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

Abstract

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.

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms

“…Alternatively, Shears et al have recently put forward the interesting idea that signaling by PP-InsPs is evolutionarily ancient, and, as a result, the molecules InsP 6 and 5PP-InsP 5 may have partially overlapping functions (7). Very similar affinities of InsP 6 and 5PP-InsP 5 were, for example, observed toward PtdInsP 3 -binding domains (59). It was proposed that more stereospecific interactions of PP-InsP messengers may have evolved more recently with the advent of PPIP5 kinases (7), which add a β-phosphoryl group to InsP 6 and 5PP-InsP 5 at the 1-position, yielding the metabolites 1PP-InsP 5 and 1,5(PP) 2 -InsP 4 , respectively.…”

Neuronal migration is mediated by inositol hexakisphosphate kinase 1 via α-actinin and focal adhesion kinase

“…IP7 mediates several physiological functions. For instance, 5-IP7 is required for insulin secretion (7), and both 5-IP7 and 1-IP7 regulate PIP3 signaling pathways (8). The three IP6Ks generate a single isomer of 5-IP7 whose pyrophosphate bond occurs at C-5, but which arise from distinct genes and mediate diverse functions.…”

Towards pharmacological intervention in inositol pyrophosphate signalling

“…Current evidence supports two different molecular mechanisms of action of PP-InsPs: association with specific ‘receptors’ [18,21–24] and the ability to non-enzymatically phosphorylate certain proteins [25,26] (since β -phosphates of PP-InsPs are transferred to a phosphoserine residue, the net result is actually protein pyrophosphorylation [25]). 5-InsP 7 , 1-InsP 7 and InsP 8 each appear to be equally effective as phosphate donors [25].…”

“…For example, Saccharomyces cerevisiae express a cyclin kinase regulator-protein that is activated by 1-InsP 7 , but not by 5-InsP 7 [18]. There is also selectivity in the inhibition by PP-InsPs of the binding of PtdIns(3,4,5) P 3 to pleckstrin-homology domains: 5-InsP 7 generally appears to be more potent than both 1-InsP 7 and InsP 8 [24]. The large number of PtdIns(3,4,5)P 3 -binding proteins that 5-InsP 7 may regulate [23,24] could contribute to PP-InsP multifunctionality.…”