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• W1963724298 abstract "Inositol phosphate-containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases inDictyostelium discoideum, Dd5P1–4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed propeller structure and interacts with G-proteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin with aSacI domain and OCRL with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by the expression of the complete protein but not by the 5-phosphatase catalytic domain. Inositol phosphate-containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases inDictyostelium discoideum, Dd5P1–4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed propeller structure and interacts with G-proteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin with aSacI domain and OCRL with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by the expression of the complete protein but not by the 5-phosphatase catalytic domain. Inositol phosphates play a role in a variety of eukaryotic cellular processes, including chemotaxis and membrane trafficking. They are regulated by a number of enzymes. The group of phosphatidylinositol 3-kinases (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PI, phosphatidylinositol; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; Ins(1, 3,4,5)P4, inositol 1,4,5-tetrakisphosphate; SH2, Src homology 2; SHIP, SH2 domain-containing inositol 5′-phosphatase; INPP, inositol polyphosphate 5-phosphatase; PH, pleckstrin homology; GAP, GTPase-activating protein 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PI, phosphatidylinositol; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; Ins(1, 3,4,5)P4, inositol 1,4,5-tetrakisphosphate; SH2, Src homology 2; SHIP, SH2 domain-containing inositol 5′-phosphatase; INPP, inositol polyphosphate 5-phosphatase; PH, pleckstrin homology; GAP, GTPase-activating protein phosphorylates the lipid substrates PI, PI(4)P, and PI(4,5)P2 at the 3-position of the inositol ring (1Cantrell D.A. J. Cell Sci. 2001; 114: 1439-1445Google Scholar). The lipid product PI(3,4,5)P3 has been strongly implicated to be important in chemotaxis in neutrophils and fibroblasts (2Servant G. Weiner O.D. Herzmark P. Balla T. Sedat J.W. Bourne H.R. Science. 2000; 287: 1037-1040Google Scholar, 3Haugh J.M. Codazzi F. Teruel M. Meyer T. J. Cell Biol. 2000; 151: 1269-1280Google Scholar). PTEN, identified as a tumor suppressor gene (4Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Google Scholar), reverses the action of PI3K by dephosphorylation of PI(3,4,5)P3 and PI(3,4)P2 at the 3-position (5Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Google Scholar). Another group of enzymes, the inositol 5-phosphatases, can remove the phosphate group at the 5-position of the inositol ring (6Erneux C. Govaerts C. Communi D. Pesesse X. Biochim. Biophys. Acta. 1998; 1436: 185-199Google Scholar, 7Woscholski R. Parker P.J. Trends Biochem. Sci. 1997; 22: 427-431Google Scholar, 8Majerus P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Google Scholar). The importance of inositol 5-phosphatase activity in PI(3,4,5)P3 regulation is demonstrated by SHIP1. In stimulated B-cells, SHIP1 accounts for the major phosphatase activity toward PI(3,4,5)P3, and inactivation of SHIP1 leads to an increased and prolonged PI(3,4,5)P3 production (9Brauweiler A. Tamir I. Dal Porto J. Benschop R.J. Helgason C.D. Humphries R.K. Freed J.H. Cambier J.C. J. Exp. Med. 2000; 191: 1545-1554Google Scholar). Other inositol 5-phosphatases have been shown to play important roles in a number of cellular processes. Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome in human (10Attree O. Olivos I.M. Okabe I. Bailey L.C. Nelson D.L. Lewis R.A. McInnes R.R. Nussbaum R.L. Nature. 1992; 358: 239-242Google Scholar), and deletion of the presynaptic inositol 5-phosphatase Synaptojanin leads to neurological abnormalities and early death of mice (11Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Google Scholar). In the social amoeba, Dictyostelium discoideum chemotaxis toward folic acid and cAMP is an essential strategy for survival (12Parent C.A. Devreotes P.N. Annu. Rev. Biochem. 