Integration of Inositol Phosphate Signaling Pathways via Human ITPK1

Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2007; 282(38):28117-25. DOI: 10.1074/jbc.M703121200
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Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.

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    • "Several inositol phosphate kinases have been shown to be physiologically reversible, that is, their relative rates of phosphorylation and dephosphorylation can be influenced by changes in cellular [ATP]/[ADP] ratios: IP6K [26], InsP5 2-kinase [31] and ITPK1 (inositol 1,3,4-trisphosphate 5/6-kinase) [47]. A previous report [25] has included the PPIP5Ks in this category. "
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    ABSTRACT: We obtained detailed kinetic characteristics - stoichiometry, reaction rates, substrate affinities, and equilibrium conditions - of human PPIP5K2, which synthesizes "high-energy" diphosphoinositol polyphosphates (PP-InsPs) by metabolizing InsP6 and 5-InsP7 to 1-InsP7 and InsP8, respectively. These data increase our insight into the PPIP5K2 reaction mechanism and clarify the interface between PPIP5K catalytic activities and cellular bio-energetic status. For example, stochiometric analysis uncovered non-productive, substrate-stimulated ATPase activity (thus, approximately 2 and 1.2 ATP molecules are utilized to synthesize each molecule of 1-InsP7 and InsP8, respectively). Impaired ATPase activity of a PPIP5K2-K248A mutant increased atomic-level insight into the enzyme's reaction mechanism. We found PPIP5K2 to be fully reversible as an ATP-synthase in vitro, but our new data contradict previous perceptions that significant "reversibility" occurs in vivo. PPIP5K2 was insensitive to physiological changes in either [AMP] or [ATP]/[ADP] ratios. Those data, together with adenine nucleotide kinetics (ATP Km = 20-40 µM), reveal how insulated PPIP5K2 is from cellular bio-energetic challenges. Finally, the specificity constants for PPIP5K2 revise upwards by one-to-two orders of magnitude the inherent catalytic activities of this enzyme, and we show its equilibrium point favors 80-90% depletion of InsP6/5-InsP7.
    Bioscience Reports 12/2012; 33(2). DOI:10.1042/BSR20120115 · 2.64 Impact Factor
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    • "Therefore, the primary objective of the present study was to examine whether the novel Ca 2+ dependent Cl -channel TMEM16A is the target for INO-4995. Studies demonstrated that intracellular Ins(3,4,5,6)P4 levels are a result of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) dephosphorylation of Ins(1,3,4,5,6)P5 and are regulated by Ins(1,3,4)P3 levels (Menniti et al., 1990; Chamberlain et al. 2007; Saiardi and Cockcroft, 2008). Enzymes that generate inositolphosphate intermediates may therefore determine the magnitude and duration of CaCC (Vajanaphanich et al., 1994; Traynor-Kaplan et al., 2010). "
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    ABSTRACT: Background and purpose: Ca(2+)-dependent Cl(-) secretion (CaCC) in airways and other tissues is due to activation of the Cl(-) channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca(2+) -activated Cl(-) channels are regulated by membrane lipid inositol phosphates, and that 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester (INO-4995) augments CaCC. Here we examined whether TMEM16A is the target for INO-4995 and if the channel is regulated by inositol phosphates. Experimental approach: The effects of INO-4995 on CaCC were examined in overexpressing HEK293, colonic and primary airway epithelial cells as well as Xenopus oocytes. We used patch clamping, double electrode voltage clamp and Ussing chamber techniques. Key results: We found that INO-4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9 nS pF(-1) in overexpressing cells. The tetrakisphosphates Ins(3,4,5,6)P(4) or Ins(1,3,4,5)P(4) and enzymes controlling levels of InsP(4) or PIP(2) and PIP(3) had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in Xenopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, Cl(-) currents were not acutely activated by INO-4995. However incubation with INO-4995 augmented 1.6- to 4-fold TMEM16A-dependent Cl(-) currents activated by ionomycin or ATP, while intracellular Ca(2+) signals were not affected. The potentiating effect of INO-4995 on transient ATP-activated TMEM16A-currents in cystic fibrosis (CF) airways was twice of that observed in non-CF airways. Conclusions and implications: These data indicate that TMEM16A is the target for INO-4995, although the mode of action appears different for overexpressed and endogenous channels. INO-4995 may be useful for the treatment of CF lung disease.
    British Journal of Pharmacology 09/2012; 168(1). DOI:10.1111/j.1476-5381.2012.02193.x · 4.84 Impact Factor
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    • "illuminate the bases for these activities (Chamberlain et al., 2007; Miller et al., 2005). On the other hand, I(1,4,5)P 3 3-kinases have been identified in mammals and Drosophila. "

    Advances in enzyme regulation 12/2009; 50(1):324-37. DOI:10.1016/j.advenzreg.2009.12.002
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