The Role of Phosphatases in Inositol Signaling Reactions

The phosphatidylinositol signaling pathway employs a host of kinases and phosphatases that form and degrade the many signaling molecules that act in this system. The complexity and robustness of the system are indicated by the fact that there are six inositol phospholipids and more than 20 soluble inositol phosphates that have been found in mammalian cells (1Majerus P.W. Annu. Rev. Biochem. 1991; 61: 225-250Crossref Scopus (348) Google Scholar). The system is present in all eukaryotic cells in one form or another. This review will focus on a few of the phosphatases as they exist in mammalian cells. In general, the inositol phosphatases are analogous to protein phosphatases in that they both tend to inhibit or terminate signaling reactions. There are specific exceptions to this general rule in both systems. Numerous inositol phosphatases have been discovered and characterized in the past 15 years. Surprisingly, in many cases the same enzymes hydrolyze phosphate from both water-soluble inositol phosphates and the corresponding lipids with the same arrangement of phosphate groups. The first 5-phosphatase enzymes studied were those that hydrolyze inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) 1The abbreviations used are: Ins, inositol; PtdIns, phosphatidylinositol; OCRL, oculocerebrorenal (Lowe) syndrome; SHIP, SH2-containing inositol 5-phosphatase.to inositol 1,4-bisphosphate (Ins-1,4-P2) (1Majerus P.W. Annu. Rev. Biochem. 1991; 61: 225-250Crossref Scopus (348) Google Scholar, 2Mitchell C.A. Brown S. Campbell J.K. Munday A.D. Speed C.J. Biochem. Soc. Trans. 1996; 24: 994-1000Crossref PubMed Scopus (54) Google Scholar, 3Drayer A.L. Pesesse X. De Smedt F. Communi D. Moreau C. Erneux C. Biochem. Soc. Trans. 1996; 24: 1001-1005Crossref PubMed Scopus (23) Google Scholar). Because the former stimulates cellular calcium mobilization whereas the latter does not this reaction serves to terminate calcium signaling. In initial attempts to purify 5-phosphatase from human platelets two peaks of activity were eluted from DEAE-cellulose (4Connolly T.M. Bross T.E. Majerus P.W. J. Biol. Chem. 1985; 260: 7868-7874Abstract Full Text PDF PubMed Google Scholar, 5Mitchell C.A. Connolly T.M. Majerus P.W. J. Biol. Chem. 1989; 264: 8873-8877Abstract Full Text PDF PubMed Google Scholar). This was the first indication that multiple 5-phosphatase enzymes exist, and the platelet enzymes were designated as types I and II. There are now numerous 5-phosphatase enzymes that have been cloned and characterized (6Majerus P.W. Genes Dev. 1996; 10: 1051-1053Crossref PubMed Scopus (50) Google Scholar). All are magnesium-dependent phosphomonoesterases. There are eight distinct mammalian enzymes that are products of different genes for which cDNA clones have been isolated (Fig.1). In addition, several of these cDNAs exist in alternatively spliced versions, the significance of which is for the most part unknown. 5-Phosphatases are also found in plants, Caenorhabditis elegans, and Drosophila,and there are four yeast genes that encode 5-phosphatase enzymes (6Majerus P.W. Genes Dev. 1996; 10: 1051-1053Crossref PubMed Scopus (50) Google Scholar,7Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Crossref PubMed Google Scholar). We have classified the 5-phosphatases according to their substrate specificity into four groups. These enzymes are designated as inositol polyphosphate 5-phosphatases as the known substrates have multiple phosphate groups. There are four known substrates for 5-phosphatases: Ins-1,4,5-P3, Ins-1,3,4,5-P4, and the lipids PtdIns-4,5-P2 and PtdIns-3,4,5-P3. 5-Phosphatases are homologous with varying degrees of sequence conservation (8Jefferson A.B. Majerus P.W. Biochemistry. 