Portal de Periódicos da CAPES

Inositol Pyrophosphates Get the Vip1 Treatment

2007; Cell Press; Volume: 129; Issue: 4 Linguagem: Inglês

10.1016/j.cell.2007.05.002

1097-4172

Sara Maria Nancy Onnebo, Adolfo Saiardi,

Pineapple and bromelain studies

Inositol pyrophosphates are unique signaling molecules implicated in the regulation of diverse cellular processes. Two new studies by Mulugu et al., 2007Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science. 2007; 316: 106-109Crossref PubMed Scopus (178) Google Scholar and Lee et al., 2007Lee Y.S. Mulugu S. York J.D. O'Shea E.K. Science. 2007; 316: 109-112Crossref PubMed Scopus (209) Google Scholar extend the biological and metabolic diversity of this class of molecules. They identify yeast Vip1 as a new inositol pyrophosphate synthase and show that the products of Vip1 activity regulate a cyclin/cyclin-dependent kinase complex. Inositol pyrophosphates are unique signaling molecules implicated in the regulation of diverse cellular processes. Two new studies by Mulugu et al., 2007Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science. 2007; 316: 106-109Crossref PubMed Scopus (178) Google Scholar and Lee et al., 2007Lee Y.S. Mulugu S. York J.D. O'Shea E.K. Science. 2007; 316: 109-112Crossref PubMed Scopus (209) Google Scholar extend the biological and metabolic diversity of this class of molecules. They identify yeast Vip1 as a new inositol pyrophosphate synthase and show that the products of Vip1 activity regulate a cyclin/cyclin-dependent kinase complex. The fully phosphorylated six-carbon ring of IP6 (phytic acid) was thought to represent the end point of inositol phosphorylation. The discovery of inositol phosphate species with seven (diphosphoinositol pentakisphosphate; PP-IP5; IP7) or eight phosphates (bis-diphosphoinositol tetrakisphosphate; [PP]2-IP4; IP8) on the inositol ring was exciting and unexpected (Menniti et al., 1993Menniti F.S. Miller R.N. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar, Stephens et al., 1993Stephens L. Radenberg T. Thiel U. Vogel G. Khoo K.H. Dell A. Jackson T.R. Hawkins P.T. Mayr G.W. J. Biol. Chem. 1993; 268: 4009-4015Abstract Full Text PDF PubMed Google Scholar). These "high-energy" molecules have been linked to a wide range of biological functions, including vesicle trafficking, apoptosis, DNA repair, telomere maintenance, and the stress response (Bennett et al., 2006Bennett M. Onnebo S.M. Azevedo C. Saiardi A. Cell. Mol. Life Sci. 2006; 63: 552-564Crossref Scopus (138) Google Scholar). Although the mechanisms of action of inositol pyrophosphates in these cellular processes remain unclear, IP7 has been shown to directly transfer the energetic β phosphate of the pyrophosphate moiety to multiple proteins, indicating that IP7 may regulate signaling pathways through a new mechanism of protein phosphorylation (Saiardi et al., 2004Saiardi A. Bhandari R. Resnick A.C. Snowman A.M. Snyder S.H. Science. 2004; 306: 2101-2105Crossref Scopus (225) Google Scholar). Two new studies published in Science identify an enzyme that catalyzes the production of inositol pyrophosphates (Mulugu et al., 2007Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science. 2007; 316: 106-109Crossref PubMed Scopus (178) Google Scholar) and a new target of regulation by IP7, a cyclin/cyclin-dependent kinase complex (Lee et al., 2007Lee Y.S. Mulugu S. York J.D. O'Shea E.K. Science. 2007; 316: 109-112Crossref PubMed Scopus (209) Google Scholar). The inositol hexakisphosphate kinases (IP6K) are the enzymes responsible for synthesis of inositol pyrophosphates (Saiardi et al., 1999Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text PDF PubMed Scopus (289) Google Scholar). They are highly conserved evolutionarily and at least one member of this enzyme family is present in all eukaryotic genomes sequenced so far (Bennett et al., 2006Bennett M. Onnebo S.M. Azevedo C. Saiardi A. Cell. Mol. Life Sci. 2006; 63: 552-564Crossref Scopus (138) Google Scholar). Inositol pyrophosphates and their kinases have been predominantly studied in the budding yeast Saccharomyces cerevisiae, particularly using mutants lacking the single yeast IP6K, Kcs1. In the kcs1 deletion mutant, the naturally occurring inositol pyrophosphates IP7 and IP8 are virtually undetectable. Surprisingly, an additional deletion of the IP7 phosphatase DDP1 in this mutant resulted in the reappearance of IP7, suggesting the existence of a second enzyme that can synthesize IP7 (York et al., 2005York S.J. Armbruster B.N. Greenwell P. Petes T.D. York J.D. J. Biol. Chem. 2005; 280: 4264-4269Crossref Scopus (123) Google Scholar). Using cell extracts prepared from yeast lacking both Kcs1 and DDP1, Mulugu et al., 2007Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science. 