摘要 : Among inositol phosphate kinases, Ins(3,4,5,6)P-4 1-kinase has been considered to be an outsider with disparate sequence, a proclaimed capacity to also phosphorylate proteins and apparent I-phosphatase activity. Such multifunction... 展开 Among inositol phosphate kinases, Ins(3,4,5,6)P-4 1-kinase has been considered to be an outsider with disparate sequence, a proclaimed capacity to also phosphorylate proteins and apparent I-phosphatase activity. Such multifunctionality, coupled with ignorance of its operational domains, complicates any mechanistic rationale behind literature reports that Ins(3,4,5,6)P, 1-kinase regulates apoptosis, salt and fluid secretion, and transcription. We have expressed poly(His)-tagged human Ins(3,4,5,6)P4 I-kinase in Sf9 insect cells and purified the enzyme using Ni-agarose chromatography. Protein kinase activity was eluted from the Ni-agarose column, but this did not co-elute with the Ins(3,4,5,6)P, 1-kinase, indicating that the protein kinase and inositol kinase activities belong to separate proteins. To pursue this conclusion, we prepared catalytically inactive mutants of the Ins(3,4,5,6)P4 I-kinase by identifying and targeting the ATP-binding site. Our strategy was based on sequence alignments suggesting homology of the Ins(3,4,5,6)P4 1-kinase with ATP-grasp metabolic enzymes. Individual mutation of four candidate MgATP-binding participants, Lys(157), Asp(281), Asp(295) and Asn(297), severely compromised Ins(3,4,5,6)P, I-kinase activity. Yet, these mutations did not affect the protein kinase activity. We conclude that the Ins(3,4,5,6)P4 I-kinase is not a protein kinase, contrary to earlier reports [e.g. Wilson, Sun, Cao and Majerus (2001) J. Biol. Chem. 276, 40998-41004]. Elimination of protein kinase activity from the enzyme's repertoire and recognition of its ATP-grasp homology together indicate that structural, functional and catalytic relationships between Ins(3,4,5,6)P4 I-kinase and other inositol phosphate kinases are closer than previously thought [Gonzalez, Schell, Letcher, Veprintsev, Irvine and Williams (2004) Mol. Cell 15, 689-701]. 收起