1996; 65: 411-440Google Scholar). Several observations suggest that phosphatidylinositol phosphates mediate chemotaxis and, in particular, the localization of the signal inside D. discoideum cells. The PH domains of a number of proteins involved in chemotaxis, including CRAC, Akt/PKB, and PhdA, have been shown to transiently localize at the leading edge of cells moving in a chemotactic gradient (13Parent C.A. Blacklock B.J. Froehlich W.M. Murphy D.B. Devreotes P.N. Cell. 1998; 95: 81-91Google Scholar, 14Meili R. Ellsworth C. Lee S. Reddy T.B. Ma H. Firtel R.A. EMBO J. 1999; 18: 2092-2105Google Scholar, 15Funamoto S. Milan K. Meili R. Firtel R.A. J. Cell Biol. 2001; 153: 795-810Google Scholar). As these PH domains bind to PI(3,4,5)P3 and PI(3,4)P2, an asymmetric lipid distribution is implicated by these observations. Inpi3k1/2-null cells, a strain with two putative PI3Ks inactivated (16Zhou K. Takegawa K. Emr S.D. Firtel R.A. Mol. Cell. Biol. 1995; 15: 5645-5656Google Scholar), the transient localization of PhdA can no longer be observed and cells show reduced chemotaxis (15Funamoto S. Milan K. Meili R. Firtel R.A. J. Cell Biol. 2001; 153: 795-810Google Scholar). On the other hand, in PTEN-null cells, a strain in which a putative 3-phosphatase is inactivated, the localization of PH-domains is prolonged and broadened and chemotaxis is also reduced (17Iijima M. Devreotes P. Cell. 2002; 109: 599-610Google Scholar, 18Funamoto S. Meili R. Lee S. Parry L. Firtel R.A. Cell. 2002; 109: 611-623Google Scholar). Inositol 5-phosphatases may play an important role in the regulation of the phosphoinositides. As this group of enzymes leads to the degradation of PI(3,4,5)P3 and at the same time formation of PI(3,4)P2, another PH-binding molecule, they can be central players in the metabolic route of these signaling molecules. Phosphoinositides have also been implicated in endocytosis in D. discoideum. Pi3k1/2-null cells are affected with respect to pinocytosis (19Buczynski G. Grove B. Nomura A. Kleve M. Bush J. Firtel R.A. Cardelli J. J. Cell Biol. 1997; 136: 1271-1286Google Scholar), suggesting a role for PI(3,4,5)P3 in this process. The inhibitors of phospholipase C, an enzyme converting PI(4,5)P2 into Ins(1,4,5)P3 and diacylglycerol, reduce the rate of phagocytosis (20Peracino B. Borleis J. Jin T. Westphal M. Schwartz J.M. Wu L. Bracco E. Gerisch G. Devreotes P. Bozzaro S. J. Cell Biol. 1998; 141: 1529-1537Google Scholar, 21Seastone D.J. Zhang L. Buczynski G. Rebstein P. Weeks G. Spiegelman G. Cardelli J. Mol. Biol. Cell. 1999; 10: 393-406Google Scholar). Because inositol 5-phosphatases can act on PI(4,5)P2, PI(3,4,5)P3, and Ins(1,4,5)P3, they are probably important in the endocytotic pathway. To investigate the role of inositol 5-phosphatases in chemotaxis and endocytosis, we cloned and characterized four D. discoideum inositol 5-phosphatases. Catalytic activity was determined, indicating that they act as inositol 5-phosphatases. Single and double gene disruptants were obtained and growth, chemotaxis, and development were studied in these knock-out strains. The first putative inositol 5-phosphatase sequence was obtained using degenerated primers complementary to the conserved motifs I and II present in inositol 5-phosphatases (see “Results”), and the PCR product was used to screen a cDNA library kindly provided by Dr. R. A. Firtel. TheD. discoideum genomic (www.sdsc.edu/mpr/dicty) and cDNA databases (www.csm.biol.tsukuba.ac.jp) were screened for other putative inositol 5-phosphatases using either the conserved motif I or II. Using Seqman from Lasergene (DNAstar), contigs were formed. Additional sequences were obtained by screening a cDNA library, kindly provided by Dr. R. H. Gomer, with a PCR fragment containing part of the catalytic domain (used primers: Dd5P2: 5P2S1 + 5P2R1; Dd5P3: 5P5S1 + 5P5R1; and Dd5P4: 5P4S1 + 5P4R1, see “Appendix A”). The longest clones obtained for Dd5P3 (4212 bp) and Dd5P4 (2795 bp) encode the complete open reading frame of 1377 and 787 amino acids, respectively. For Dd5P2, the longest clone only encoded for the amino acids 1–599. In combination with data from the genomic data base, a complete open reading frame of 1794 amino acids was constructed. The longest clone obtained for Dd5P1 encodes for amino acids 118–678; the missing part of the 5′-open reading frame was obtained from the data base. A comparison of the sequence obtained from cDNA clones with the genomic data base sequences revealed the presence of one intron in each inositol 5-phosphatase gene (Dd5P1: nucleotides 397–499; Dd5P2: nucleotides 53–187; Dd5P3: nucleotides 241–384; and Dd5P4: nucleotides 1357–1622) (GenBankTM accession numbers AY184992, AY184993, AY184994, and AY184995, respectively). BLAST, Smart, Pfam, and Expasy programs were used to analyze the obtained sequences. Alignments are made using Megalign (Lasergene) and Genedoc followed by optimizing by eye. The D. discoideumstrains AX3 (wild type), DH1 (uracil auxotroph wild type), and the mutant strains described were grown in HG5 medium supplemented with 10 μg/ml blasticidin S or G418 when necessary. Selection for presence of the pyr5/6 cassette was obtained using the uracil-deficient FM medium. When grown in shaking culture, the cell density was kept below 6.106 cells/ml. Growth on bacterial lawns was studied using Klebsiella aerogens grown on 3 × LP plates (8.3 mmlactose, 2 mm KH2PO4, 2 mmNa2HPO4, 3 g/liter bactopeptone, 15 g/liter agar). To determine whether cells were capable to aggregate properly, they were put on non-nutrient agar plates (11 mm KH2PO4, 2.8 mm Na2HPO4, 15 g/liter agar) at three different cell densities (2 × 106, 4 × 105, and 8 × 104 cells/cm2). Chemotaxis toward cAMP was studied using the small-droplet chemotaxis assay (22Konijn T.M. Experientia. 