FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2084824425> ?p ?o ?g. }

• W2084824425 endingPage "39610" @default.
• W2084824425 startingPage "39604" @default.
• W2084824425 abstract "Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an important second messenger in signaling pathways in organisms ranging from yeast to mammals, but the regulation of PI(4,5)P2 levels remains unclear. Here we present evidence that PI(4,5)P2 levels in Saccharomyces cerevisiae are down-regulated by the homologous and functionally redundant proteins TAX4 and IRS4. The EPS15 homology domain-containing proteins TAX4 and IRS4 bind and activate the PI(4,5)P 5-phosphatase INP51 via an Asn-Pro-Phe motif in INP51. Furthermore, the INP51-TAX4/IRS4 complex negatively regulates the cell integrity pathway. Thus, TAX4 and IRS4 are novel regulators of PI(4,5)P2 and PI(4,5)P2-dependent signaling. The interaction between TAX4/IRS4 and INP51 is analogous to the association of EPS15 with the 5-phosphatase synaptojanin 1 in mammalian cells, suggesting that EPS15 is an activator of synaptojanin 1. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an important second messenger in signaling pathways in organisms ranging from yeast to mammals, but the regulation of PI(4,5)P2 levels remains unclear. Here we present evidence that PI(4,5)P2 levels in Saccharomyces cerevisiae are down-regulated by the homologous and functionally redundant proteins TAX4 and IRS4. The EPS15 homology domain-containing proteins TAX4 and IRS4 bind and activate the PI(4,5)P 5-phosphatase INP51 via an Asn-Pro-Phe motif in INP51. Furthermore, the INP51-TAX4/IRS4 complex negatively regulates the cell integrity pathway. Thus, TAX4 and IRS4 are novel regulators of PI(4,5)P2 and PI(4,5)P2-dependent signaling. The interaction between TAX4/IRS4 and INP51 is analogous to the association of EPS15 with the 5-phosphatase synaptojanin 1 in mammalian cells, suggesting that EPS15 is an activator of synaptojanin 1. Phosphoinositides are conserved from yeast to mammals as second messengers. They mediate the signal transduction involved in many different cellular processes and thereby comprise a complex signaling system (1Martin T.F. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (449) Google Scholar). One of the more thoroughly studied phosphoinositides is phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). 1The abbreviations used are: PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; EH, EPS115 homology; GAP, GTPase-activating protein; HA, hemagglutinin A; MAP, mitogen-activated protein; TOR, target of rapamycin; TORC, TOR complex; TRITC, tetramethylrhodamine isothiocyanate; ts, temperature sensitive. 1The abbreviations used are: PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; EH, EPS115 homology; GAP, GTPase-activating protein; HA, hemagglutinin A; MAP, mitogen-activated protein; TOR, target of rapamycin; TORC, TOR complex; TRITC, tetramethylrhodamine isothiocyanate; ts, temperature sensitive. PI(4,5)P2 was originally shown to be cleaved by phospholipase C to generate the two second messengers inositol-1,4,5-phosphate and diacylglycerol (2Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3311) Google Scholar). More recently, uncleaved PI(4,5)P2 has also been shown to act as a second messenger (3Liscovitch M. Chalifa V. Pertile P. Chen C.S. Cantley L.C. J. Biol. Chem. 1994; 269: 21403-21406Abstract Full Text PDF PubMed Google Scholar). Uncleaved PI(4,5)P2 acts by binding conserved domains in target proteins, such as the pleckstrin homology domain (4Gascard P. Sauvage M. Sulpice J.C. Giraud F. Biochemistry. 1993; 32: 5941-5948Crossref PubMed Scopus (19) Google Scholar, 5Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (674) Google Scholar, 6Itoh T. Koshiba S. Kigawa T. Kikuchi A. Yokoyama S. Takenawa T. Science. 2001; 291: 1047-1051Crossref PubMed Scopus (387) Google Scholar). In mammalian cells, PI(4,5)P2 signaling is important in regulating vesicular transport, the organization of the actin cytoskeleton, and the regulation of ion channels (7Cremona O. De Camilli P. J. Cell Sci. 2001; 114: 1041-1052Crossref PubMed Google Scholar, 8Yin H.L. Janmey P.A. Annu. Rev. Physiol. 