1996; 35: 7890-7894Crossref PubMed Scopus (60) Google Scholar). They are defined by two signature motifs: (F/I)WXGDXN(F/Y)R and (R/N)XP(S/A)(W/Y)(C/T)DR(I/V)(L/I) as shown in Fig. 1. These are separated by 60–75 amino acids except for 5-phosphatase I, where the motifs are 103 amino acids apart. Group I 5-phosphatases hydrolyze only the water-soluble substrates Ins-1,4,5-P3 and Ins-1,3,4,5-P4. They are the most active against these substrates of all 5-phosphatases and probably function as the enzymes that terminate calcium signaling. The platelet type I enzyme is a representative of this group, which includes members cloned from dog thyroid and placenta (9Laxminarayan K.M. Chan B.K. Tetaz T. Bird P.I. Mitchell C.A. J. Biol. Chem. 1994; 269: 17305-17310Abstract Full Text PDF PubMed Google Scholar, 10Verjans B. De Smedt F. Lecocq R. Vanweyenberg V. Moreau C. Erneux C. Biochem. J. 1994; 300: 85-91Crossref PubMed Scopus (46) Google Scholar). These enzymes are membrane-associated presumably through isoprenylation (11Jefferson A.B. Majerus P.W. J. Biol. Chem. 1995; 270: 9370-9377Crossref PubMed Scopus (117) Google Scholar, 12De Smedt F. Boom A. Pesesse X. Schiffmann S.N. Erneux C. J. Biol. Chem. 1996; 271: 10419-10424Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 5-Phosphatase I in platelets is in a stoichiometric complex with pleckstrin that regulates its activity (13Auethavekiat V. Abrams C.S. Majerus P.W. J. Biol. Chem. 1997; 272: 1786-1790Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). When platelets are stimulated with thrombin, pleckstrin is rapidly phosphorylated on serine and threonine residues by protein kinase C, and this activates the 5-phosphatase. Another signaling molecule, 14-3-3ε protein, is also found in this complex (14Campbell J.K. Gurung R. Romero S. Speed C.J. Andrews R.K. Berndt M.C. Mitchell C.A. Biochemistry. 1997; 36: 15363-15370Crossref PubMed Scopus (40) Google Scholar). Cells stably transfected with an antisense cDNA for 5-phosphatase I underexpress the enzyme, have elevated levels of Ins-1,4,5-P3 and elevated basal intracellular calcium levels, and display a transformed phenotype further supporting the hypothesis that group I enzymes control calcium signaling (15Speed C.J. Little P.J. Hayman J.A. Mitchell C.A. EMBO J. 1996; 15: 4852-4861Crossref PubMed Scopus (51) Google Scholar). Group II enzymes hydrolyze all four 5-phosphatase substrates although with varying catalytic efficiency. Platelet type II 5-phosphatase belongs to this group and was the first 5-phosphatase cloned (16Ross T.S. Mitchell C.A. Jefferson A.B. Majerus P.W. J. Biol. Chem. 1991; 266: 20283-20289Abstract Full Text PDF PubMed Google Scholar). OCRL-1 is the X chromosome-encoded gene that when mutated causes Lowe syndrome or oculocerebrorenal dystrophy (17Attree O. Olivos I.M. Okabe I. Bailey L.C. Nelson D.L. Lewis R.A. McInnes R.R. Nussbaum R.L. Nature. 1992; 358: 239-242Crossref PubMed Scopus (402) Google Scholar). Platelet 5-phosphatase II and OCRL-1 are 51% identical over a span of 744 amino acids and OCRL-1 is a 5-phosphatase (18Zhang X. Jefferson A.B. Auethavekiat V. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4853-4856Crossref PubMed Scopus (221) Google Scholar). Lowe syndrome is characterized by renal tubular acidosis, cataracts, and mental retardation; renal failure and retinal degeneration develop inexorably by early adult life (19Charnas L.R. Nussbaum R.L. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. 7th Ed. The Metabolic and Molecular Bases of Inherited Disease. 3. McGraw-Hill Inc., New York1995: 3705-3716Google Scholar). Both of these enzymes have about 300 amino acids of nonhomologous sequence N-terminal to the 5-phosphatase catalytic domain. These extra sequences are not required for enzyme activity and presumably provide for localization or association with other proteins. Both of these enzymes are membrane-associated without membrane-spanning domains. 5-Phosphatase II is isoprenylated (11Jefferson A.B. Majerus P.W. J. Biol. Chem. 1995; 270: 9370-9377Crossref PubMed Scopus (117) Google Scholar) and is bound to mitochondria and plasma membrane (20Speed C.J. Matzaris M. Bird P.I. Mitchell C.A. Eur. J. Biochem. 