Properties of the Inositol 3,4,5,6-Tetrakisphosphate 1-Kinase Purified from Rat Liver

Abstract

Inositol 3,4,5,6-tetrakisphosphate is a novel intracellular signal that regulates calcium-dependent chloride conductance (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097). The molecular mechanisms that regulate the cellular levels of this signal are not characterized. To pursue this problem we have now studied the 1-kinase that deactivates inositol 3,4,5,6-tetrakisphosphate. The enzyme was purified from rat liver 1600-fold with a 1% yield. The native molecular mass was determined to be 46 kDa by gel filtration. The Km values for inositol 3,4,5,6-tetrakisphosphate and ATP were 0. 3 and 10.6 microM, respectively. The kinase was unaffected by either protein kinase A or protein kinase C. Increases in Ca2+ concentration from 0.1 to 1-2 microM inhibited activity by 10-20%. Most importantly, inositol 1,3,4-trisphosphate was shown to be a potent (Ki = 0.2 microM), specific, and competitive inhibitor of the 1-kinase. Our new kinetic data show that typical receptor-dependent adjustments in cellular levels of inositol 1,3,4-trisphosphate provide a mechanism by which the concentration of inositol 3,4,5,6-tetrakisphosphate is dependent on changes in phospholipase C activity. These conclusions also provide a new perspective to our understanding of the physiological importance of the pathway of inositol phosphate turnover initiated by the inositol 1,4, 5-trisphosphate 3-kinase.

Mammalian inositol polyphosphate multikinase synthesizes inositol 1,4,5-trisphosphate and an inositol pyrophosphate

“…Thus, the ability of mIPMK to phosphorylate at positions 1, 3, and 6 might account for such activities reported with a variety of substrates that include some that we have not directly examined ourselves. For instance, Shears and associates (29)(30)(31) reported enzymatic activity in rat that phosphorylates various inositol phosphates at 1, 3, 5, and 6 positions and might reflect mIPMK activity.…”

Regulation of Ca2+‐dependent Cl− conductance in a human colonic epithelial cell line (T84): cross‐talk between Ins(3,4,5,6)P4 and protein phosphatases

“…This is particularly significant from a cell-signalling perspective, because the cellular levels of Ins (3,4,5,6)PÚ are controlled by occupation of appropriate cell-surface receptors. For example, the concentration of Ins (3,4,5,6)PÚ in TÞÚ cells increases from 1 ìÒ to as much as 10-15 ìÒ following PLC activation (Vajanaphanich et al 1995), due to modification of the poise of the Ins(1,3,4,5,6)PÛ 1-phosphatase-Ins(3,4,5,6)PÚ 1_kinase substrate cycle (Shears, 1997;Tan et al 1997). …”

Neural tube defects in mice with reduced levels of inositol 1,3,4-trisphosphate 5/6-kinase

“…In mammalian cells, ITPK1 uses inositol 1,3,4-trisphosphate [I(1,3,4)P 3 ] as a substrate to generate inositol 1,3,4,5-tetrakisphosphate (IP 4 ) and inositol 1,3,4,6-P 4 (14), which is further phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (IP 5 ) by a 5-kinase (15,16) and then to IP 6 by a 2-kinase (17,18). ITPK1 also can phosphorylate inositol 3,4,5,6-P 4 [I(3,4,5,6)P 4 ] at the 1-position (19,20), as well as function as a phosphatase and as an isomerase (21)(22)(23). Overexpression of ITPK1 leads to increased levels of IP 4 isomers, IP 5 , and IP 6 , and depletion of ITPK1 by RNAi results in decreased levels of IP 4 isomers, IP 5 , and IP 6 (18).…”