Adolfo Saiardi

Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.

Inositol (1,4,5) trisphosphate (Ins(1,4,5)P(3)) is a well-known messenger molecule that releases calcium from intracellular stores. Homologues with up to six phosphates have been characterized and recently, homologues with seven or eight phosphate groups, including pyrophosphates, have been identified. These homologues are diphosphoinositol pentakisphosphate (PP-InsP(5)/InsP(7)) and bis(diphospho)inositol tetrakisphosphate (bis-PP-InsP(4)/InsP(8)) [1], the rapid turnover of which [2] is regulated by calcium [2] and adrenergic receptor activity [3]. It has been proposed that the high-energy pyrophosphates might participate in protein phosphorylation [4]. We have purified InsP(6) kinase [5] and PP-InsP(5) kinase [6], both of which display ATP synthase activity, transferring phosphate to ADP. Here, we report the cloning of two mammalian InsP(6) kinases and a yeast InsP(6) kinase. Furthermore, we show that the yeast protein, ArgRIII, is an inositol-polyphosphate kinase that can convert InsP(3) to InsP(4), InsP(5) and InsP(6). We have identified a new family of highly conserved inositol-polyphosphate kinases that contain a newly identified, unique consensus sequence.

Inositol Pyrophosphates Mediate Chemotaxis in Dictyostelium via Pleckstrin Homology Domain-PtdIns(3,4,5)P3 Interactions

Inositol phosphates are well-known signaling molecules, whereas the inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7/IP7) and bis-diphosphoinositol tetrakisphosphate (InsP8/IP8), are less well characterized. We demonstrate physiologic regulation of Dictyostelium chemotaxis by InsP7 mediated by its competition with PtdIns(3,4,5)P3 for binding pleckstrin homology (PH) domain-containing proteins. Chemoattractant stimulation triggers rapid and sustained elevations in InsP7/InsP8 levels. Depletion of InsP7 and InsP8 by deleting the gene for InsP6 kinase (InsP6K/IP6K), which converts inositol hexakisphosphate (InsP6/IP6) to InsP7, causes rapid aggregation of mutant cells and increased sensitivity to cAMP. Chemotaxis is mediated by membrane translocation of certain PH domain-containing proteins via specific binding to PtdIns(3,4,5)P3. InsP7 competes for PH domain binding with PtdIns(3,4,5)P3 both in vitro and in vivo. InsP7 depletion enhances PH domain membrane translocation and augments downstream chemotactic signaling activity.

The Inositol Hexakisphosphate Kinase Family CATALYTIC FLEXIBILITY AND FUNCTION IN YEAST VACUOLE BIOGENESIS

Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323–1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP6) to diphosphoinositol pentakisphosphate, a “high energy” candidate regulator of cellular trafficking. We have now studied the significance of InsP6 kinase activity inSaccharomyces cerevisiae by disrupting the kinase gene. These ip6kΔ cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60–80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP6kinases were identified; inositol pentakisphosphate (InsP5) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP4), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP5(K m = 1.2 μm) and InsP6(K m = 6.7 μm) compete for phosphorylation in vivo. This is the first time a PP-InsP4 synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP6. Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.

Inositol pyrophosphates regulate endocytic trafficking.

The high energy potential and rapid turnover of the recently discovered inositol pyrophosphates, such as diphosphoinositol-pentakisphosphate and bis-diphosphoinositol-tetrakisphosphate, suggest a dynamic cellular role, but no specific functions have yet been established. Using several yeast mutants with defects in inositol phosphate metabolism, we identify dramatic membrane defects selectively associated with deficient formation of inositol pyrophosphates. We show that this phenotype reflects specific abnormalities in endocytic pathways and not other components of membrane trafficking. Thus, inositol pyrophosphates are major regulators of endocytosis.

Inositol polyphosphate multikinase (ArgRIII) determines nuclear mRNA export in Saccharomyces cerevisiae.