2007; 316: 106-109Crossref PubMed Scopus (178) Google Scholar biochemically purified a second inositol pyrophosphate synthase, which they call Vip1. Protein sequence analysis revealed two conserved domains in Vip1: an amino-terminal ATP-grasp superfamily domain with IP6 kinase activity and a histidine acid-phosphatase domain in the C terminus (Mulugu et al., 2007Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science. 2007; 316: 106-109Crossref PubMed Scopus (178) Google Scholar). The generation of yeast lacking Kcs1, DDP1, and Vip1 resulted in ablation of IP7, which could be restored by reintroducing Vip1, confirming that Vip1 produces IP7 in vivo (Mulugu et al., 2007Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science. 2007; 316: 106-109Crossref PubMed Scopus (178) Google Scholar). The most important finding of the Mulugu et al. work is that Vip1 synthesizes a form of IP7 that is distinct from the isomers 5PP-IP5 produced by human IP6K1 (which may have similar activity to yeast Kcs1). The IP7 generated by Vip1 has the pyrophosphate moiety located at either the C4 or C6 position (4/6PP-IP5) producing enantiomers that cannot be distinguished (Figure 1). Vip1 and Kcs1 together produce IP8, but it is unclear if Vip1 is equivalent to the previously isolated IP7-kinase activity (Huang et al., 1998Huang C.F. Voglmaier S.M. Bembenek M.E. Saiardi A. Snyder S.H. Biochemistry. 1998; 37: 14998-15004Crossref PubMed Scopus (42) Google Scholar). Insight into the physiological role of Vip1 comes from studies of Asp1, its ortholog in fission yeast Schizosaccharomyces pombe, which regulates actin-related protein (Arp) complexes. Functional analyses revealed that IP6 kinase activity is essential for maintaining cellular integrity, normal growth, and genetic interactions with Arp complex components. Mulugu and colleagues indisputably demonstrate that Vip1 is required to produce the IP7 present in yeast lacking both Kcs1 and DDP1. Given that yeast lacking Kcs1 have nearly undetectable levels of inositol pyrophosphates, how much of the total IP7 in cells is synthesized by Vip1? This might be determined by analyzing the single mutant lacking Vip1 and double mutants deficient in both Vip1 and DDP1. The most interesting characteristic of Vip1 is its dual domain architecture, although the specificity of the phosphatase domain has yet to be determined. In a companion study, Lee et al., 2007Lee Y.S. Mulugu S. York J.D. O'Shea E.K. Science. 2007; 316: 109-112Crossref PubMed Scopus (209) Google Scholar elegantly demonstrate that 4/6PP-IP5, the form of IP7 generated by Vip1, but not 5PP-IP5, the form generated by Kcs1, is a regulator of the Pho80-Pho85 cyclin/cyclin-dependent kinase (CDK) complex. This CDK complex is a key component of the PHO phosphate response pathway in yeast. Using a biochemical approach, Lee et al., 2007Lee Y.S. Mulugu S. York J.D. O'Shea E.K. Science. 2007; 316: 109-112Crossref PubMed Scopus (209) Google Scholar sought to identify cellular components that control the phosphate starvation response initiated when cells are grown in media low in phosphate. In S. cerevisiae, the Pho80-Pho85 cyclin-CDK complex is a key regulator of the PHO signaling pathway. Under high-phosphate conditions, the Pho80-Pho85 complex is active and phosphorylates the transcription factor Pho4, which is then exported from the nucleus into the cytoplasm. In contrast, when cells are starved of phosphate, the Pho80-Pho85 complex is inhibited, leading to nuclear accumulation of dephosphorylated Pho4 and transcription of PHO genes. The Pho81 CDK inhibitor is constitutively bound to the Pho80-Pho85 complex and is required for its inhibition in response to phosphate limitation. Analysis of fractionated extracts from phosphate-starved cells led Lee et al. to identify a small molecule capable of inhibiting the Pho80-Pho85 kinase activity in a Pho81-dependent manner. This molecule is IP7. Importantly, inactivation of Pho80-Pho85 by IP7 is specific because inositol and many of its phosphorylated derivatives did not affect kinase activity (Lee et al., 2007Lee Y.S. Mulugu S. York J.D. O'Shea E.K. Science. 