The Ins(1,3,4)P3 5/6-kinase/Ins(3,4,5,6)P4 1-kinase is not a protein kinase.
Among inositol phosphate kinases, Ins(3,4,5,6)P4 1-kinase has been considered to be an outsider with disparate sequence, a proclaimed capacity to also phosphorylate proteins and apparent 1-phosphatase activity. Such multifunctionality, coupled with ignorance of its operational domains, complicates any mechanistic rationale behind literature reports that Ins(3,4,5,6)P4 1-kinase regulates apoptosis, salt and fluid secretion, and transcription. We have expressed poly(His)-tagged human Ins(3,4,5,6)P4 1-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)P4 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 1-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, Lys157, Asp281, Asp295 and Asn297, severely compromised Ins(3,4,5,6)P4 1-kinase activity. Yet, these mutations did not affect the protein kinase activity. We conclude that the Ins(3,4,5,6)P4 1-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 1-kinase and other inositol phosphate kinases are closer than previously thought [Gonzalez, Schell, Letcher, Veprintsev, Irvine and Williams (2004) Mol. Cell 15, 689-701].
Qian
Xun
X
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, DHSS, Research Triangle Park, PO Box 12233, NC 27709, USA.
Mitchell
Jennifer
J
Wei
Sung-Jen
SJ
Williams
Jason
J
Petrovich
Robert M
RM
Shears
Stephen B
SB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D017124:Conserved Sequence; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C103909:inositol-tetrakisphosphate 1-kinase; C056875:myo-inositol-trisphosphate 6-kinase