1970; 26: 367-369Google Scholar). RNA was isolated from cells grown on plates or starved on non-nutrient plates for 3 or 6 h or from tight aggregates, slugs, or culminants using RNeasy Mini kit from Qiagen. Equal amounts of RNA (∼40 μg) were loaded on the gel. The probes used to screen the cDNA library were used for hybridization of inositol 5-phosphatase RNA. For Dd5P1, the PCR product of primer 6BIr + 6BIf was used. DNA was labeled with [α-32P]dCTP using the random primer method (High Prime, Roche Molecular Biochemicals). Blots were incubated overnight at 65 °C in hybridization solution (0.5 mNaH2PO4/Na2HPO4, pH 7.0, 7% SDS, 0.2 mg/ml bovine serum albumin). Stringency of the final washes was 0.1 mNaH2PO4/Na2HPO4, pH 7.0, 1% SDS at 65 °C. Bands were visualized using a PhosphorImager. Gene inactivation was obtained by replacing part of the gene by the bsr orpyr5/6 cassette. The cassettes were inserted into NdeI/NsiI site of Dd5P1,NdeI/EcoRI (nucleotide 1210) site ofDd5P2, AccI (nucleotide 1648)/BglII site of Dd5P3, and NcoI (nucleotide 887)/NdeI site of Dd5P4. Orientation of the cassette was opposite to the orientation ofDd5P1(bsr and pyr),Dd5P2(bsr), and Dd5P3(bsr) and similar to the orientation of Dd5P2(pyr) andDd5P4(bsr and pyr). The place of insertion of the selection marker is indicated by an asterisk in Fig. 1. Single gene inactivation was performed with AX3 cells, whereas double gene inactivation was performed with DH1 cells. Potential knock-outs were screened by PCR and confirmed by Southern and/or Northern blots. As initiation from an internal ATG does not occur, at the most the N-terminal part of the protein will be expressed. A truncated RNA transcript could be observed for strains in whichDd5P2, Dd5P3, or Dd5P4 was inactivated. The size corresponded with the expected size for transcription of the first part of the gene. Therefore, the catalytic inositol 5-phosphatase domain is not transcribed and not expressed. The inositol 5-phosphatase catalytic domains were cloned into the BamHI site (Dd5P1, Dd5P2, and Dd5P4) orXhoI/PstI site (Dd5P3) of pRSETB (Invitrogen) using primers shown in “Appendix A” (Dd5P1: 1OES2 + 1OER1; Dd5P2: 2OES1 + 2OER1; Dd5P3: 5OES2 + 5OER2; and Dd5P4: 4OES1 + 4OER1). The catalytic domains were expressed in E. coli BL21DE3 (pLysS) (Novagen). The expression of the proteins was induced at 22 °C using 1 mm isopropyl-1-thio-β-d-galactopyranoside at a cell density of A 600 nm = 0.4–0.6 in 2× YT (16 g/liter Trypton peptone, 10 g/liter yeast extract, 0.09m NaCl). After 3–5 h of induction, 0.5–3.0 liters of cell culture were harvested, resuspended in binding buffer (20 mm Tris-HCl, pH 7.9, 0.5 m NaCl, 5 mm imidazole), lysed with lysozyme (0.5 g/liter) and phenylmethylsulfonyl fluoride (1 mm), and ultrasonificated. Soluble proteins were purified from supernatant by the binding of the His tag to nickel-nitrilotriacetic acid and elution of the protein (binding buffer with 60, 100, 80, and 60 mm imidazole for Dd5P1, Dd5P2, Dd5P3, and Dd5P4, respectively). The purified inositol 5-phosphatase catalytic domains were analyzed by 10% SDS-PAGE followed by immunostaining with Penta-His Antibody (Qiagen) and Anti-mouse IgG peroxidase conjugate (Sigma). Bands were visualized using chemiluminescence blotting substrate POD (Roche Molecular Biochemicals) and 1-min exposure to film. Prestained Protein Marker, Broad Range (New England Biolabs) was used to determine the size. To express proteins in D. discoideum, the desired DNA fragment was cloned into an extrachromosomal vector containing a neomycin cassette (pMB74 or pAH2). This results in expressing the protein from actin 15 promoter. To express the full-length Dd5P4 inDd5P4− cells, the obtained full-length cDNA clone was inserted into the BglII site of pMB74. To clone the inositol 5-phosphatase catalytic domain, the domain was excised from the pRSETB vector (see above) with BamHI andBglII and cloned into the BglII site of pAH2. The RhoGAP domain was obtained by PCR using primer OE4GAPs1 (see “Appendix A”) and the universal T7 primer with cDNA as template. The PCR product was digested withBamHI/XhoI and cloned into theBamHI/XhoI site of pRSETB. The insert was sequenced and cloned into the BglII site of pAH2. Activity toward Ins(1,4,5)P3 and Ins(1,3,4,5)P4 was determined as described previously (23Erneux C. Takazawa K. Verjans B. Methods Neurosci. 1994; 18: 312Google Scholar). 5 μl (diluted) of enzyme was added to the reaction mixture (50 mm HEPES, pH 7.4, 2 mm MgCl2, 48 mmβ-mercaptoethanol, 1 mg/ml bovine serum albumin), containing either 100 μmIns(1,4,5)P3/[3H]Ins(1,4,5)P3 or 10 μmIns(1,3,4,5)P4/[3H]Ins(1,3,4,5)P4. After 15 min of incubation the reaction was stopped and the products were separated on Dowex columns. Activity toward PI(4,5)P2 and PI(3,4,5)P3 was determined as described previously (24Giuriato S. Payrastre B. Drayer A.L. Plantavid M. Woscholski R. Parker P. Erneux C. Chap H. J. Biol. Chem. 1997; 272: 26857-26863Google Scholar). 50 μg of phosphatidyl serine and 10 μg of [32P]PI(3,4,5)P3 (see below for preparation method) or 20 μg of phosphatidyl serine and 10 μg of PI(4,5)P2/[3H]PI(4,5)P2 were resuspended in 50 μl of 50 mm Tris-HCl, pH 7.4. Vesicles were formed by sonication, and 1 μl of 1 mMgCl2 and 60 μl of (diluted) enzyme were added. After 30 min, the reaction was stopped and the lipids were extracted. Lipids were separated by TLC and visualized by exposure to film or PhosphorImager. In the case of the PI(4,5)P2 assay, the spots were visualized using EN3Hance spray (PerkinElmer Life Sciences) and scraped from the plates (Silica Gel 60, Merck) and radioactivity was measured. [32P]PI(3,4,5)P3 was prepared by making vesicles containing 50 μg of PI(4,5)P2 and 100 μg of phosphatidyl serine and incubation with PI3K (purified from baculovirus using an expression vector kindly provided by B. Vanhaesebroeck (25Vanhaesebroeck B. Stein R.C. Waterfield M.D. Cancer Surv. 1996; 27: 249-270Google Scholar)) and 70 μCi [γ-32P]ATP (3000 Ci/mmol) in reaction buffer (50 mm Tris-HCl, pH 7.4, 1.5 mmdithiothreitol, 100 mm NaCl, 0.5 mm EDTA, 5 mm MgCl2, 100 μm ATP). After 30 min, the reaction was stopped and lipids were extracted. The first potential inositol 5-phosphatase inD. discoideum was identified by performing a PCR with degenerated primers. The obtained PCR product was used as probe for cDNA library screening. The gene found using this method was calledDd5P1 (D. discoideum 5-phosphatase 1) and codes for a protein of 678 amino acids (see “Appendix B”). To identify other putative inositol 5-phosphatases in D. discoideum, we searched the D. discoideum data base for sequences showing homology to the conserved motifs I and II, WXGDXN(Y/F)R and P(A/S)W(C/T)DR(I/V)L respectively, which are characteristic for inositol 5-phosphatases (6Erneux C. Govaerts C. Communi D. Pesesse X. Biochim. Biophys. Acta. 1998; 1436: 185-199Google Scholar). Using the partial sequence obtained from the data base, complete sequences were obtained from cDNA library screens. Three putative inositol 5-phosphatases were identified (see “Appendix B”), coding for proteins consisting of 1800, 1377, and 787 amino acids, respectively. A comparison of the sequence obtained from cDNA clones with the genomic data base sequences revealed the presence of one intron in each inositol 5-phosphatase gene. The position of the introns is indicated by a triangle in Fig.1. Alignment of the catalytic domain of the fourD. discoideum inositol 5-phosphatases with other inositol 5-phosphatase catalytic domains shows a high degree of identity between the amino acid sequence of the four proteins and type II inositol 5-phosphatases (Fig. 2) (see “Appendix C”). All of the four proteins contain the conserved amino acids present in motifs I and II that are important for inositol 5-phosphatase activity (26Communi D. Lecocq R. Erneux C. J. Biol. Chem. 1996; 271: 11676-11683Google Scholar, 27Jefferson A.B. Majerus P.W. Biochemistry. 1996; 35: 7890-7894Google Scholar, 28Tsujishita Y. Guo S. Stolz L.E. York J.D. Hurley J.H. Cell. 2001; 105: 379-389Google Scholar). Also, the amino acids strongly conserved in other regions of the inositol 5-phosphatase domains are present in all four D. discoideum inositol 5-phosphatases, suggesting that they are active inositol 5-phosphatases. The four inositol 5-phosphatases differ with respect to their domain composition (Fig. 1). Dd5P1 shows the least complex composition containing only the inositol 5-phosphatase domain. A BLAST analysis with the catalytic domain shows the highest score (Expect value = 2e−54) with the catalytic domain of human SHIP2 (30% identity, 44% similarity) (29Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Google Scholar). This score is clearly higher than scores for non-SHIP-like proteins with the highest score for inositol polyphosphate 5-phosphatase II of Mus musculus (Expect value = 7e−37). The homology with SHIP2 does not expand to the N-terminal part of the protein, because the SH2 domain present in SHIP2 is not present in Dd5P1. Besides the inositol 5-phosphatase domain, Dd5P2 contains a region homologous to RCC1 (“Appendix D”) (30Ohtsubo M. Kai R. Furuno N. Sekiguchi T. Sekiguchi M. Hayashida H. Kuma K. Miyata T. Fukushige S. Murotsu T. Genes Dev. 1987; 1: 585-593Google Scholar). The structure of RCC1 has been solved, revealing a seven-bladed propeller with each blade comprising by four β-sheets (31Renault L. Nassar N. Vetter I. Becker J. Klebe C. Roth M. Wittinghofer A. Nature. 