2007; 316: 109-112Crossref PubMed Scopus (209) Google Scholar). That Vip1 is the IP6 kinase involved in controlling the PHO pathway was shown by assaying extracts from phosphate-starved cells lacking Kcs1 and Vip1 for their ability to inactivate Pho80-Pho85-Pho81. Only extracts from cells lacking Kcs1 but containing Vip1 were able to inhibit Pho80-Pho85 in a Pho81-dependent manner. Furthermore, the IP7 (4/6PP-IP5) synthesized by Vip1 inhibits the Pho80-Pho85 complex, whereas the 5PP-IP5 isomer synthesized by human IP6K1 does not (Lee et al., 2007Lee Y.S. Mulugu S. York J.D. O'Shea E.K. Science. 2007; 316: 109-112Crossref PubMed Scopus (209) Google Scholar). Interestingly, IP7 levels increased in response to phosphate limitation, leading the authors to conclude that when cells are starved of phosphate, the IP7 concentration increases and thus inhibits the Pho80-Pho85-Pho81 complex. However, in our laboratory, using a different yeast strain and slightly different experimental conditions, we observed a decrease in IP7 cellular concentrations in response to phosphate starvation (A.S., unpublished data), suggesting that more characterization of this response may be necessary. Lee et al. convincingly provide a link between Vip1-mediated inositol pyrophosphate signaling and phosphate metabolism. However, previous reports have also linked Kcs1 and its IP6K family members to phosphate metabolism, suggesting that both 5PP-IP5 and 4/6PP-IP5 have roles in these processes. Indeed, the first clue to a relationship between inositol pyrophosphate signaling and phosphate metabolism came from the discovery that PiUS, a stimulator of inorganic phosphate uptake, was in fact IP6K2 (Bennett et al., 2006Bennett M. Onnebo S.M. Azevedo C. Saiardi A. Cell. Mol. Life Sci. 2006; 63: 552-564Crossref Scopus (138) Google Scholar). Then it was shown that inorganic phosphate uptake in yeast lacking Kcs1 is less efficient than in wild-type cells. Yeast lacking Kcs1 also have increased expression of the PHO phosphate response genes and reduced intracellular polyphosphate levels (Auesukaree et al., 2005Auesukaree C. Tochio H. Shirakawa M. Kaneko Y. Harashima S. J. Biol. Chem. 2005; 280: 25127-25133Crossref PubMed Scopus (78) Google Scholar, Bennett et al., 2006Bennett M. Onnebo S.M. Azevedo C. Saiardi A. Cell. Mol. Life Sci. 2006; 63: 552-564Crossref Scopus (138) Google Scholar). Moreover, yeast lacking Kcs1 constitutively express the acid phosphatase PHO5, which is activated by Pho4, indicating that the IP7 synthesized by Kcs1 also influences the PHO transcriptional pathway (Auesukaree et al., 2005Auesukaree C. Tochio H. Shirakawa M. Kaneko Y. Harashima S. J. Biol. Chem. 2005; 280: 25127-25133Crossref PubMed Scopus (78) Google Scholar). These observations may reflect different aspects of IP7 function in phosphate sensing. It is possible that 4/6PP-IP5 mainly regulates the PHO pathway by protein binding, whereas 5PP-IP5 primarily functions as a phosphate donor. The two new studies raise several exciting possibilities that could advance our understanding of inositol pyrophosphates and their roles in many different aspects of cell signaling. Certainly, the notion that two structurally different IP7 molecules can have distinct cellular roles is intriguing and will no doubt guide the way to more pioneering work. In the past decade, a large number of inositol pyrophosphate species, not only IP7 and IP8, have been discovered as well as several inositol pyrophosphate synthases and phosphatases. Further studies into these fascinating signaling molecules may result in the discovery of other species, perhaps even pyrophosphorylated inositol lipids.