The ARGRIII gene of Saccharomyces cerevisiae encodes a transcriptional regulator that also has inositol polyphosphate multikinase (ipmk) activity [Saiardi et al. (1999) Curr. Biol. 9, 1323–1326]. To investigate how inositol phosphates regulate gene expression, we disrupted the ARGRIII gene. This mutation impaired nuclear mRNA export, slowed cell growth, increased cellular [InsP3] 170‐fold and decreased [InsP6] 100‐fold, indicating reduced phosphorylation of InsP3 to InsP6. Levels of diphosphoinositol polyphosphates were decreased much less dramatically than was InsP6. Low levels of InsP6, and considerable quantities of Ins(1,3,4,5)P4, were synthesized by an ipmk‐independent route. Transcriptional control by ipmk reflects that it is a pivotal regulator of nuclear mRNA export via inositol phosphate metabolism.

Identification and characterization of a novel inositol hexakisphosphate kinase

The inositol pyrophosphate disphosphoinositol pentakisphosphate (PP-InsP3/InsP7) is formed in mammals by two recently cloned inositol hexakiphosphate kinases, InsP6K1 and InsP6K2 (Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323–1326). We now report the identification, cloning, and characterization of a third InsP7 forming enzyme designated InsP6K3. InsP6K3 displays 50 and 45% sequence identity to InsP6K1 and InsP6K2, respectively, with a smaller mass (46 kDa) and a more basic character than the other two enzymes. InsP6K3 is most enriched in the brain where its localization resembles InsP6K1 and InsP6K2. Intracellular disposition discriminates the three enzymes with InsP6K2 being exclusively nuclear, InsP6K3 predominating in the cytoplasm, and InsP6K1 displaying comparable nuclear and cytosolic densities.

Inositol hexakisphosphate kinase-2, a physiologic mediator of cell death

Diphosphoinositol pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate contain pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by a family of three inositol hexakisphosphate kinases (InsP6K). In this study we establish one of the InsP6Ks, InsP6K2, as a physiologic mediator of cell death. Overexpression of wild-type InsP6K2 augments the cytotoxic actions of multiple cell stressors in diverse cell lines, whereas transfection with a dominant negative InsP6K2 decreases cell death. During cell death, InsP6 kinase activity is enhanced, and intracellular InsP7 level is augmented. Deletion of InsP6K2 but not the other forms of InsP6K diminishes cell death, suggesting that InsP6K2 is the major InsP6 kinase involved in cell death. Cytotoxicity is associated with a translocation of InsP6K2 from nuclei to mitochondria, whereas the intracellular localization of the other isoforms of the enzyme does not change. The present study provides compelling evidence that endogenous InsP6K2, by generating InsP7, provides physiologic regulation of the apoptotic process.

Mammalian inositol polyphosphate multikinase synthesizes inositol 1,4,5-trisphosphate and an inositol pyrophosphate

Using a consensus sequence in inositol phosphate kinase, we have identified and cloned a 44-kDa mammalian inositol phosphate kinase with broader catalytic capacities than any other member of the family and which we designate mammalian inositol phosphate multikinase (mIPMK). By phosphorylating inositol 4,5-bisphosphate, mIPMK provides an alternative biosynthesis for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. mIPMK also can form the pyrophosphate disphosphoinositol tetrakisphosphate (PP-InsP4) from InsP5. Additionally, mIPMK forms InsP4 from Ins(1,4,5)P3 and InsP5 from Ins(1,3,4,5)P4.

GRAB: A Physiologic Guanine Nucleotide Exchange Factor for Rab3a, which Interacts with Inositol Hexakisphosphate Kinase

Diphosphoinositol-pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate (InsP8) possess pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by recently identified InsP6 kinases designated InsP6K1 and InsP6K2. We now report the identification, cloning, and characterization of a novel protein, GRAB (guanine nucleotide exchange factor for Rab3A), which interacts with both InsP6K1 and Rab3A, a Ras-like GTPase that regulates synaptic vesicle exocytosis. GRAB is a physiologic GEF (guanine nucleotide exchange factor) for Rab3A. Consistent with a role of Rab3A in synaptic vesicle exocytosis, GRAB regulates depolarization-induced release of dopamine from PC12 cells and nicotinic agonist-induced hGH release from bovine adrenal chromaffin cells. The association of InsP6K1 with GRAB fits with a role for InsP7 in vesicle exocytosis.