1995; 234: 216-224Crossref PubMed Scopus (24) Google Scholar). OCRL-1 is not modified by any lipid moiety and is found on the surface of lysosomes (21Zhang X. Hartz P. Philip E. Racusen L.C. Majerus P.W. J. Biol. Chem. 1998; 273: 1574-1582Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) although in lymphocytes it was reported to be bound to the Golgi apparatus (22Suchy S.F. Olivos-Glander I.M. Nussbaum R.L. Hum. Mol. Genet. 1995; 4: 2245-2250Crossref PubMed Scopus (126) Google Scholar). In preliminary studies using renal proximal tubule cell lines from a patient with Lowe syndrome, lysosomal enzymes appear to be missorted. Extracts of these cells are deficient in PtdIns-4,5-P2 and PtdIns-3,4,5-P3 hydrolytic activity, whereas the corresponding water-soluble inositol phosphates are hydrolyzed normally, indicating that OCRL-1 is mainly a lipid phosphatase (21Zhang X. Hartz P. Philip E. Racusen L.C. Majerus P.W. J. Biol. Chem. 1998; 273: 1574-1582Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). There is considerable evidence that PtdIns-4,5-P2 is required for budding of membrane vesicles from lysosomes, and the accumulation of this lipid may afford abnormally increased trafficking of enzymes from lysosomes to the extracellular space (23De Camilli P. Emr J. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (658) Google Scholar). Disruption of OCRL-1 in mice leads to no abnormal phenotype, and 5-phosphatase II-deficient mice also appear normal. When these mutants were interbred, deficiency of both enzymes resulted in an embryonic lethal phenotype (24Janne P.A. Suchy S.F. Bernard D. MacDonald M. Crawley J. Grinberg A. Wynshaw-Boris A. Westphal H. Nussbaum R.L. J. Clin. Invest. 1998; 101: 2042-2053Crossref PubMed Scopus (138) Google Scholar). This result suggests that the enzymes have overlapping functions in mice, although apparently not in humans, because OCRL-1-deficient proximal tubule cell lines from a Lowe syndrome patient express 5-phosphatase II and still have a severe defect in PtdIns-4,5-P2 metabolism. Thus, these cells accumulate PtdIns-4,5-P2, presumably on lysosomes, and lysosomal enzymes accumulate extracellularly. Perhaps the lifelong leakage of enzymes from lysosomes leads to tissue damage and eventually causes renal failure and blindness. Synaptojanin (25McPherson P.S. Czernik A.J. Chilcote T.J. 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Commun. 1998; 247: 116-122Crossref PubMed Scopus (22) Google Scholar) are related group II enzymes that participate in synaptic vesicle trafficking. These enzymes occur in several alternatively spliced forms of uncertain significance (30Seet L.-F. Cho S. Hessel A. Dumont D.J. Biochem. Biophys. Res. Commun. 1998; 247: 116-122Crossref PubMed Scopus (22) Google Scholar). Synaptojanin forms complexes with dynamin and amphiphysin to promote synaptic vesicle recycling. Synaptojanin contains C-terminal proline-rich regions that can bind to SH3 domains. Synaptojanins contain a domain that is homologous to the yeast proteinSacI, which suppresses mutations in yeast Sec14 phosphatidylinositol transfer protein (31Sha B. Phillips S.E. Bankaitis B.A. Luo M. Nature. 1998; 391: 506-510Crossref PubMed Scopus (228) Google Scholar). The two synaptojanins are over 50% identical in the SacI and 5-phosphatase domains. Group III enzymes hydrolyze only substrates with a 3-position phosphate group (i.e. Ins-1,3,4,5-P4 and PtdIns-3,4,5-P3). There are two such enzymes designated as SHIP and SHIP2 (32Kavanaugh W.M. Pot D.A. Chin S.M. Deuter-Reinhard M. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (560) Google Scholar, 34Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar). They are 50% identical in amino acid sequence but have very different tissue distributions. They contain the N-terminal SH2 domain and phosphotyrosine binding domains and upon activation of receptors form complexes with cellular signaling proteins. SHIP is expressed only in cells of hematopoietic origin and participates in signaling through essentially all hematopoietic cytokine receptors including the erythropoietin, interleukin-3, and colony-stimulating factor receptors, as well as various mast cell and B and T cell receptors (35Lioubin M.N. Algate P.A. Tsai S. Calrberg K. Aebersold A. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (377) Google Scholar). SHIP forms complexes with the cellular signaling molecules Grb2 and Shc and with the ITIM motif on receptor-ligated FcγRII (36Osborne M.A. Zenner G. Lubinus M. Zhang X. Songyang Z. Cantley L.C. Majerus P.W. Burn P. Kochan J.P. J. Biol. Chem. 1996; 271: 29271-29278Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 37Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (643) Google Scholar, 38Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). The enzyme hydrolyzes the signaling molecule PtdIns-3,4,5-P3 that is presumed to stimulate signaling by activating Akt, protein kinase C, and other targets. Thus, SHIP has a negative function and terminates signals. The targeted disruption of SHIP in mice leads to excessive cytokine signaling, myeloid proliferation, and death from infiltration and consolidation of the lungs by myeloid cells (39Helgason C.D. Damen J.E. Rosten P. Grewal R. Sorensen P. Chappel S.M. Borowski A. Jirik F. Krystal G. Humphries R.K. Genes Dev. 1998; 12: 1610-1620Crossref PubMed Scopus (481) Google Scholar). SHIP2 is expressed widely in nonhematopoietic cells and serves a similar function inhibiting cellular responses to insulin, epidermal growth factor, and platelet-derived growth factor (40Habib T. Hejna J.A. Moses R.E. Decker S.J. J. Biol. Chem. 1998; 273: 18605-18609Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Group IV 5-phosphatases remain poorly characterized and are an activity that only hydrolyzes PtdIns-3,4,5-P3 (41Jackson S.P. Schoenwaelder S.M. Matzaris M. Brown S. Mitchell C.A. EMBO J. 1995; 14: 4490-4500Crossref PubMed Scopus (74) Google Scholar) and forms complexes with PtdIns 3-kinase. We have recently isolated a cDNA encoding a Group IV enzyme (as shown in Fig. 1). In contrast to the large 5-phosphatase gene family, there is a single gene that encodes inositol polyphosphate 1-phosphatase. It is a member of a magnesium-dependent, lithium-inhibited gene family that is characterized by a common structural fold (42York J.D. Ponder J.W. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5149-5153Crossref PubMed Scopus (155) Google Scholar). The enzyme hydrolyzes Ins-1,4-P2 and Ins-1,3,4-P3and does not act on lipid substrates. Inositol monophosphatase is another member of this family, although it does not share significant amino acid identity except in the active site region (43York J.D. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9548-9552Crossref PubMed Scopus (43) Google Scholar). Lithium is used to treat manic depressive disease. A popular though unsubstantiated theory is that monophosphatase is the target of lithium action and serves to trap inositol monophosphates. This could deplete cells of free inositol leading to inhibition of inositol signaling reactions. The recent targeted deletion of 1-phosphatase inDrosophila suggests that lithium may inhibit this enzymein vivo (44Acharya J.K. Labarca P. Delgado R. Jalink K. Zuker C.S. Neuron. 1998; 20: 1219-1229Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The 1-phosphatase-deficient flies have a “shaker” phenotype and dramatic defects in synaptic transmission. Treatment of wild-type flies with lithium produces phenocopies of the knockout flies, indicating that lithium inhibits 1-phosphatase in vivo. Treatment of 1-phosphatase knockout flies with lithium had no further effect. Responsiveness to lithium in patients with bipolar disorder has been linked to 1-phosphatase (45Steen V.M. Lovlie R. Osher Y. Belmaker R.H. Berle J.O. Gulbrandsen A.