2003; 65: 761-789Crossref PubMed Scopus (567) Google Scholar, 9Hilgemann D.W. Feng S. Nasuhoglu C. Science's STKE. 2001; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2001/111/re19)PubMed Google Scholar). In the model organism Saccharomyces cerevisiae, PI(4,5)P2 is also essential and is produced by MSS4, the sole phosphatidylinositol 4-phosphate 5-kinase in yeast (10Desrivieres S. Cooke F.T. Parker P.J. Hall M.N. J. Biol. Chem. 1998; 273: 15787-15793Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 11Homma K. Terui S. Minemura M. Qadota H. Anraku Y. Kanaho Y. Ohya Y. J. Biol. Chem. 1998; 273: 15779-15786Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). One target of PI(4,5)P2 signaling in S. cerevisiae is the GDP/GTP exchange factor ROM2. ROM2 contains a pleckstrin homology domain that binds PI(4,5)P2 in the plasma membrane, thus mediating the localization of ROM2 (12Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (184) Google Scholar, 13Audhya A. Emr S.D. Dev. Cell. 2002; 2: 593-605Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). ROM2 has two homologs, ROM1 and TUS1, which also harbor putative pleckstrin homology domains (12Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (184) Google Scholar, 14Schmelzle T. Helliwell S.B. Hall M.N. Mol. Cell. Biol. 2002; 22: 1329-1339Crossref PubMed Scopus (107) Google Scholar). ROM2 and its homologs are components of the cell integrity pathway necessary for the cellular response to cell wall damage induced by stress such as heat shock (14Schmelzle T. Helliwell S.B. Hall M.N. Mol. Cell. Biol. 2002; 22: 1329-1339Crossref PubMed Scopus (107) Google Scholar, 15Bickle M. Delley P.A. Schmidt A. Hall M.N. EMBO J. 1998; 17: 2235-2245Crossref PubMed Scopus (161) Google Scholar). The response to cell wall damage includes a reorganization of the actin cytoskeleton and an up-regulation of cell wall synthesis (16Igual J.C. Johnson A.L. Johnston L.H. EMBO J. 1996; 15: 5001-5013Crossref PubMed Scopus (223) Google Scholar, 17Delley P.A. Hall M.N. J. Cell Biol. 1999; 147: 163-174Crossref PubMed Scopus (244) Google Scholar, 18Jung U.S. Levin D.E. Mol. Microbiol. 1999; 34: 1049-1057Crossref PubMed Scopus (356) Google Scholar). Activation of the cell integrity pathway is mediated by its most upstream component, the cell wall sensor WSC1, which signals to ROM2 (19Verna J. Lodder A. Lee K. Vagts A. Ballester R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13804-13809Crossref PubMed Scopus (318) Google Scholar, 20Philip B. Levin D.E. Mol. Cell. Biol. 2001; 21: 271-280Crossref PubMed Scopus (245) Google Scholar). Subsequently, ROM2 stimulates the exchange of GDP to GTP in the Rho GTPase RHO1, thereby activating RHO1 (12Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (184) Google Scholar). The GTPase-activating protein (GAP) SAC7 converts RHO1-GTP into the inactive GDP-bound form (21Schmidt A. Bickle M. Beck T. Hall M.N. Cell. 1997; 88: 531-542Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). RHO1 in its active (GTP-bound) form has several effectors such as the glucan synthase FKS1 and the PKC1-MAP kinase cascade (22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (394) Google Scholar, 23Drgonova J. Drgon T. Tanaka K. Kollar R. Chen G.C. Ford R.A. Chan C.S. Takai Y. Cabib E. Science. 1996; 272: 277-279Crossref PubMed Scopus (302) Google Scholar, 24Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 25Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (305) Google Scholar). Activation of the MAP kinase MPK1 induces transcription of genes involved in cell wall biosynthesis such as CHS3 encoding chitin synthase III, which is important for cell wall repair (16Igual J.C. Johnson A.L. Johnston L.H. EMBO J. 1996; 15: 5001-5013Crossref PubMed Scopus (223) Google Scholar, 18Jung U.S. Levin D.E. Mol. Microbiol. 