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A Conserved Family of Enzymes That Phosphorylate Inositol Hexakisphosphate

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Science  06 Apr 2007:
Vol. 316, Issue 5821, pp. 106-109
DOI: 10.1126/science.1139099

Abstract

Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.

Activation of phosphoinositide-specific phospholipase C (PLC) generates an ensemble of intracellular inositol phosphate (IP) second messengers involved in diverse cellular processes (13). Among these is inositol 1,4,5-trisphosphate, which functions as a regulator of calcium release (4) and as a precursor to more highly phosphorylated IP molecules, including inositol tetrakis- (IP4), pentakis- (IP5), hexakis- (IP6), and inositol pyrophosphates (PP-IPs) (5). The PP-IPs have more phosphates than IP6 (6, 7) and were chemically identified as diphosphoryl inositol species (7, 8). Cloning of the inositol pyrophosphate synthase designated IP6 kinase (IP6K in metazoans and Kcs1 in budding yeast) (9, 10) provided a molecular basis for the synthesis of PP-IP5 (also known as IP7 or InsP7) through the coupled activities of PLC and inositol phosphate kinases (IPKs) (1113). Loss of IP6K results in pleiotropic cellular defects including aberrant DNA recombination, vacuolar morphology, gene expression, chemotaxis, osmotic stress, protein phosphorylation, and telomere length (5, 14). Cells also have a second IP6 kinase detected in kcs1 deficient cells whose molecular identity has yet to be reported (15, 16).

We used a biochemical approach to purify and clone the remaining IP6 kinase activity. To reduce interference by contaminating IP6 kinase and PP-IP pyrophosphatase activities, initial protein extracts were prepared from kcs1Δ ddp1Δ–deficient cells and then fractionated on a series of ion-exchange chromatography columns (tables S1 and S2). IP6 kinase activity was enriched by a factor of more than 3000. Partially purified peak fractions were separated by SDS–polyacrylamide gel electrophoresis, and individual protein bands were excised and subjected to microsequence analysis. Numerous proteins were identified, so we obtained and purified individual candidate proteins from the tandem-affinity purification (TAP)–tagged yeast collection and tested each for kinase activity (17). Of the 40 proteins examined, only the purified TAP-tagged Vip1 protein showed IP6 kinase activity (Fig. 1A). We inserted the entire coding sequence of Vip1 into a glutathione S-transferase (GST) expression plasmid and produced the protein in bacteria (which lack endogenous IP6 kinase activity). Purified recombinant GST-Vip1 also had IP6 kinase activity (Fig. 1A). These data indicate that Vip1 functions as an intrinsic inositol pyrophosphate synthase or IP6 kinase.

Fig. 1.

Characterization of inositol hexakisphosphate IP6 kinase enzyme, Vip1. (A) IP6 kinase activity of purified yeast TAP-Vip1 and the production of IP7 (left). Dose-dependent (ng of protein added) activity of recombinant GST-Vip1 (middle) and sensitivity of IP7 and IP8 products of TAP-Kcs1 and GST-Vip1 to the diphospho-inositol phosphatase GST-Ddp1 (right). (B) Schematic of the dual-domain structure of Vip1 and Asp1. Evolutionarily conserved residues from the ATP-grasp (IP6K) and putative acid phosphatase domains from either budding or fission yeast proteins are shown. (C) Function of Vip1 to regulate PP-IP5 and IP6 levels in cells. Loss of vip1 in kcs1 ddp1–null yeast ablated PP-IP5 production and increased IP6 by a factor of 2, as monitored by HPLC analysis of 3H-inositol–labeled mutant yeast strains (10 mM AP increased linearly to 1.7 M AP over 12 min, followed by an isocratic flow at 1.7 M AP for 48 min) and inline radioactivity detection. Synthesis of PP-IP5 was restored in cells expressing either GFP-tagged full-length Vip1 (kΔdΔvΔ + pVip1) or kinase-only (kΔdΔvΔ + pvip1H548A), but not kinase-deficient protein (kΔ dΔ vΔ + pvip1D487A).

The protein information annotation for Vip1 (Saccharomyces Genome Database), and its Schizosaccharomyces pombe ortholog Asp1 (18), identified two regions with relevance to inositol signaling enzymes (Fig. 1B): a “rimK” or adenosine triphosphate (ATP)–grasp superfamily domain SSF56059 (residues 200 to 525) and a histidine acid-phosphatase or phytase PF00328 domain (residues 530 to 1025). The ATP-grasp fold is present in certain inositol signaling kinases (19), and the acid-phosphatase fold is present in microbial and fungal phytase enzymes that hydrolyze IP6 to release inorganic phosphate (Pi) and IPs (20). Sequence alignments of Vip1 or Asp1 genes revealed that orthologs having both amino- and carboxyl-terminal domains were present in all eukaryotic genomes from yeast to man (figs. S1 and S2). Thus, the Vip1 and Asp1 proteins define a distinct class of IP6 kinase with a conserved dual-domain structure.

