title: Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain.
abstract: The inositol pyrophosphates are multifunctional signalling molecules. One of the families of enzymes that synthesize the inositol pyrophosphates are the Vip1/PPIP5Ks (PP-InsP5 kinases). The kinase domains in Vip1/PPIP5Ks have been mapped to their N-terminus. Each of these proteins also possess a phosphatase-like domain of unknown significance. In the present study, we show that this phosphatase-like domain is not catalytically active. Instead, by using SPR (surface plasmon resonance) to study protein binding to immobilized lipid vesicles, we show that this domain is specialized for binding PtdIns(3,4,5)P3 (PPIP5K1 K(d)=96 nM; PPIP5K2 K(d)=705 nM). Both PtdIns(3,4)P2 and PtdIns(4,5)P2 are significantly weaker ligands, and no significant binding of PtdIns(3,5)P2 was detected. We confirm the functional importance of this domain in inositol lipid binding by site-directed mutagenesis. We present evidence that the PtdIns(3,4,5)P3-binding domain is an unusual hybrid, in which a partial PH (pleckstrin homology) consensus sequence is spliced into the phosphatase-like domain. Agonist-dependent activation of the PtdIns 3-kinase pathway in NIH 3T3 cells drives translocation of PPIP5K1 from the cytosol to the plasma membrane. We have therefore demonstrated receptor-regulated compartmentalization of inositol pyrophosphate synthesis in mammalian cells.
authors.affiliation: Inositol Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA.
authors.forename: Nikhil A
authors.initials: NA
authors.lastname: Gokhale
authors.forename: Angelika
authors.initials: A
authors.lastname: Zaremba
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Shears
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D016384:Consensus Sequence; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D020349:Surface Plasmon Resonance
chemical_list: D008024:Ligands; D018129:Phosphatidylinositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C040985:phosphatidylinositol receptors; D017856:Phosphotransferases (Phosphate Group Acceptor); C407349:diphosphoinositol pentakisphosphate kinase