1998; 392: 97-101Google Scholar). RCC1 is known to bind Ran, a small G-protein present at high concentrations in the nucleus. The binding of RCC1 to Ran leads to an increased rate of exchange of GDP for GTP, thus acting as a guanine exchange factor for Ran (32Bischoff F.R. Ponstingl H. Nature. 1991; 354: 80-82Google Scholar). The highest BLAST score of the catalytic domain of Dd5P2 is shown with the catalytic domain of mouse inositolpolyphosphate 5-phosphataseb (INPP5b), a RhoGAP domain containing protein (41% identity, 59% similarity) (33Matzaris M. O'Malley C.J. Badger A. Speed C.J. Bird P.I. Mitchell C.A. J. Biol. Chem. 1998; 273: 8256-8267Google Scholar). The long C terminus of Dd5P2 (∼1000 amino acids) does not represent a known domain structure. Three poly(N) stretches and a poly(D/E) stretch are present in this C-terminal part of the protein. These kind of stretches are not unusual in D. discoideum proteins. The other two inositol 5-phosphatases identified in D. discoideum are homologous to human inositol 5-phosphatases. Dd5P3 resembles the synaptojanin-like proteins found in both human and yeast with the highest BLAST score of the catalytic domain with that of INP52p from fission yeast (37% identity, 59% similarity) (34Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Google Scholar). Similar to human Synaptojanin and yeast INP52p, Dd5P3 has aSacI-like domain including the conserved RXNCXDCLDRTN motif (35Hughes W.E. Cooke F.T. Parker P.J. Biochem. J. 2000; 350: 337-352Google Scholar, 36Guo S. Stolz L.E. Lemrow S.M. York J.D. J. Biol. Chem. 1999; 274: 12990-12995Google Scholar) in front of the inositol 5-phosphatase domain (see “Appendix E”). The SacI domains of Synaptojanin and INP52p have been shown to remove the phosphate group of PI(4)P, PI(3)P, and at a low rate, both phosphates of PI(3,5)P2(36). The long C-terminal part of Dd5P3 does not have any homology with known domains and consists of poly(Q) and poly(N) repeats. Dd5P4 is homologous to human OCRL (10Attree O. Olivos I.M. Okabe I. Bailey L.C. Nelson D.L. Lewis R.A. McInnes R.R. Nussbaum R.L. Nature. 1992; 358: 239-242Google Scholar) and INPP5b (37Mitchell C.A. Connolly T.M. Majerus P.W. J. Biol. Chem. 1989; 264: 8873-8877Google Scholar, 38Jefferson A.B. Majerus P.W. J. Biol. Chem. 1995; 270: 9370-9377Google Scholar), consisting of a inositol 5-phosphatase domain followed by a RhoGAP domain (see “Appendix F”). The catalytic domain has the highest BLAST score with the catalytic domain of human INPP5b (44% identity, 60% similarity). RhoGAP domains are known to catalyze the GTPase activity of Rho proteins. The crystal structure of human RhoGAP has been solved indicating a role for two conserved amino acids, Arg-85p50RhoGAP and Asn-194p50RhoGAP, in GAP-activated GTP hydrolysis (39Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Google Scholar). The role of these amino acids is supported by mutational analysis (39Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Google Scholar, 40Graham D.L. Eccleston J.F. Lowe P.N. Biochemistry. 1999; 38: 985-991Google Scholar, 41Harris T.W. Hartwieg E. Horvitz H.R. Jorgensen E.M. J. Cell Biol. 2000; 150: 589-600Google Scholar). Mutational analysis has also shown that the conserved Arg is not predominantly involved in the binding of Rho proteins (40Graham D.L. Eccleston J.F. Lowe P.N. Biochemistry. 1999; 38: 985-991Google Scholar). In Dd5P4, the Arg and the Asn are substituted by an Ile and Gln, respectively. This observation may suggest that Dd5P4 does not exhibit high RhoGAP activity, but may still bind Rho proteins. To determine the transcription levels of the D. discoideuminositol 5-phosphatases, Northern blot analysis was performed. Very low transcription levels were observed for the four inositol 5-phosphatases in all stages of the D. discoideum life cycle (Fig.3). Dd5P1 and Dd5P3were equally transcribed in all stages with the exception of the even lower transcription of Dd5P3 in the vegetative stage. The levels of transcription of Dd5P2 were higher during aggregation than during growth and multicellular development. The transcription of Dd5P4 was relatively high during growth, decreased during aggregation, and returned to almost vegetative levels in the multicellular stages. Furthermore, a smaller transcript of 2.5 