1999; 34: 1049-1057Crossref PubMed Scopus (356) Google Scholar, 26Garcia-Rodriguez L.J. Trilla J.A. Castro C. Valdivieso M.H. Duran A. Roncero C. FEBS Lett. 2000; 478: 84-88Crossref PubMed Scopus (72) Google Scholar). The ROM2-MAP kinase-signaling pathway is also an effector branch of the TOR signaling network. The TOR signaling network contains two structurally and functionally distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2) (27Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar). TORC2 regulates the polarization of the actin cytoskeleton, and this regulation is via the ROM2-MAP kinase pathway (21Schmidt A. Bickle M. Beck T. Hall M.N. Cell. 1997; 88: 531-542Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 27Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar, 28Helliwell S.B. Schmidt A. Ohya Y. Hall M.N. Curr. Biol. 1998; 8: 1211-1214Abstract Full Text Full Text PDF PubMed Google Scholar, 29Schmidt A. Kunz J. Hall M.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13780-13785Crossref PubMed Scopus (214) Google Scholar). Interestingly, the sole phosphatidylinositol 4-phosphate 5-kinase MSS4 is required for the polarization of the actin cytoskeleton, and overexpression of MSS4 restores growth in conditional TORC2 mutants (10Desrivieres S. Cooke F.T. Parker P.J. Hall M.N. J. Biol. Chem. 1998; 273: 15787-15793Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 11Homma K. Terui S. Minemura M. Qadota H. Anraku Y. Kanaho Y. Ohya Y. J. Biol. Chem. 1998; 273: 15779-15786Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 27Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar, 30Helliwell S.B. Howald I. Barbet N. Hall M.N. Genetics. 1998; 148: 99-112Crossref PubMed Google Scholar). However, the link between PI(4,5)P2 and TORC2 in the regulation of ROM2 and, ultimately, the actin cytoskeleton is not understood. The phosphoinositide 5-phosphatases INP51, INP52, and INP53 (also known as SJL1–3) mediate the turnover of PI(4,5)P2 and are implicated in several cellular processes such as cell wall biosynthesis and the organization of the actin cytoskeleton (31Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Crossref PubMed Google Scholar, 32Stefan C.J. Audhya A. Emr S.D. Mol. Biol. Cell. 2002; 13: 542-557Crossref PubMed Scopus (187) Google Scholar). Heat shock has been observed to induce an increase in PI(4,5)P2 levels, suggesting the existence of a mechanism regulating the levels of this phosphoinositide (10Desrivieres S. Cooke F.T. Parker P.J. Hall M.N. J. Biol. Chem. 1998; 273: 15787-15793Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). However, the nature of this regulatory mechanism and, more specifically, the possible regulation of the phosphoinositide 5-phosphatases are not well understood. In mammalian cells, the INP family orthologue synaptojanin 1 interacts with several proteins involved in endocytosis in nerve terminals (33Haffner C. Takei K. Chen H. Ringstad N. Hudson A. Butler M.H. Salcini A.E. Di Fiore P.P. De Camilli P. FEBS Lett. 1997; 419: 175-180Crossref PubMed Scopus (132) Google Scholar, 34McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (491) Google Scholar, 35Haffner C. Di Paolo G. Rosenthal J.A. de Camilli P. Curr. Biol. 2000; 10: 471-474Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To investigate the regulation of PI(4,5)P2 turnover in S. cerevisiae, we focused on INP51, INP52, and INP53 and their role in PI(4,5)P2 signaling linked to the cell integrity pathway and the TORC2 pathway. Here we present evidence for two novel positive regulators of the PI(4,5)P2 5-phosphatase INP51, the redundant EPS15 homology (EH) domain-containing proteins TAX4 and IRS4. INP51 associates with TAX4 or IRS4 to form a complex important for the turnover of PI(4,5)P2 linked to the cell integrity pathway. Strains and Media—The S. cerevisiae strains used in this study are listed in Table I. All strains were isogenic derivatives of TB50. Rich medium (yeast extract, peptone, and dextrose (YPD)) and minimal medium (synthetic dextrose (SD)) were as described previously (36Guthrie C. Fink G.R. Methods in Enzymology. 