Our analyses indicated that the kinase domain of Vip1 might reside within the aminoterminal portion of the protein. Therefore we expressed a deletion mutant GST-Vip11-535 (residues 1 to 535) and performed kinase reactions. GST-Vip11-535 had IP6 kinase activity that equaled or exceeded that of full-length Vip1 (fig. S3A). With full-length or truncated Vip1 proteins, we observed that IP6 kinase activity was pH dependent, with optimum activity occurring at pH 6.2 (fig. S3B). These data demonstrate that the ATP-grasp domain of Vip1 is sufficient to encode the IP6 kinase activity.

To determine whether Vip1 functioned to produce IP7 in cells, we analyzed IP production in yeast mutants. The predominant IPs observed in kcs1 ddp1 null cells (kΔ dΔ) are IP6 and PP-IP5 (Fig. 1C) (16). Deletion of Vip1 in this background (vip1 kcs1 ddp1 triple null, kΔ dΔ vΔ) resulted in the ablation of PP-IP5 synthesis and increased the amount of IP6 (Fig. 1C). Production of PP-IP5 could be restored by introduction of a plasmid encoding the VIP1 promoter and coding sequence (Fig. 1C). We also identified that Vip1 was required to synthesize PP-IP4β in cells (fig. S3C). These observations are consistent with Vip1 functioning as a cellular inositol pyrophosphate synthase that phosphorylates both IP6 and I(1,2,3,4,5)P5 and indicate that Vip1 encodes activities previously designated as Ids1 (16) and Ips1 (15).

To identify catalytic residues in the kinase domain of Vip1, we used structure-based threading software [Protein Homology/analogY Recognition Engine (PHYRE) server, www.sbg.bio.ic.ac.uk/phyre]. Within the ATP-grasp domain, we identified aspartate 487 as a putative activesite catalytic residue (fig. S1B) and, indeed, substitution with alanine (Vip1D487A) rendered the mutant kinase defective in vivo (Fig. 1C) and in vitro. Similar analysis of the acid-phosphatase domain identified histidine 548 and 993 as invariant residues (fig. S2B). Substitution of H548 in Vip1 with alanine (Vip1H548A) did not appear to alter IP6 kinase activity in vitro (fig. S3A). Reintroduction of Vip1H548A into kcs1 ddp1 vip1 mutant yeast cells restored PP-IP5 and IP6 levels to those observed in kcs1 ddp1 mutant cells (Fig. 1C). These data identify a key catalytic residue for kinase activity and are consistent with Vip1 functioning as a regulator of IP6 and PP-IP5 levels in cells.

To determine the molecular structure of the Vip1 kinase reaction product, samples were subjected to nuclear magnetic resonance (NMR) analysis. Comparison of proton-decoupled phosphorus NMR spectra of high-performance liquid chromatography (HPLC)–purified IP6 standard (Fig. 2A) or PP-IP5 Vip1 product (Fig. 2B) revealed the appearance of five singlet peaks of monophosphates and two upfield phosphorus-phosphorus–coupled doublets corresponding to the β- and α-phosphates of a pyrophosphate moiety. The spectrum observed for the Vip1 PP-IP5 product is nearly identical to the reported spectrum for 4-PP-I(1,2,3,5,6)P5 or its enantiomer 6-PP-I(1,2,3,4,5)P5 purified from Dictyostelium (21). Two-dimensional 1H–31P–NMR correlation analysis was reported, allowing for unequivocal assignment of the pyrophosphate (21). We performed NMR analysis on the PP-IP5 product of human IP6K, which is reported to have a similar activity to that of Kcs1 (14, 16, 22). The spectrum of IP7 produced from an HPLC-purified IP6K reaction was distinct from Vip1 product (Fig. 2C). Tentative assignment was again made based on this spectrum's appearing to be similar to that reported for 5-PP-I(1,2,3,4,6)P5 (23). Overall, our NMR analysis confirms that the IP7 produced by Vip1 is structurally distinct from that produced by the IP6K class of proteins, and that Vip1 appears to harbor D-4 or D-6 kinase activity, or both (fig. S3A).

Fig. 2.

