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• W1995517360 abstract "In examining the protein kinase components of mitogen-activated protein (MAP) kinase (MAPK) cascades that regulate the c-Jun N-terminal kinase (JNK) in Drosophila S2 cells, we previously found that distinct upstream kinases were involved in responses to sorbitol and lipopolysaccharide. Here we have extended that analysis to the possible MAPK kinase kinase kinases (MAP4Ks) in the JNK pathway. Fray, a putative Drosophila MAP4K, provided a major contribution to JNK activation by sorbitol. To explore the possible link to JNK in mammalian cells, we isolated and characterized OSR1 (oxidative stress-responsive 1), one of two human Fray homologs. OSR1 is a 58-kDa protein of 527 amino acids that is widely expressed in mammalian tissues and cell lines. Of potential regulators surveyed, endogenous OSR1 is activated only by osmotic stresses, notably sorbitol and to a lesser extent NaCl. However, OSR1 did not increase the activity of coexpressed JNK, nor did it activate three other MAPKs, p38, ERK2, and ERK5. A two-hybrid screen implicated another Ste20p family member, the p21-activated protein kinase PAK1, as an OSR1 target. OSR1 phosphorylated threonine 84 in the N-terminal regulatory domain of PAK1. Replacement of threonine 84 with glutamate reduced the activation of PAK1 by an active form of the small G protein Cdc42, suggesting that phosphorylation by OSR1 modulates the G protein sensitivity of PAK isoforms. In examining the protein kinase components of mitogen-activated protein (MAP) kinase (MAPK) cascades that regulate the c-Jun N-terminal kinase (JNK) in Drosophila S2 cells, we previously found that distinct upstream kinases were involved in responses to sorbitol and lipopolysaccharide. Here we have extended that analysis to the possible MAPK kinase kinase kinases (MAP4Ks) in the JNK pathway. Fray, a putative Drosophila MAP4K, provided a major contribution to JNK activation by sorbitol. To explore the possible link to JNK in mammalian cells, we isolated and characterized OSR1 (oxidative stress-responsive 1), one of two human Fray homologs. OSR1 is a 58-kDa protein of 527 amino acids that is widely expressed in mammalian tissues and cell lines. Of potential regulators surveyed, endogenous OSR1 is activated only by osmotic stresses, notably sorbitol and to a lesser extent NaCl. However, OSR1 did not increase the activity of coexpressed JNK, nor did it activate three other MAPKs, p38, ERK2, and ERK5. A two-hybrid screen implicated another Ste20p family member, the p21-activated protein kinase PAK1, as an OSR1 target. OSR1 phosphorylated threonine 84 in the N-terminal regulatory domain of PAK1. Replacement of threonine 84 with glutamate reduced the activation of PAK1 by an active form of the small G protein Cdc42, suggesting that phosphorylation by OSR1 modulates the G protein sensitivity of PAK isoforms. Cell growth and differentiation are precisely regulated by complex systems involving protein kinase cascades. A family of these cascades contain the pleiotropic mitogen-activated protein kinases (MAPKs). 1The abbreviations used are: MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MAP4K, MAPK kinase kinase kinases; ERK, extracellular signal-regulated kinase; MEKK, MAPK/ERK kinase kinase; JNK, c-Jun N-terminal kinase; PAK, p21-activated protein kinase; GCK, germinal center kinase; GST, glutathione S-transferase; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; dsRNA, double-stranded RNA; MBP, myelin basic protein; RT, reverse transcriptase. 