194. Academic Press, New York1991Google Scholar). PCR cassettes were used to generate gene deletions and for tagging with HA, Myc, or the tandem affinity purification tag, as described (37Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2236) Google Scholar, 38Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4168) Google Scholar, 39Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1421) Google Scholar). Yeast transformation was performed by the lithium acetate procedure (40Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Correct integration was verified by PCR, and tagged proteins were tested for their functionality.Table IStrains used in this studyStrainGenotypeTB50aMATaleu2-3,112 ura3-52 trp1 his3 rme1 HMLaPA66-2ATB50awsc1::HIS3MX6TS124-2ATB50arom2::kanMX4SF8-2DTB50asac7::kanMX4SF36-2BTB50ator2::kanMX4/YCplac33::tor2-21tsHM44-1CTB50α inp51::HIS3MX6HM47-1CTB50aINP51-HA3-kanMX4HM52-2DTB50α inp51::HIS tor2::kanMX4/YCplac33::tor2-21tsHM59-2CTB50ainp51::HIS rom2::kanMX4HM64-1BTB50ainp51::HIS wsc1::HIS3MX6HM77-1BTB50ainp51::HIS sac7::kanMX4HM90-1ATB50airs4::kanMX4HM92-1ATB50α TAX4-MYC13-HIS3MX6HM93-1ATB50α tax4::HIS3MX6HM96-1BTB50aINP51-HA3-kanMX4 TAX4-MYC13-HIS3MX6HM102-3BTB50aINP51ΔNPF-HA3-HIS3MX6HM104-2DTB50α IRS4-MYC13-HIS3MX6HM112-2CTB50α tax4::HIS3MX6 irs4::kanMX4HM119-2BTB50aINP51-HA3-kanMX4 IRS4-MYC13-HIS3MX6HM122-9BTB50atax4::HIS3MX6 irs4::kanMX4 sac7::kanMX4HM125-1ATB50α tax4::HIS3MX6 irs4::kanMX4 tor2::kanMX4/YCplac33::tor2-21tsHM127-3BTB50aINP51ΔNPF-HA3-HIS3MX6 TAX4-MYC13-HIS3MX6HM132-2DTB50aINP51ΔNPF-HA3-HIS3MX6 IRS4-MYC13-HIS3MX6 Open table in a new tab Spot Assay—Logarithmically growing cells were harvested and resuspended in 10 mm Tris-HCl (pH 7.4). The resuspended cells were diluted in a 10-fold dilution series. 3 μl of each dilution (10×, 100×, 1,000× and 10,000× diluted) were spotted on a YPD plate. Growth was scored after 2 days at 30 or 37 °C. INP51 Purification—Cells expressing a tagged or untagged (mock purification) version of INP51 were grown in 5 liters of YPD to an A600 of 0.8 at 30 °C, harvested by centrifugation, and washed with cold water before resuspension in lysis buffer. The lysis buffer used to prepare cell extracts contained phosphate-buffered saline, 5% glycerol. 0.5% Tween 20, phosphatase inhibitors (10 mm NaF, 10 mm NaN3, 10 mmp-nitrophenylphosphate, 10 mm sodium pyrophosphate, 10 mm β-glycerophosphate, and 1 mm phenylmethylsulfonyl fluoride), and protease inhibitor mixture tablets (Roche Applied Science). Cell lysate was obtained by glass bead lysis. Cell lysates containing ∼700 mg of protein were cleared with a 5-min, 500 × g spin, diluted with lysis buffer to 10 mg/ml, and subsequently passed over an ion exchange resin (SP-Sepharose). The resin was washed twice with lysis buffer and twice with lysis buffer containing 50 mm potassium acetate. Bound proteins were eluted with lysis buffer containing 600 mm potassium acetate. The eluate was precleared over a protein A-Sepharose (Amersham Biosciences) column prior to the addition of anti-HA (12CA5) cross-linked to protein ASepharose beads. After 5 times washing with lysis buffer, immunoprecipitated proteins were visualized by silver staining (41Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7814) Google Scholar). Analysis of protein bands by mass spectrometry was performed as described (27Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar). Immunoprecipitation—Yeast extracts from cells (500-ml culture in YPD grown to A600 of 0.8 at 30 °C) expressing tagged proteins of interest were prepared as described above. An aliquot of extract containing 10 mg of protein was adjusted to 1 ml with lysis buffer plus inhibitors. For immunoprecipitations, 20 μl of anti-HA (12CA5) or anti-Myc (9E10) cross-linked to protein A-Sepharose beads were added and mixed for 4 h at 4 °C. Beads were collected by centrifugation, washed five times with 1 ml of lysis buffer, and resuspended in 5× SDS-polyacrylamide gele sample buffer for electrophoresis. After SDS-PAGE, the proteins were blotted onto nitrocellulose membranes, blocked in 5% dry milk powder in 1× phosphate-buffered saline and 0.1% Nonidet P-40, and incubated with primary antibody anti-HA (clone 12CA5) or anti-Myc (clone 9E10) (1:10000 in blocking solution). Subsequently tagged proteins were detected using horseradish peroxidase-conjugated goat anti-mouse secondary antibodies and ECL reagents (Amersham Biosciences). Phosphoinositide Analysis—Yeast cells were diluted 5 × 104/ml in SD medium lacking inositol containing 10 μC/ml [3H]inositol and grown to a density of 2–4 × 106/ml (12–16 h). 