the cyclic AMP-dependent protein kinase increased activity 1.5-fold, whereas phosphorylation of isoform B decreased activity by 45%. Phosphorylation of either isoform A or B by protein kinase C resulted in a 70% reduction of calcium/calmodulin-stimulated activity. Differential expression and regulation of the two inositol 1,4,5-trisphosphate 3-kinase isoforms provides multiple mechanisms for regulating the cytosolic level of inositol 1,4,5-trisphosphate in cells.
authors.affiliation: Department of Pharmacology and Cancer Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. jcg8w@virginia.edu
authors.forename: P J
authors.initials: PJ
authors.lastname: Woodring
authors.forename: J C
authors.initials: JC
authors.lastname: Garrison
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002478:Cells, Cultured; D002850:Chromatography, Gel; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats
chemical_list: D002147:Calmodulin; D007527:Isoenzymes; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium