Catalytic properties of inositol trisphosphate kinase: activation by Ca 2+ and calmodulin

Abstract

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P₃) is an important second-messenger molecule that mobilizes Ca²⁺ from intracellular stores in response to the occupancy of receptor by various Ca²⁺ -mobilizing agonists. The fate of Ins-1,4,5-P₃ is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P₃ to Ins-1,3,4,5-P₄, whereas the latter forms Ins-1,4-P₂. Recent studies suggest that Ins-1,3,4,5-P₄ might modulate the entry of Ca²⁺ from an extracellular source. In the current report, we describe the partial purification of the 3-kinase [ ~ 400-fold purified, specific activity = 0.12 μmol/(min · mg)] from the cytosolic fraction of bovine brain and studies of its catalytic properties. We found that the 3-kinase activity is significantly activated by the Ca²⁺/calmodulin complex. Therefore, we propose that Ca²⁺ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P₃ forms a complex with calmodulin, and that the Ca²⁺/calmodulin complex stimulates the conversion of Ins-1,4,5-P₃, an intracellular Ca²⁺ mobilizer, to Ins-1,3,4,5-P₄, an extracellular Ca²⁺ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-³² P]Ins-1,3,4,5-P₄ and [γ-³²P]ATP by thin-layer chromatography. Using this new assay method, we evaluated kinetic parameters (K_m for ATP = 40 μm, K_m for Ins-1,4,5-P₃ = 0.7 μm, K_i for ADP - 12 μm) and divalent cation specificity (Mg²⁺ > > Mn²⁺ > Ca²⁺) for the 3-kinase. Studies with various inositol polyphosphates indicate that the substrate-binding site is quite specific to Ins-1,4,5-P₃. Nevertheless, Ins-2,4,5-P₃ could be phosphorylated at a velocity approximately 1/20-1/30 that of Ins-1,4,5-P₃.—Ryu, S. H.; Lee, S. Y.; Lee, K.-Y.; Rhee, S. G. Catalytic properties of inositol trisphosphate kinase: activation by Ca²⁺ and calmodulin. FASEB J. 1: 388-393; 1987.