2-ml aliquots were taken and mixed with 2 ml of MeOH. Cells were pelleted by centrifugation and processed as described previously (42Dove S.K. Cooke F.T. Douglas M.R. Sayers L.G. Parker P.J. Michell R.H. Nature. 1997; 390: 187-192Crossref PubMed Scopus (392) Google Scholar). Differential Centrifugation—Differential centrifugation was performed as described previously (43Kaiser C.A. Chen E.J. Losko S. Methods Enzymol. 2002; 351: 325-338Crossref PubMed Scopus (36) Google Scholar). Indirect Immunofluorescence—Logarithmically growing cells containing INP51-HA, TAX4-Myc, or IRS4-Myc were fixed for 2 h in the growth medium supplemented with formaldehyde (3.7% final) and potassium phosphate buffer (100 mm final, pH 6.5). Cells were washed and resuspended in sorbitol buffer (1.2 m sorbitol and 100 mm potassium phosphate, pH 6.5). Cell walls were digested for 45 min at 37 °C in sorbitol buffer supplemented with β-mercaptoethanol (20 mm final) and zymolyase 20T (12.5 mg/ml; Seigagaku Corporation). Spheroblasts were fixed on poly-l-lysine-coated glass slides and permeabilized with PBT (53 mm Na2HPO4, 13 mm NaH2PO4, 75 mm NaCl, 1% bovine serum albumin, and 0.1% Triton X-100). Immunofluorescence directed against the HA epitope was performed by application of a primary antibody anti-HA (clone 12CA5) or anti-Myc (clone 9E10) at a dilution of 1:1,000 in PBT for 2 h and, subsequently, the application of a Cy3-conjugated rabbit anti-mouse IgG (Molecular Probes) diluted 1:1,000 in PBT for 90 min. Washed cells were examined with a Zeiss Axiophot microscope (100× objective). Immunofluorescent detection of FKS1 was performed as described previously (17Delley P.A. Hall M.N. J. Cell Biol. 1999; 147: 163-174Crossref PubMed Scopus (244) Google Scholar). MAP Kinase Activation Assay—YPD cultures of logarithmically growing cells at 24 or 39 °C were harvested, and cell extracts were prepared as described previously (44Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Protein concentrations of extracts were determined by using a Bradford assay (Bio-Rad). Samples were denatured by the addition of 5× SDS-PAGE sample loading buffer and heating at 95 °C for 5 min. A total of 25 μg of protein (for MPK1 protein detection) or 40 μg of protein (for phosphorylated MAP kinase detection) was loaded for standard SDS-PAGE (10% acrylamide) and Western blot. For immunodetection, a goat anti-MPK1 antibody (clone yN-19, 1:1000 dilution; Santa Cruz Biotechnology) and a rabbit antiphospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (1:1000; Cell Signaling) were used. The anti-MPK1 and anti-phospho-MAP kinase were verified to specifically recognize activated MPK1 under heat stress conditions (data not shown). Secondary antibodies were horseradish peroxidase-conjugated anti-goat (anti-MPK1) or anti-rabbit (anti-phospho-MAP kinase) secondary antibody and detection by ECL reagents (Amersham Biosciences). The nitrocellulose membrane used for the Western blot was stained with Coomassie Blue for the visualization of total protein. Actin Staining—Logarithmically growing cells were fixed in formaldehyde (3.7%) and potassium phosphate buffer (100 mm, pH 6.5) and stained with TRITC-phalloidin (Sigma) to visualize actin, as described previously (45Benedetti H. Raths S. Crausaz F. Riezman H. Mol. Biol. Cell. 1994; 5: 1023-1037Crossref PubMed Scopus (237) Google Scholar). Chitin Staining—1 ml of cells at an A600 of 0.5 were collected by centrifugation and