kilobases was observed in the multicellular stages. To determine whether the four putative inositol 5-phosphatases identified in D. discoideum can function as inositol 5-phosphatases, the catalytic activity and specificity of the inositol 5-phosphatase domains were studied in vitro. The catalytic domains were expressed as His tag fusion proteins in E. coli and purified. The protein of the expected size could be detected by Western blot analysis for Dd5P1–3 (Fig. 4). The Western blot analysis performed for Dd5P4 showed a band at a higher position than expected (62 instead of 55 kDa), but the purified protein did show inositol 5-phosphatase activity. Unfortunately, because of the very low expression levels obtained for the catalytic domain of Dd5P1, no activity could be determined for this protein. The purified catalytic domains of Dd5P2, Dd5P3, and Dd5P4 were incubated with the phospholipids PI(4,5)P2 and PI(3,4,5)P3 and the water-soluble inositol phosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Substrate degradation was quantified using thin-layer chromatography for the phospholipids and ion-exchange chromatography for the water-soluble inositol phosphates. The results are summarized in Fig.5 demonstrating good degradation of PI(4,5)P2 by all inositol 5-phosphatases, whereas PI(3,4,5)P3 is degraded predominantly by Dd5P2 and also by Dd5P4 but not by Dd5P3. Good degradation of the water-soluble inositol phosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4 is observed for Dd5P2, Dd5P3, and Dd5P4, showing the best degradation of Ins(1,4,5)P3 by Dd5P3, whereas Ins(1,3,4,5)P4is best degraded by Dd5P2 (Fig. 5 C). Comparing the activity of the four enzymes for each substrate indicates the relatively preferred substrates (Table I). The protein with the highest activity toward Ins(1,4,5)P3 is the catalytic domain of Dd5P3. This protein, compared with the other proteins, is poor in dephosphorylation of lipid-soluble substrates. This substrate specificity is almost opposite to the specificity of the homologue INP51p, which degrades PI(4,5)P2 but does not hydrolyze Ins(1,4,5)P3. Dd5P2, in contrast, is a very good PI(3,4,5)P3- and Ins(1,3,4,5)P4-metabolizing enzyme.Table ISubstrate specificity of Dd5P1–4 towards different substratesEnzymesRelative activityIns(1,4,5) P3Ins(1,3,4,5) P4PI(4,5) P2PI(3,4,5) P3Dd5P1NDNDNDNDDd5P2++++++++++++Dd5P3++++++++/−Dd5P4++++Catalytic domains of Dd5P1–4 were expressed in E. coli,purified, and incubated with the four substrates. Every additional “+” indicates an additional doubling in activity. ND, not determined because of low expression levels. Open table in a new tab Catalytic domains of Dd5P1–4 were expressed in E. coli,purified, and incubated with the four substrates. Every additional “+” indicates an additional doubling in activity. ND, not determined because of low expression levels. Upon starvation, D. discoideum wild-type cells show chemotaxis toward the cAMP secreted by other starving cells. The formed aggregate develops into a migrating slug or fruiting body. The spores of the fruiting body are resistant to severe conditions and maturate into single amoeba under better conditions. To get an indication of the function of inositol 5-phosphatases inD. discoideum, the four identified inositol 5-phosphatase genes have been knocked out. The effect of gene inactivation on chemotaxis has been investigated by determining the response of cells toward different cAMP concentrations using the small-droplet chemotaxis assay. Single gene inactivation of either Dd5P3 orDd5P4 or double gene inactivation of Dd5P2 andDd5P3 (Dd5P2/3−) did not negatively affect chemotaxis, whereas single disruption ofDd5P1 or Dd5P2 slightly improved chemotaxis (Fig.6). Also, the double disruption of eitherDd5P1 and Dd5P2(Dd5P1/2−) or Dd5P1 andDd5P3 (Dd5P1/3−) resulted in slightly improved chemotaxis. Growth and development of the cells were also studied. The single gene inactivation of Dd5P1,Dd5P2, or Dd5P3 did not result in any observable difference in growth rate in axenic medium or on bacterial lawns (data not shown). The single disruptants Dd5P1−,Dd5P2−, and Dd5P3− were deposited on non-nutrient agar plates or grown on bacterial lawns to study the development of the cells. Aggregation of the cells proceeded at a rate comparable to wild-type cells. Slug formation was also normal as well as the formation of fruiting bodies. The spores that were formed were viable (data not shown). Also,Dd5P1/2−,Dd5P1/3−, orDd5P2/3− cells showed no defect in growth or development. The single disruption of Dd5P4 did affect growth and development. The growth rate in axenic medium was significantly lower for Dd5P4 cells compared with wild-type AX3 cells (Fig.7). Whereas the doubling time of