1The abbreviations used are: MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MAP4K, MAPK kinase kinase kinases; ERK, extracellular signal-regulated kinase; MEKK, MAPK/ERK kinase kinase; JNK, c-Jun N-terminal kinase; PAK, p21-activated protein kinase; GCK, germinal center kinase; GST, glutathione S-transferase; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; dsRNA, double-stranded RNA; MBP, myelin basic protein; RT, reverse transcriptase. These enzymes play critical roles in transducing signals from extracellular stimuli, including hormones, growth factors, and environmental stresses, throughout the cell (1Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (864) Google Scholar, 2Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 3Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2864) Google Scholar, 4Chen Z. Gibson T.B. Robinson F. Silvestro L. Pearson G. Xu B. Wright A. Vanderbilt C. Cobb M.H. Chem. Rev. 2001; 101: 2449-2476Crossref PubMed Scopus (779) Google Scholar). The core modules of MAPK cascades are composed of three sequentially acting protein kinases, a MAPK activated by a MAPK kinase (MAP2K), which is activated by a MAPK kinase kinase (MAP3K). In mammals, the most studied MAPKs are ERK1/2, the c-Jun N-terminal kinase (JNK), p38, and ERK5. Ste20p is the yeast MAP4K protein that activates the MAP3K in the pheromone-responsive MAPK cascade of the budding yeast mating pathway (5Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar, 6Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (344) Google Scholar). In the past several years, numerous protein kinases with catalytic domains closely related to that of Ste20p have been identified and constitute the Ste20p family. Based on structure and regulation, two subfamilies have been defined, the p21-activated kinase (PAK) subfamily and the larger germinal center kinase (GCK) subfamily (7Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). The PAKs include the six enzymes nearest in characteristics to Ste20p itself; each contains a C-terminal catalytic domain and an N-terminal regulatory domain with a small G protein binding motif. PAKs have been shown not only to activate MAPKs (primarily JNK and p38) but also to influence disassembly of the actin cytoskeleton and apoptosis (8Harden N. Lee J. Loh H.Y. Ong Y.M. Tan I. Leung T. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 1896-1908Crossref PubMed Scopus (174) Google Scholar, 9Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (602) Google Scholar, 10Frost J.A. Khokhlatchev A. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28253-28260Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11Abo A. Qu J. Cammarano M.S. Dan C. Fritsch A. Baud V. Belisle B. Minden A. EMBO J. 1998; 17: 6527-6540Crossref PubMed Scopus (311) Google Scholar, 12Gnesutta N. Qu J. Minden A. J. Biol. Chem. 2001; 276: 14414-14419Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 13Pandey A. Dan I. Kristiansen T.Z. Watanabe N.M. Voldby J. Kajikawa E. Khosravi-Far R. Blagoev B. Mann M. Oncogene. 2002; 21: 3939-3948Crossref PubMed Scopus (101) Google Scholar, 14Yang F. Li X. Sharma M. Zarnegar M. Lim B. Sun Z. J. Biol. Chem. 2001; 276: 15345-15353Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The GCKs are distinct from PAKs in that they have N-terminal catalytic domains followed by C-terminal putative regulatory regions without conserved G protein binding sites. The 28 known human GCK-related kinases are classified in eight subdivisions and have diverse and much less well characterized functions (7Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). Some, like Ste20p and PAKs, regulate the JNK and p38 MAPK pathways. Those reported to activate JNK include the GCK-IV subfamily members, MINK, NIK, HGK, TNIK; GCK-I subfamily members, GCK, HPK1, GLK; and the GCK-V subfamily member, SLK (15Dan I. Watanabe N.M. Kobayashi T. Yamashita-Suzuki K. Fukagaya Y. Kajikawa E. Kimura W.K. Nakashima T.M. Matsumoto K. Ninomiya-Tsuji J. Kusumi A. FEBS Lett. 2000; 469: 19-23Crossref PubMed Scopus (55) Google Scholar, 16Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (216) Google Scholar, 17Yao Z. Zhou G. Wang X.S. Brown A. Diener K. Gan H. Tan T.H. J. Biol. Chem. 1999; 274: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 18Pombo C.M. Kehrl J.H. Sánchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar, 19Hu M.C. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (192) Google Scholar, 20Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N.J. Pawson T. Woodgett J.R. Iscove N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (196) Google Scholar, 21Fu C.A. Shen M. Huang B.C. Lasaga J. Payan D.G. Luo Y. J. Biol. Chem. 1999; 274: 30729-30737Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22Ling P. Yao Z. Meyer C.F. Wang X.S. Oehrl W. Feller S.M. Tan T.H. Mol. Cell Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar, 23Diener K. Wang X.S. Chen C. Meyer C.F. Keesler G. Zukowski M. Tan T.H. Yao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9687-9692Crossref PubMed Scopus (116) Google Scholar, 24Sabourin L.A. Seale P. Wagner J. Rudnicki M.A. Mol. Cell. Biol. 2000; 20: 684-696Crossref PubMed Scopus (92) Google Scholar). Those reported to activate p38 include the GCK-VI subfamily member SPAK and the GCK-VIII subfamily members TAO1 and TAO2 (25Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (115) Google Scholar, 26Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 27Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). However, some have no apparent connection to known MAPK pathways. These include the GCK-II subfamily member MST1, the GCK-III subfamily members MST3 and MST4, the GCK-V subfamily member LOK, and the GCK-III subfamily member SOK-1 (28Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 29Qian Z. Lin C. Espinosa R. LeBeau M. Rosner M.R. J. Biol. Chem. 2001; 276: 22439-22445Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 30Dan I. Ong S.E. Watanabe N.M. Blagoev B. Nielsen M.M. Kajikawa E. Kristiansen T.Z. Mann M. Pandey A. J. Biol. Chem. 2002; 277: 5929-5939Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 31Kuramochi S. Moriguchi T. Kuida K. Endo J. Semba K. Nishida E. Karasuyama H. J. Biol. Chem. 1997; 272: 22679-22684Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 32Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (133) Google Scholar). Similar to PAKs, some GCKs have been reported to regulate F-actin structure, cell spreading, and apoptosis (21Fu C.A. Shen M. Huang B.C. Lasaga J. Payan D.G. Luo Y. J. Biol. Chem. 1999; 274: 30729-30737Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 24Sabourin L.A. Seale P. Wagner J. Rudnicki M.A. Mol. Cell. Biol. 2000; 20: 684-696Crossref PubMed Scopus (92) Google Scholar, 33Tsutsumi T. Ushiro H. Kosaka T. Kayahara T. Nakano K. J. Biol. Chem. 2000; 275: 9157-9162Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 34Ong S.H. Hadari Y.R. Gotoh N. Guy G.R. Schlessinger J. Lax I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6074-6079Crossref PubMed Scopus (263) Google Scholar, 35Nishigaki K. Thompson D. Yugawa T. Rulli K. Hanson C. Cmarik J. Gutkind J.S. Teramoto H. Ruscetti S. J. Biol. Chem. 2003; 278: 13520-13530Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 36Ura S. Masuyama N. Graves J.D. Gotoh Y. Genes Cells. 2001; 6: 519-530Crossref PubMed Scopus (100) Google Scholar). Among novel functions that have been found, SPAK is reported to regulate the Na-K-Cl cotransporter (NKCC1), and a role in cell cycle control has been inferred for Stk10, which is a novel polo-like kinase (PLK) kinase (37Dowd B.F. Forbush B. J. Biol. Chem. 2003; 278: 27347-27353Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 38Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 39Walter S.A. Cutler Jr., R.E. Martinez R. Gishizky M. Hill R.J. J. Biol. Chem. 2003; 278: 18221-18228Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We previously examined the MAPK cascade components used by two agents to stimulate JNK in Drosophila S2 cells (40Chen W. White M.A. Cobb M.H. J. Biol. Chem. 2002; 277: 49105-49110Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). To extend these studies here, we have examined the potential involvement of putative MAP4Ks in regulating Drosophila JNK. We found that when the Ste20p relative Fray was knocked down using RNA interference, JNK activity stimulated by sorbitol decreased markedly, whereas ablation of other putative MAP4Ks decreased JNK activity little (CG4527) or not at all. These results suggested that Fray