washed with water. Cells were then incubated in a solution with 0.5 mg/ml calcofluor white (Sigma) for 5 min to visualize chitin. To remove residual calcofluor white, cells were washed twice with water. Cells were examined with a Zeiss Axiophot microscope (100× objective). Disruption of INP51 Restores Growth of a tor2 Mutant—To understand further how the turnover of PI(4,5)P2 is important for PI(4,5)P2 signaling, we tested whether the phosphoinositide 5-phosphatase INP51, INP52, or INP53 antagonizes MSS4 signaling. We asked if the lack of any INP gene mimics MSS4 overexpression in suppressing a TOR2 signaling defect. A deletion of each INP gene was introduced into a temperaturesensitive tor2 (tor2ts) mutant defective in the organization of the actin cytoskeleton. The double mutants were grown at permissive (30 °C) and restrictive (37 °C) temperatures to determine whether an INP mutation can suppress the tor2ts mutation. Deletion of only INP51, INP52, or INP53 conferred no growth defect at any temperature (data not shown). Interestingly, the deletion of INP51, but not the deletion of INP52 or INP53, suppressed the growth defect of tor2ts cells (data not shown). The inp51 tor2ts cells grew almost as well as wild-type cells and as well as tor2ts cells overexpressing MSS4 (Fig. 1). This result suggests that INP51, but not INP52 or INP53, antagonizes the role of MSS4 in the TORC2 signaling pathway. Curiously, although inp51 suppressed the growth defect of tor2ts, it did not appear to suppress the actin defect of the tor2ts mutant (data not shown). INP51 Synthetically Interacts with Mutations Affecting the Cell Integrity Pathway—The finding that an inp51 deletion suppresses the growth defect of a tor2ts mutant is similar to the previous observation that activation of the cell integrity pathway suppresses the growth defect of a tor2ts mutant (15Bickle M. Delley P.A. Schmidt A. Hall M.N. EMBO J. 1998; 17: 2235-2245Crossref PubMed Scopus (161) Google Scholar) and suggests that INP51 may antagonize the cell integrity pathway. Thus, we investigated whether an inp51 mutation interacts with mutations affecting components of the cell integrity pathway. Specifically, we asked if an inp51 deletion suppresses or enhances the effect of mutations in WSC1, ROM2, SAC7, or MPK1. We observed that the deletion of INP51 suppresses the growth defects of wsc1 and rom2 cells at 37 and 30 °C, the restrictive temperatures of these mutants, respectively (Fig. 2, A and B). Conversely, the combination of inp51 with sac7 conferred a synthetic growth defect (Fig. 2C). Finally, the growth defect of an mpk1 mutant grown at restrictive temperature (38 °C) was not suppressed by INP51 deletion (data not shown). The above results show that INP51 genetically interacts with at least some components of the cell integrity pathway. Deletion of INP51 suppresses mutations in positive components of the pathway (WSC1 and ROM2) and causes a synthetic defect when combined with a mutation in a negative element of the pathway (SAC7). The nature of these interactions suggests that the phosphoinositide phosphatase INP51 negatively regulates signaling through the cell integrity pathway. This conclusion is consistent with the previous conclusions (13Audhya A. Emr S.D. Dev. Cell. 2002; 2: 593-605Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 27Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar, 30Helliwell S.B. Howald I. Barbet N. Hall M.N. Genetics. 1998; 148: 99-112Crossref PubMed Google Scholar, 46Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Abstract Full Text PDF PubMed Google Scholar) that the phosphoinositide kinase MSS4 and PI(4,5)P2 act positively on the TORC2 and cell integrity pathways. INP51 Associates with the EH Domain-containing Proteins TAX4 and IRS4 —In mammalian cells, the kinases and phosphatases that determine PI(4,5)P2 levels are regulated by interacting proteins (33Haffner C. Takei K. Chen H. Ringstad N. Hudson A. Butler M.H. Salcini A.E. Di Fiore P.P. De Camilli P. FEBS Lett. 1997; 419: 175-180Crossref PubMed Scopus (132) Google Scholar, 47Malecz N. McCabe P.C. Spaargaren C. Qiu R. Chuang Y. Symons M. Curr. Biol. 2000; 10: 1383-1386Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 48Song W. Zinsmaier K.E. Neuron. 