Alterations in an inositol phosphate code through synergistic activation of a G protein and inositol phosphate kinases

Abstract

In mammals, many cellular stimuli evoke a response through G protein activation of phospholipase C, which results in the lipidderived production of inositol 1,4,5-trisphosphate (IP3). Although it is well established that IP3 is converted to numerous inositol phosphates (IPs) and pyrophosphates (PP-IPs) through the action of up to six classes of inositol phosphate kinases (IPKs), it is not clear that these metabolites are influenced by G protein signaling. Here we report that activation of G␣q leads to robust stimulation of IP3 to IP8 metabolism. To expose flux through these pathways, genetic perturbation was used to alter IP homeostasis. Coupled expression of a constitutively active G␣qQL and one or more IPK gene products synergistically generated dramatic changes in the patterns of intracellular IP messengers. Many distinct IP profiles were observed through the expression of different combinations of IPKs, including changes in previously unappreciated pools of IP 5 and IP6, two molecules widely viewed as stable metabolites. Our data link the activation of a trimeric G protein to a plethora of metabolites downstream of IP3 and provide a framework for suggesting that cells possess the machinery to produce an IPK-dependent IP code. We imply, but do not prove, that agonist-induced alterations in such a code would theoretically be capable of enhancing signaling complexity and specificity. The essential roles for IPKs in organism development and cellular adaptation are consistent with our hypothesis that such an IP code may be relevant to signaling pathways.Gq ͉ metabolomics ͉ phospholipase C ͉ signal transduction ͉ G protein-coupled receptor I n mammals, hundreds of G protein-coupled receptors (GPCR) and receptor tyrosine kinases (RTKs) are present and link to numerous intracellular signaling modules that, in combination, enable selective cellular responses. One of the most commonly activated signaling modules is the generation of the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and 1,2-diacylglycerol by the action of phospholipase C isozymes (PLCs) on phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (1-5). The ubiquity of PLC activation in response to a wide range of stimuli supports its central role in signaling, but also emphasizes that the production of two messengers alone, IP 3 and diacylglycerol, is insufficient to encode the breadth of specific cellular responses necessary for survival and adaptation. Therefore, it has been postulated that further signaling diversity could theoretically arise from the conversion of IP 3 to numerous inositol tetrakisphosphate (IP 4 ), inositol pentakisphosphate (IP 5 ), inositol hexakisphosphate (IP 6 ), and inositol pyrophosphate (PP-IP) molecules (2, 6-9). The production of these regulators occurs through the action of up to six classes of evolutionarily conserved IP kinase (IPK) gene products that include: IPMK/IPK2 (10-14), IPK1 (15-17), , ITPK/IP56K (21, 22), IP6K/ IHPK (23-25), and VIP1 (25-27) (Fig. 1). Genetic and biochemical studies of IPKs have provided v...

Structural Analysis and Detection of Biological Inositol Pyrophosphates Reveal That the Family of VIP/Diphosphoinositol Pentakisphosphate Kinases Are 1/3-Kinases

“…1 H-decoupled 31 P NMR spectra were recorded at 202.3 MHz with a spectral window of 12,143.3 Hz digitized into 96,000 data points (digital resolution of 0.253 Hz/point), a 60°pulse flip angle (20 s), and a 4.96-s total repeat time. Inverse decoupled difference spectra were recorded as 1 H-detected 31 P-decoupled heteronuclear NMR experiments, as previously described (27,28).…”

“…It has previously (12,15,16,31) been determined that the IP6K and VIP/PPIP5K enzymes work in concert to generate (PP) 2 -IP 4 . Thus, when IP 6 was incubated with both human IP6K and ScVip1, three products accumulated (labeled peaks i, ii, and iii in Fig.…”

“…4 by Two-dimensional NMR-Provided that milligram quantities of pure inositol phosphates can be prepared, NMR can be used to analyze their structure (32,33). We used 31 P and 1 H one-and two-dimensional NMR analysis to determine the structures of the two inositol pyrophosphates (PP-IP 5 and (PP) 2 -IP 4 ) produced by the VIP1/PPIP5K family (see "Experimental Procedures" and Fig. 3).…”

“…Indeed, the VIP/PPIP5K family appears to account for a distinct PP-IP 5 kinase activity that was observed initially in experiments with cell lysates (17,18) and subsequently in yeast mutants lacking Kcs1 (19). Previous one-dimensional 31 P NMR studies demonstrated that yeast Vip1 phosphorylates IP 6 to a novel isomer of PP-IP 5 that is different from the 5-PP-IP 5 produced by recombinant human IP6K (12). However, the isomeric nature of this new PP-IP 5 isomer could not be unequivocally established by one-dimensional NMR (it was tentatively suggested to be 4/6-PP-IP 5 (12)).…”

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

“…9E). A more precise spatiotemporal understanding of the inter-relationships between P i and InsP 8 in vivo requires additional information that is not readily obtained: a quantitative resolution of their separation into different cellular pools (2,43). Furthermore, differential levels of expression of PPIP5K1 versus PPIP5K2 could contribute to quantitative and qualitative cell type-specific differences in InsP 8 responses to extracellular [P i ] because this anion only stimulates the kinase domain of PPIP5K2 and not that in PPIP5K1 (Fig.…”