was the major MAP4K regulating the JNK pathway in response to sorbitol in S2 cells. We then wished to determine whether mammalian Fray homologs were MAP4Ks upstream of JNK. The kinases most closely related to Fray in the human genome are OSR1 (oxidant stress-responsive protein 1) and SPAK (Ste20/SPS1-related, proline alanine-rich kinase). SPAK has been reported to activate p38 but not JNK (25Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (115) Google Scholar). Human OSR1 had been isolated but not characterized. Here, we report the characterization of human OSR1 and an initial analysis of its biochemical functions. Cloning, Subcloning, Mutagenesis, and Plasmids—Total RNA prepared from HeLa cells was subjected to RT-PCR with a pair of primers spanning the complete human OSR1 cDNA synthesized based on the sequence in the NCBI data base. The 1.6-kb RT-PCR products were cloned into the GST-tagged bacterial expression vector pGEX-KG. This plasmid was used as the template for subsequent subcloning. Full-length OSR1 cDNA was also subcloned into p3XFLAG-CMV and pCMV5-Myc. Fragments encoding OSR1-(1–433), OSR1-(1–344), OSR1-(1–291), and OSR1-(345–527) were amplified by PCR and subcloned into pGEX-KG, pRSET (His6 tag), pCMV5-Myc, and pVJL11 as indicated. Kinase-dead mutants of OSR1 (OSR1KR) and fragments were generated by mutating lysine 46 in the ATP binding pocket to arginine. All constructs were transformed into the bacterial strain TG-1 and grown at 30 °C to reduce the frequency of mutations. All clones were sequenced to confirm correct amplification. The plasmids pCEP4-HA-ERK2, pSRα-HA-JNK1, pCEP4-HA-p38α, and pCEP4-HA-ERK5 were described previously (41Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Constructs encoding pCMV-Myc-V12Cdc42, pCMV-Myc-PAK1 (rat sequence), pCMV-Myc-PAK1 H83L/H86L, pCMV-Myc-PAK1 L107F, and GST-PAK1-(1–231), -(1–231) H83L/H86L, -(1–231) L107F, -(232–544) D406A (kinase-dead), -(1–132), -(75–132), -(147–231), and -(1–544) K298A (kinase-dead) were described previously (10Frost J.A. Khokhlatchev A. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28253-28260Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Plasmids encoding GST-PAK1-(1–92), -(86–231), -(1–100), and -(101–231) were constructed by inserting the appropriate PCR products into pGEX-KG. Mutations, including T84A, T84E, and T109A/T113A PAK1, were generated with the QuikChange site-directed mutagenesis kit (Stratagene). Proteins and Antibodies—All GST and His6 epitope tagged fusion proteins were expressed in the bacterial strain BLR(DE3)pLys (Novagen). Cells were grown at 30 °C to A600 = 0.5–0.6, and protein expression was induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside at 30 °C for 4–6 h before harvest. Proteins were purified on glutathioneagarose or Ni2+-nitrilotriacetic acid-agarose, respectively, as described by the manufacturers. Myelin basic protein (MBP) was purchased from Sigma. GST-c-Jun, GST-MEF2C, GST-MEKK1-(30–220), and the GST-MEK1 proline-rich insert (residues 265–301) were as described (42English J.M. Pearson G. Baer R. Cobb M.H. J. Biol. Chem. 1998; 273: 3854-3860Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 43Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 44Dang A. Frost J.A. Cobb M.H. J. Biol. Chem. 1998; 273: 19909-19913Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The HA antibody (12CA5) was from Berkeley Antibody, and the anti-Myc antibody (9E10) was from the National Cell Culture Center, and both were used at a dilution of 1:1000 for immunoblotting. The monoclonal anti-FLAG antibody was from Sigma and was used at 1:4000. The anti-Lamin A/C antibody was from Santa Cruz Biotechnology and was used at 1:1000. The polyclonal anti-OSR1 serum (U5438) was raised against His6-OSR1-(345–527) and was used at 1:8000. Cell Culture and Transfection—HEK 293 and HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mm l-glutamine, and 100 units/ml penicillin/streptomycin at 37 °C under 10% CO2. HEK 293 cells were transfected using calcium phosphate as