wild-type cells is ∼15 h in shaking culture, the doubling time of Dd5P4-deficient cells is 41 h. The growth rate on bacterial lawns was affected as well for Dd5P4− cells. Amoeba were deposited on a bacterial lawn and incubated at 22 °C. Approximately 90% wild-type amoeba formed visible plaques within 4 days, whereas <1% Dd5P4− cells formed visible plaques after 9 days. The development of the Dd5P4 cells when grown on bacterial lawns was affected as well. The inactivation of Dd5P4 lead to the formation of multiple-tipped aggregates (Fig. 8).Figure 8Development of wild-type AX3,Dd5P4 −, andDd5P4 −/Dd5P4 on K. aerogens. Pictures were taken of tipped aggregates using ×40 magnification.View Large Image Figure ViewerDownload (PPT) The overexpression of the full-length Dd5P4 restored the defects ofDd5P4− cells in growth and development, confirming that the disruption of Dd5P4 was indeed responsible for the observed defects. Growth rate in axenic medium was comparable to the growth rate of wild-type cells (AX3) with a doubling time of 15 h (Fig. 7). When grown on bacterial plates, ∼90% of the amoeba formed visible plaques within 4 days, and the multiple-tipped phenotype observed for theDd5P4− cells could not be observed anymore (Fig. 8). Overexpression of the inositol 5-phosphatase catalytic domain or the RhoGAP domain of Dd5P4 in the Dd5P4 knock-out strains (Dd5P4−/cat5P4 andDd5P4−/GAP5P4, respectively) did not restore any of the defects. As can be seen in Fig. 7, growth rate in axenic medium was not enhanced by overexpression of either one of the domains. Also, growth rate and development on bacterial lawns were not restored (data not shown). The implicated role of phosphoinositide molecules in signal localization has lead to an increased interest in metabolizing enzymes such as inositol 5-phosphatases. The amount of data on mammalian inositol 5-phosphatases has expanded rapidly over the last few years, showing important functions for inositol 5-phosphatases in several processes (6Erneux C. Govaerts C. Communi D. Pesesse X. Biochim. Biophys. Acta. 1998; 1436: 185-199Google Scholar). Human inositol 5-phosphatases are divided in two groups. Type I inositol 5-phosphatases only convert the water-soluble substrates Ins(1,4,5)P3 and Ins(1,3,4,5)P4. They do not convert lipid substrates. Type II inositol 5-phosphatases do convert phosphoinositides (e.g. PI(4,5)P2 and PI(3,4,5)P3), and in most cases, they also convert water-soluble substrates. The insertions present in Type I enzymes may prevent the enzymes from binding to the membrane surface, which may explain the differences in substrate specificity between Types I and II inositol 5-phosphatases (28Tsujishita Y. Guo S. Stolz L.E. York J.D. Hurley J.H. Cell. 2001; 105: 379-389Google Scholar). The insertions present in Type I inositol 5-phosphatases are not present in either one of the D. discoideum inositol 5-phosphatases. As expected by this sequence analysis, the D. discoideum 5-phosphatases Dd5P2, Dd5P3, and Dd5P4 catalyze the dephosphorylation of both water-soluble and lipid substrates, which classify them as Type II inositol 5-phosphatases. The D. discoideum genome has been sequenced to near completion, making it unlikely that more inositol 5-phosphatases containing the motifs WXGDXN(Y/F)R and P(A/S)W(C/T)DR(I/V)L are present. Therefore, probably no Type I and only four Type II inositol 5-phosphatases are present in D. discoideum. Type II inositol 5-phosphatases can be divided in three subgroups on the basis of domain composition. SHIP1 represents the group of SH2 domain containing inositol 5-phosphatases Synaptojanin, theSacI domain containing enzymes, and OCRL, the RhoGAP domain containing inositol 5-phosphatases (Fig. 1). Mammals contain all three groups of proteins. Six non-mammalian organisms containing putative inositol 5-phosphatases have been sequenced completely so far. The metazoa C. elegans and D. melanogaster lack inositol 5-phosphatases containing a SH2 domain, but both contain one Synaptojanin-like protein ((41) and Q9W296, respectively) and one OCRL-like protein (O17590 and O46049, respectively). The diversity in the plant Arabidopsis thaliana, the yeast strainsSaccharomyces cerevisiae and Schizosaccharomyces pombe, and the microsporidia Encephalitozoon cuniculiis even less. The inositol 5-phosphatases of A. thalianacontain only WD40 repeats as additional domains (e.g. Q9SKB7 and O80560), whereas in yeast only Synaptojanin-like proteins are present (28Tsujishita Y. Guo S. Stolz L.E. York J.D. Hurley J.H. Cell. 2001; 105: 379-389Google Scholar, 34Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Google Scholar). The one inositol 5-phosphatase ofE. cuniculi (CAD25856) only contains the inositol 5-phosphatase catalytic