2003; 40: 665-667Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 49Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar, 50Weernink P.A. Meletiadis K. Hommeltenberg S. Hinz M. Ishihara H. Schmidt M. Jakobs K.H. J. Biol. Chem. 2004; 279: 7840-7849Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 51Chong L.D. Traynor-Kaplan A. Bokoch G.M. Schwartz M.A. Cell. 1994; 79: 507-513Abstract Full Text PDF PubMed Scopus (594) Google Scholar). An indication that the level of PI(4,5)P2 is regulated in S. cerevisiae has been suggested previously by the observation that PI(4,5)P2 levels increase upon heat shock (10Desrivieres S. Cooke F.T. Parker P.J. Hall M.N. J. Biol. Chem. 1998; 273: 15787-15793Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). However, proteins regulating such kinases and phosphatases in S. cerevisiae remain to be identified. To identify potential INP51 regulatory proteins, we opted to isolate INP51-interacting proteins by using a biochemical approach described previously (27Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar). A functional, epitope-tagged version of INP51 (INP51-HA) was constructed and purified from yeast cell extracts as described under “Experimental Procedures.” Two homologous proteins were co-purified with INP51, IRS4, and an uncharacterized protein encoded by the open reading frameYJL083w that we named TAX4 (Fig. 3). The interaction between INP51 and TAX4 or IRS4 was confirmed by coimmunoprecipitation using epitope-tagged versions of TAX4 (TAX4Myc) and IRS4 (IRS4-Myc), as described under “Experimental Procedures” (Fig. 4). We did not observe interaction between TAX4 and IRS4 by coimmunoprecipitation of the heterologously tagged versions (TAX4-TAP and IRS4-Myc) of these proteins (data not shown). IRS4 was identified previously in a screen for mutants defective in rDNA silencing (52Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google Scholar). Untagged TAX4 has an apparent molecular mass of 77 kDa and a predicted size of 68.7 kDa. Untagged IRS4 is observed as two bands between 70 and 75 kDa, slightly larger than the predicted size of 68.8 kDa. TAX4 and IRS4 have an overall identity of 31% and contain a C-terminal EH domain. The EH domains of TAX4 and IRS4 are 64% identical. The EH domain, conserved from yeast to human, is a protein-protein interaction domain of ∼100 amino acids that interacts specifically with short motifs containing an asparagine-proline-phenylalanine (NPF) core (53Wong W.T. Schumacher C. Salcini A.E. Romano A. Castagnino P. Pelicci P.G. Di Fiore P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9530-9534Crossref PubMed Scopus (135) Google Scholar, 54Salcini A.E. Confalonieri S. Doria M. Santolini E. Tassi E. Minenkova O. Cesareni G. Pelicci P.G. Di Fiore P.P. Genes Dev. 1997; 11: 2239-2249Crossref PubMed Scopus (286) Google Scholar). INP51 has a C-terminal NPF motif (55Wendland B. Emr S.D. Riezman H. Curr. Opin. Cell Biol. 1998; 10: 513-522Crossref PubMed Scopus (150) Google Scholar), amino acids 932–934, which is important for the interaction between INP51 and TAX4 or IRS4. The absence of the NPF motif and amino" @default.
• W2084824425 created "2016-06-24" @default.
• W2084824425 creator A5003476561 @default.
• W2084824425 creator A5023356262 @default.
• W2084824425 creator A5032034269 @default.
• W2084824425 creator A5057556751 @default.
• W2084824425 date "2004-09-01" @default.
• W2084824425 modified "2023-09-30" @default.
• W2084824425 title "Negative Regulation of Phosphatidylinositol 4,5-Bisphosphate Levels by the INP51-associated Proteins TAX4 and IRS4" @default.
• W2084824425 cites W1499601028 @default.
• W2084824425 cites W1538592956 @default.
• W2084824425 cites W1542805579 @default.
• W2084824425 cites W1695245462 @default.
• W2084824425 cites W1864334043 @default.