described (45Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). HeLa cells were transfected using FuGENE 6 following the manufacturer's protocol (Roche Applied Science). Drosophila S2 cells were cultured, and RNA interference was as described previously (40Chen W. White M.A. Cobb M.H. J. Biol. Chem. 2002; 277: 49105-49110Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Fractionation and Immunofluorescence—Cell fractionation was as described (41Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Immunofluorescence was as described (46Robinson F.L. Whitehurst A.W. Raman M. Cobb M.H. J. Biol. Chem. 2002; 277: 14844-14852Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) with the following changes. HeLa cells were grown to confluence and starved in medium with 0.5% FBS overnight. Cells on coverslips were fixed in 2% paraformaldehyde for 10 min, permeabilized in cold methanol at –20 °C for 10 min, and then incubated with anti-OSR1 U5438 antibody (1:800). After incubation with anti-rabbit secondary antibody (Alexa, 1:3000), OSR1 localization was observed using a Zeiss Axioskop 2 plus fluorescent microscope. Preparation of Tissue and Cell Lysates and Immunoblotting—Cultured cells or tissues from a 13-month-old mouse (provided by David Russell, Department of Molecular Genetics) were homogenized and lysed in Triton X-100 lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 0.5 mm sodium orthovanadate, 20 μg/ml aprotinin, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). Insoluble material was sedimented in a microcentrifuge for 15 min at 4 °C. Protein concentration was measured by Bradford assay using bovine serum albumin as standard. Thirty μg of soluble protein from each sample was resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk (40Chen W. White M.A. Cobb M.H. J. Biol. Chem. 2002; 277: 49105-49110Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) for 1 h at room temperature and then incubated with the appropriate antibody. Immunoprecipitation, in Vitro Kinase Assays, and Phosphoamino Acid Analysis—Lysate protein (300 μg) was incubated with the indicated antibody for 1 h at 4 °C and then with 30 μl of a 50% slurry of protein A-Sepharose beads for 1 h. After three washes with detergent buffer (0.25 m Tris, pH 7.4, 1 m NaCl, 0.1% Triton X-100, 0.1% sodium deoxycholate) and one with 10 mm HEPES (pH 7.6), beads were incubated with indicated substrates in 50 μl of 1× kinase buffer (20 mm HEPES, pH 7.6, 5 μm ATP (5 μCi of [γ-32P]ATP), 10 mm MgCl2, 10 mm β-glycerol phosphate) at 30 °C for 30 min for kinase assays. Purified proteins were incubated with indicated substrates in 30 μlof1× kinase buffer at 30 °C for 30 min. One-dimensional phosphoamino acid analysis was performed as described (47Robbins D.J. Cobb M.H. Mol. Biol. Cell. 1992; 3: 299-308Crossref PubMed Scopus (55) Google Scholar). Yeast Two-hybrid Analysis—A neonatal mouse brain cDNA library (gift from Mark Henkemeyer, Center for Developmental Biology) in plasmid pGADGH was screened as described. 2B.-H. Lee, X. Min, B-e. Xu, H. Shu, S. Chen, and M. H. Cobb, submitted. MAP4Ks and Activation of JNK in S2 Cells—In an earlier study, we examined the components of the protein kinase cascades that control JNK activity in response to lipopolysaccharide and sorbitol in Drosophila S2 cells (40Chen W. White M.A. Cobb M.H. J. Biol. Chem. 2002; 277: 49105-49110Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Both agents used both of the MAP2Ks MEK4 and MEK7 to activate JNK. Although lipopolysaccharide required a single MAP3K (DTAK), sorbitol employed four MAP3Ks to stimulate JNK activity. We have completed the examination of the likely kinase components of the MAPK module by testing the potential involvement of putative MAP4Ks in regulating Drosophila JNK. Based on sequence alignments and a consideration of the published classification of the fly protein kinases (48Morrison D.K. Murakami M.S. Cleghon V. J. Cell Biol. 2000; 150: F57-F62Crossref PubMed Scopus (137) Google Scholar), six putative