PPIP5K1 modulates ligand competition between diphosphoinositol polyphosphates and PtdIns(3,4,5)P₃ for polyphosphoinositide-binding domains

We describe new signalling consequences for PPIP5K1 (diphosphoinositol pentakisphosphate kinase type 1)-mediated phosphorylation of InsP₆ and 5-InsP₇ to 1-InsP₇ and InsP₈. In NIH 3T3 cells, either hyperosmotic stress or receptor activation by PDGF (platelet-derived growth factor) promoted translocation of PPIP5K1 from the cytoplasm to the plasma membrane. The PBD1 (polyphosphoinositide-binding domain) in PPIP5K1 recapitulated that translocation. Mutagenesis of PBD1 to reduce affinity for PtdIns(3,4,5)P₃ prevented translocation. Using surface plasmon resonance, we found that PBD1 association with vesicular PtdIns(3,4,5)P₃ was inhibited by InsP₆ and diphosphoinositol polyphosphates. However, the inhibition by PPIP5K1 substrates (IC₅₀: 5-InsP₇=5 μM and InsP₆=7 μM) was substantially more potent than that of the PPIP5K1 products (IC₅₀: InsP₈=32 μM and 1-InsP₇=43 μM). This rank order of ligand competition with PtdIns(3,4,5)P₃ was also exhibited by the PH (pleckstrin homology) domains of Akt (also known as protein kinase B), GRP1 (general receptor for phosphoinositides 1) and SIN1 (stress-activated protein kinase-interaction protein 1). We propose that, in vivo, PH domain binding of InsP₆ and 5-InsP₇ suppresses inappropriate signalling (‘noise’) from stochastic increases in PtdIns(3,4,5)P₃. That restraint may be relieved by localized depletion of InsP₆ and 5-InsP₇ at the plasma membrane following PPIP5K1 recruitment. We tested this hypothesis in insulin-stimulated L6 myoblasts, using mTOR (mechanistic/mammalian target of rapamycin)-mediated phosphorylation of Akt on Ser⁴⁷³ as a readout for SIN1-mediated translocation of mTORC (mTOR complex) 2 to the plasma membrane [Zoncu, Efeyan and Sabatini (2011) Nat. Rev. Mol. Cell Biol. 12, 21–35]. Knockdown of PPIP5K1 expression was associated with a 40% reduction in Ser⁴⁷³ phosphorylation. A common feature of PtdIns(3,4,5)P₃-based signalling cascades may be their regulation by PPIP5K1.