domain. In prokaryotes, no inositol 5-phosphatases have been identified. The four inositol 5-phosphatases in D. discoideum show a high diversity in domain composition compared with the evolutionary position of this organism between plants and yeast. A homologue of OCRL (Dd5P4) and a homologue of Synaptojanin (Dd5P3) were identified. Also, a unique combination of a RCC1-like domain in front of the inositol 5-phosphatase domain was identified (Dd5P2). This combination of domains has not been found in any other protein reported or present in the GenBankTM so far. Knock-out strains with one or a combination of two inactive genes for Dd5P1, Dd5P2, and Dd5P3 show no defects in growth and development, suggesting a redundancy among these proteins. Chemotaxis toward cAMP is similar or slightly improved in all of the inositol 5-phosphatase knock-out cell lines compared with wild type. Recently, it has been shown that the 3-phosphatase PTEN is the major PI(3,4,5)P3/PI(3,4)P2-degrading enzyme. It has been suggested that inositol 5-phosphatases add an addition layer of regulation of these molecules, fine-tuning the chemotactic signal (42Comer F.I. Parent C.A. Cell. 2002; 109: 541-544Google Scholar). Our results support the role of the group of inositol 5-phosphatases as a minor negative regulator of chemotaxis in D. discoideum. It will be interesting to study the effects of either overexpression or inactivation of the inositol 5-phosphatases in a PTEN-null background. Inactivation of Dd5P4 resulted in defects in growth and development. The reduced growth rate, either in axenic culture or grown on bacterial plates, suggests a role for Dd5P4 in endocytosis. Development is also affected as the cells form multiple-tipped aggregates. The relative high transcription levels of Dd5P4 at vegetative and multicellular stages support the role of Dd5P4 in growth and development. Knock-out strains of PI3K1 + 2 are defective in chemotaxis, growth, and development (15Funamoto S. Milan K. Meili R. Firtel R.A. J. Cell Biol. 2001; 153: 795-810Google Scholar, 16Zhou K. Takegawa K. Emr S.D. Firtel R.A. Mol. Cell. Biol. 1995; 15: 5645-5656Google Scholar). The cells grow slowly on bacterial lawns and in axenic medium and form multiple-tipped aggregates resembling the phenotype of Dd5P4. No enzyme activity measurements have been reported for the PI3K in D. discoideum, but they have been suggested to catalyze the formation of PI(3)P, PI(3,4)P2, and PI(3,4,5)P3on basis of sequence homology. Our results support a role for PI(3,4,5)P3 and/or PI(3,4)P2 in growth and development; Dd5P4 regulates the levels of these phospholipids by degradation of PI(3,4,5)P3 and production of PI(3,4)P2. On the other hand, the effect of inactivation of Dd5P4 could also be assigned to its action on either PI(4,5)P2 or Ins(1,4,5)P3, two signaling molecules also implicated in endocytosis (20Peracino B. Borleis J. Jin T. Westphal M. Schwartz J.M. Wu L. Bracco E. Gerisch G. Devreotes P. Bozzaro S. J. Cell Biol. 1998; 141: 1529-1537Google Scholar, 21Seastone D.J. Zhang L. Buczynski G. Rebstein P. Weeks G. Spiegelman G. Cardelli J. Mol. Biol. Cell. 1999; 10: 393-406Google Scholar). In addition, the RhoGAP domain could be responsible for the function of Dd5P4 in endocytosis. Rho proteins have been shown to play a role in both development and growth. The defects in growth and development ofDd5P4− cells can be rescued by overexpression of the full-length protein. These defects can not be restored by transfection of D. discoideum cells with an expression vector containing either the inositol 5-phosphatase catalytic domain or the RhoGAP domain. Although we can not exclude that the domains are not properly expressed or folded, the fact that the inositol 5-phosphatase catalytic domain expressed in E. coli is catalytically active would suggest that inositol 5-phosphatase activity is not sufficient to restore the defects ofDd5P4−cells. It is possible that the RhoGAP and inositol 5-phosphatase catalytic domain act together to perform its function in growth. The binding of a Rho protein to the RhoGAP domain could affect the inositol 5-phosphatase activity. This would lead to a direct interaction between the Rho and phosphoinositide pathways. It would be interesting to see if and which Rho protein binds to the GAP domain of Dd5P4. We are indebted to all of the teams involved in the Dictyostelium sequencing projects. We thank L. Drayer and H. Otsuka for their contribution in cloning and analyzing Dd5P1." @default.
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• W1963724298 title "A Diverse Family of Inositol 5-Phosphatases Playing a Role in Growth and Development in Dictyostelium discoideum" @default.
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