• W2084824425 cites W1913497294 @default.
• W2084824425 cites W1915945057 @default.
• W2084824425 cites W1934941689 @default.
• W2084824425 cites W1956143364 @default.
• W2084824425 cites W1963724298 @default.
• W2084824425 cites W1966072209 @default.
• W2084824425 cites W1969473585 @default.
• W2084824425 cites W1973782344 @default.
• W2084824425 cites W1975631260 @default.
• W2084824425 cites W1977951612 @default.
• W2084824425 cites W1984545702 @default.
• W2084824425 cites W1985670865 @default.
• W2084824425 cites W1997093700 @default.
• W2084824425 cites W1997204703 @default.
• W2084824425 cites W2005188492 @default.
• W2084824425 cites W2010307209 @default.
• W2084824425 cites W2014440721 @default.
• W2084824425 cites W2017133620 @default.
• W2084824425 cites W2033133245 @default.
• W2084824425 cites W2033165803 @default.
• W2084824425 cites W2034092404 @default.
• W2084824425 cites W2038696130 @default.
• W2084824425 cites W2038727077 @default.
• W2084824425 cites W2040518083 @default.
• W2084824425 cites W2040838736 @default.
• W2084824425 cites W2041531078 @default.
• W2084824425 cites W2042013644 @default.
• W2084824425 cites W2051526561 @default.
• W2084824425 cites W2056701485 @default.
• W2084824425 cites W2062634275 @default.
• W2084824425 cites W2065220166 @default.
• W2084824425 cites W2066618807 @default.
• W2084824425 cites W2068432705 @default.
• W2084824425 cites W2071468845 @default.
• W2084824425 cites W2078388853 @default.
• W2084824425 cites W2078594804 @default.
• W2084824425 cites W2079122560 @default.
• W2084824425 cites W2079981145 @default.
• W2084824425 cites W2082426787 @default.
• W2084824425 cites W2086636140 @default.
• W2084824425 cites W2098100103 @default.
• W2084824425 cites W2099406730 @default.
• W2084824425 cites W2100057937 @default.
• W2084824425 cites W2113221012 @default.
• W2084824425 cites W2117111749 @default.
• W2084824425 cites W2120077272 @default.
• W2084824425 cites W2123298030 @default.
• W2084824425 cites W2126061110 @default.
• W2084824425 cites W2128511563 @default.
• W2084824425 cites W2137096941 @default.
• W2084824425 cites W2142161768 @default.
• W2084824425 cites W2142437744 @default.
• W2084824425 cites W2142939427 @default.
• W2084824425 cites W2143233645 @default.
• W2084824425 cites W2149676146 @default.
• W2084824425 cites W2151117299 @default.
• W2084824425 cites W2152600551 @default.
• W2084824425 cites W2152741987 @default.
• W2084824425 cites W2163724657 @default.
• W2084824425 cites W2164850267 @default.
• W2084824425 cites W2325991334 @default.
• W2084824425 cites W59209658 @default.
• W2084824425 cites W93651929 @default.
• W2084824425 doi "https://doi.org/10.1074/jbc.m405589200" @default.
• W2084824425 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15265867" @default.
• W2084824425 hasPublicationYear "2004" @default.
• W2084824425 type Work @default.
• W2084824425 sameAs 2084824425 @default.
• W2084824425 citedByCount "22" @default.
• W2084824425 countsByYear W20848244252012 @default.
• W2084824425 countsByYear W20848244252013 @default.
• W2084824425 countsByYear W20848244252014 @default.
• W2084824425 countsByYear W20848244252015 @default.
• W2084824425 countsByYear W20848244252016 @default.
• W2084824425 countsByYear W20848244252017 @default.
• W2084824425 countsByYear W20848244252019 @default.
• W2084824425 countsByYear W20848244252020 @default.
• W2084824425 countsByYear W20848244252022 @default.
• W2084824425 crossrefType "journal-article" @default.
• W2084824425 hasAuthorship W2084824425A5003476561 @default.
• W2084824425 hasAuthorship W2084824425A5023356262 @default.
• W2084824425 hasAuthorship W2084824425A5032034269 @default.
• W2084824425 hasAuthorship W2084824425A5057556751 @default.
• W2084824425 hasBestOaLocation W20848244251 @default.

• next