Identification of Vip1 PP-IP5 product by 31P NMR analysis and inositol heptakisphosphate kinase activity. Proton-decoupled phosphorus NMR spectrum of 2 mg of purified (A) IP6 standard, (B) Vip1 product PP-IP5, or (C) human IP6K1 PP-IP5 product. All analyses were performed on HPLC-purified samples at pH 5.8. Structures of relevant IP6 and IP7 species are shown: 4-PP-I(1,2,3,5,6)P5 and 6-PP-I(1,2,3,4,5)P5 are non-superimposable mirror images (enantiomers). Resonances have been assigned based on similarity to previous reports (21, 23). P, phosphate; PP, pyrophosphate. (D) Characterization of Vip1 and human IP6K IP7 kinase activity. Purified recombinant GST-Vip1 fusion proteins (ng of protein added are shown) were incubated with radiolabeled 32PP-IPs, ATP at pH 6.2, and reactants were visualized after separation by polyethyleneimine cellulose–thin-layer chromatography (left). All three kinase active forms of Vip1 showed robust activity and were able to convert 5-PP-I(1,2,3,4,6)P5 to 4,5-PP2-IP4 (IP8) at neutral pH. Recombinant human GST IP6K fusion protein was also examined and was found to phosphorylate 4-PP-IP5 to 4,5-PP2-IP4 (right). For simplicity, we have assigned the product of the Vip1 reactions to be the D-4 species. Given our analysis, we cannot exclude that these may also be the enantiomer species harboring a D-6 pyrophosphate. (E) Schematic diagram of IP6 metabolism pathway in Saccharomyces cerevisiae and production of IP7 and IP8 through the actions of Vip1 and Kcs1. Kcs1 functions as a D-5 kinase that may phosphorylate IP6 and 4-PP-IP5 substrates. Vip1 functions as an IP6 kinase to generate 4- or 6-PP-IP5 and phosphorylates the IP7 product of Kcs1 to form IP8. Ddp1 is an inositol pyrophosphate phosphatase that dephosphorylates IP7 or IP8 to IP6.

Given the specificities of the Vip1 and IP6K (Kcs1) kinases, we sought to examine their roles as IP7 kinases capable of generating IP8 (PP2-IP4), which has been reported in various eukaryotic cells (7, 8, 21, 24, 25). In IP6 kinase reactions with both GST-Vip1 and purified TAP-Kcs1, we observed the formation of IP7 and IP8, which were hydrolyzed to IP6 in the presence of the inositol pyrophosphatase Ddp1 (Fig. 1A). Incubation of 5-PP-IP5 along with ATP and recombinant GST-Vip1, GST-Vip11-535, or GST-Vip1H548A protein, resulted in the production of PP2-IP4, consistent with Vip1 functioning as a 5-PP-IP5 kinase capable of generating 4,5-PP2-I(1,2,3,6)P4 or 5,6-PP2-I(1,2,3,4)P4,or both (Fig. 2D). Similarly, when we incubated radiolabeled 4-PP-IP5 or 6-PP-IP5, ATP and recombinant human GST-IP6K, we observed conversion to PP2-IP4 product (Fig. 2D). These data indicate that Vip1 and IP6K (Kcs1) are the enzymes required for the synthesis of IP8 or PP2-IP4 from IP6 (Fig. 2E).

Clues into possible functional roles for the IP6 kinase activity of Vip1 came from studies of the S. pombe ortholog, Asp1, which may participate in actin cytoskeleton and cellular morphology through the regulation of actin-related protein (Arp) complexes (18). Arp2/3 multiprotein complexes are involved in cellular signaling–dependent control of nucleation of branched-actin-filament networks required for regulating cell motility and endocytosis (26, 27). Activation of Arp2/3 requires additional nucleation-promoting factors such as Wiskott-Aldrich syndrome protein (WASP) (28, 29).

We therefore examined whether these phenotypes depended on IP6 kinase activity. Wild-type and asp1 null strains were transformed with thiamine-regulated (nmt1 promoter) GFP-tagged expression plasmids encoding empty vector control (p3X), full-length protein (pASP1), or proteins with mutations in the kinase (pasp1D333A) or putative acid-phosphatase (pasp1H397A) domains. The temperature-sensitive growth and morphological defects exhibited by asp1 null cells were rescued by low-level expression (medium with thiamine) of either pASP1 or pasp1H397A, consistent with a role for IP6 kinase in mediating these phenotypes (Fig. 3, A and B). When these strains were induced to high-level expression (medium lacking thiamine), the kinase-only protein, pasp1H397A, prevented cell growth at both normal and high temperatures (Fig. 3, A and B). Cells overexpressing kinase-only protein underwent rapid lysis when analyzed by light microscopy (Fig. 3B). These data indicate that loss of IP6 kinase activity accounts for asp1 temperature sensitivity and morphological defects and that too much IP6 kinase is cytotoxic.