-K. Pharmacogenetics. 1998; 8: 259-268PubMed Google Scholar). Thus 1-phosphatase is abona fide target for the therapeutic action of lithium, and other inhibitors of this enzyme might be used in the treatment of this disease. The inositol polyphosphate 4-phosphatases are a family of enzymes implicated in the regulation of PtdIns 3-kinase signaling. These magnesium-independent phosphatases catalyze the hydrolysis of the 4-position phosphate of the second messenger, PtdIns-3,4-P2, yielding the product PtdIns-3-P (46Norris F.A. Majerus P.W. J. Biol. Chem. 1994; 269: 8716-8720Abstract Full Text PDF PubMed Google Scholar). 4-Phosphatase cDNA clones have been isolated that are derived from two 4-phosphatase genes. 4-Phosphatase type I and II cDNAs encode proteins that are 37% identical and contain the conserved motif CKSAKDRT (47Norris F.A. Atkins R.C. Majerus P.W. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar). This motif contains the active site sequence for other magnesium-independent phosphatases Y. J.A. M.A. J.E. Biochemistry. 1994; PubMed Scopus Google Scholar, G. J.E. Biochemistry. 1994; 269: Scholar). The conserved is the catalytic for phosphatases F.A. Auethavekiat V. Majerus P.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google and of this to serine in the of 4-phosphatase (46Norris F.A. Majerus P.W. J. Biol. Chem. 1994; 269: 8716-8720Abstract Full Text PDF PubMed Google Scholar). Both 4-phosphatase I and II are alternatively spliced in the of proteins with domains their (47Norris F.A. Atkins R.C. Majerus P.W. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar). 4-Phosphatase that with the C-terminal of 4-phosphatase I and II of activity from human and that these are enzymes for the of this second F.A. Auethavekiat V. Majerus P.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). of human platelets with or calcium in of 4-phosphatase I by by the F.A. Atkins R.C. Majerus P.W. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This of 4-phosphatase with the accumulation of of stimulated human This result 4-phosphatase is in the regulation of this lipid second F.A. Atkins R.C. Majerus P.W. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (50) Google Scholar). A recent suggests a for an inositol phosphate phosphatase in the of a protein by was shown to have sequence to the 4-phosphatase active site motif F.A. T.S. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). is into the of host cells by type III system and has been shown to to the of S. by and R. S. T.S. Mol. 1997; PubMed Scopus Google Scholar). encodes a phosphatase that hydrolyzes many but not all inositol phosphates including inositol yielding the product inositol has been shown to by PtdIns-3,4,5-P3 inhibition of the and of cells with S. in a of levels L. A. C. T. R. J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). of the active site to serine phosphatase of cells with a S. that the to levels R. S. T.S. Mol. 1997; PubMed Scopus Google Scholar). of with S. in or indicating that enzyme activity for These that by disruption of the inositol of the host the phosphatase is related to it has a substrate A human gene designated as was recently shown to be an inositol phosphatase T. J.E. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). It has been that this enzyme suppresses cell by PtdIns-3,4,5-P3 K. Science. 1998; PubMed Google Scholar). In of this enzyme that it has substrate specificity similar to A mammalian phosphatase of substrate specificity has been cloned recently and designated as or multiple inositol polyphosphate phosphatase A. Biochem. J. 1997; PubMed Scopus Google Scholar). has no to any other inositol for inositol phosphate phosphatases in cellular signaling have been discovered of these enzymes will further the of intracellular signaling.