Fig. 3.

A role for IP6 kinase activity in regulating cell morphology, growth, and ARP complex function. (A) Asp1 IP6 kinase activity regulates cell growth of fission yeast. Loss of IP6 kinase inhibited growth at increased temperatures (left two panels). Overproduction of Asp1 IP6 kinase-only (pasp1H397A) protein (without thiamine) was toxic to cell growth at normal and elevated temperatures (middle and right two panels). Appropriate strains were serially diluted, spotted onto solid medium, and grown at the indicated temperature. (B) IP6 kinase activity regulates cell morphology, vacuole biogenesis, and cell lysis. Cells lacking Asp1 kinase activity (asp1 Δ + 3X or asp1 Δ + aspD333A) had rounded, vacuolated, and irregular cell morphology as compared with kinase-competent cells (asp1 Δ + ASP1, asp1 Δ + asp1H397A). Overproduction of IP6 kinase-only protein (without thiamine) resulted in the inhibition of cell growth and promoted cellular lysis (ASP1 + pasp1H397A). (Insets) Zoomed images. (C) Toxicity induced by IP6 kinase-only overexpression and suppression by loss of arp3. Cells overproducing asp1H397A fail to grow at 30°C (Arp3+); however, they were able to grow when shifted to a cold temperature (arp3). In contrast, when arp3 function is lost (19°C), cells overexpressing Asp1 IP6 kinase-dead protein (pasp1D333A) were unable to grow, and this defect is rescued by restoring Arp3 function (30°C). (D) Expression of Asp1 IP6 kinase-dead protein (pasp1D333A) results in Arp3-dependent defects in cell-wall deposition, as shown by microscopic analysis of cells stained by calcofluor white. Staining of cells overexpressing full-length Asp1 show normal cell wall synthesis regardless of Arp3 function. (E) Requirement of Vip1 IP6 kinase activity for genetic interactions with ARP complex member, Las17. A temperature-induced growth defect was observed upon loss of both Vip1 and Las17 (vΔlΔ, top), the WASP component of the ARP complex. This synthetic growth defect was rescued in the vip1 las17 double-mutant strain by restoring Vip1 IP6 kinase activity (bottom).

To probe whether ARP complex function was required for the observed effects, we examined the expression of wild-type or mutant ASP1 proteins in a cold-sensitive mutant Arp3 strain (arp3 cl). The toxicity induced by overexpression of IP6 kinase-only activity (pasp1H397A) was dependent on a functional Arp3 protein, as judged by growth at 19°C (arp3) versus death at 30°C (Arp3+) (Fig. 3C). Furthermore, when Arp3 function was lost, the overexpression of kinase-deficient Asp1 was now lethal, as determined by a failure of pasp1D333A-transformed cells to grow under induced conditions at 19°C (Fig. 3C). Analysis of these cells using the cell-wall stain calcofluor white (CFW) revealed that overproduction of kinase-deficient Asp1 resulted in aberrant appearance of cell-wall material at the growing ends rather than at the septum midline (Fig. 3D). We also examined interactions between Vip1 and Las17 (the yeast WASP protein) in budding yeast cell growth. Deletion of vip1 alone did not cause temperature sensitivity; however, when combined with a loss in las17, the double mutant was growth-compromised at high temperatures (Fig. 3E). The temperature-sensitive growth was rescued by restoring Vip1 IP6 kinase activity (Fig. 3E). Overall, our functional analyses indicate that IP6 kinase activity of Asp1 and Vip1 is required for maintaining cellular integrity, temperature-dependent growth, rod-shape morphology, and genetic interactions with ARP complex components.

We identified Vip1 and Asp1 as members of a class of IP6 kinase that is responsible for producing signaling molecules 4-PP-IP5 or 6-PP-IP5, or both, that regulate cell function and yeast phosphate-responsive signaling (30). We show that the Vip1 and Kcs1 (IP6K) kinases generate distinct inositol pyrophosphate products and that both can function as IP7 kinases, together capable of converting IP6 to IP8 (PP2-IP4). The dual-domain structure of the Vip1 class of enzymes is evolutionarily conserved, raising the possibility that there may be interplay between these two regions of the proteins to control intracellular signaling pathways.

Supporting Online Material

www.sciencemag.org/cgi/content/full/316/5821/106/DC1

Materials and Methods

Figs. S1 to S3

Tables S1 and S2

References

References and Notes

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