MAP4Ks, CG11228, DPAK, DPAK3, DMSN, CG4527, and Fray, were found to be expressed in S2 cells as determined by PCR analysis (data not shown). The expression of each of these was reduced using RNA interference, and the effect of the loss of each singly on activation of JNK by sorbitol was then examined (Fig. 1, A and B; data not shown). When expression of Fray, a Drosophila GCK-VI kinase family member, was knocked down, JNK activity stimulated by sorbitol decreased significantly. Reduction in expression of one of the other putative MAP4Ks, CG4527, decreased sorbitol-stimulated JNK activation to a small but reproducible extent. Reducing expression of CG4527 caused a further reduction in the residual JNK activation remaining upon suppression of Fray (Fig. 1C). These results suggested that Fray was the major MAP4K regulating the JNK pathway in response to osmotic stress in S2 cells. Interestingly, Fray is required for normal axonal ensheathment during fly development (49Leiserson W.M. Harkins E.W. Keshishian H. Neuron. 2000; 28: 793-806Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Structure and Expression of OSR1—We wished to learn whether mammalian homologs of Fray, SPAK and OSR1, also regulate the JNK pathway. SPAK has been reported to activate p38 but not JNK (25Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (115) Google Scholar). Thus, we focused on analyzing OSR1, which has not been characterized previously as a protein. Human OSR1 contains 527 amino acids with a predicted molecular mass of 58 kDa. We isolated a cDNA encoding OSR1 by RT-PCR using HeLa RNA. It has a conserved Ste20p-like protein-serine/threonine kinase domain at its N terminus with a C-terminal region of undefined function. The OSR1 kinase domain has the highest identity to the kinase domain of SPAK (89%), Drosophila Fray (74%), and the Caenorhabditis elegans Y59A8B.23 gene product (71%). These four enzymes comprise the GCK-VI subfamily of Ste20p protein kinases. Two small regions of similarity were found in the C-terminal regions of GCK-VI subfamily members, which were named PF1 and PF2 domains. They may represent regulatory or targeting elements (25Johnston A.M. Naselli G. Gonez L.J. Martin R.M. Harrison L.C. DeAizpurua H.J. Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (115) Google Scholar). A putative caspase 3 cleavage site (DEFD) is present at the end of the PF1 domain of OSR1 (Fig. 2). To detect the expression of OSR1, a rabbit polyclonal antibody was generated against the C-terminal, poorly conserved region. The specificity of the anti-OSR1 antibody was confirmed by immunoblotting and immunoprecipitating overexpressed Myc-OSR1 in HEK-293 cells (data not shown). With this antiserum, a 58-kDa protein was recognized in all mouse tissues examined except thymus, including heart, spleen, liver, kidney, lung, testis, large intestine, small intestine, and stomach. OSR1 was also detected in mammalian cell lines including HEK 293, HeLa, PC3, BT20, HI299, SW480, 2721, and Cos-1, which were derived from a variety of tissues including kidney, cervix, ovary, prostate, breast, lung, and colon (Fig. 3A). Interestingly, although the same amount of protein was analyzed from each lysate, little or no OSR1 was detected in the pancreatic beta cell line INS-1 or the mouse fibroblast lines C2C12 or 3T3, suggesting some tissue specificity to its expression. The wide expression of OSR1 is consistent with the broad transcription of OSR1 mRNA as deduced from Northern blotting (50Tamari M. Daigo Y. Nakamura Y. J. Hum. Genet. 1999; 44: 116-120Crossref PubMed Scopus (52) Google Scholar). To detect the subcellular localization of endogenous OSR1, proteins from soluble, particulate, and nuclear fractions derived from HeLa cells were immunoblotted with anti-OSR1 and anti-lamin A/C antibodies. OSR1 was detected in all three fractions, whereas lamin A/C was detected only in the nuclear fraction (Fig. 3B). Endogenous OSR1 detected by immunofluorescence was distributed throughout HeLa cells, consistent with the fractionation data (Fig. 3C). Protein Kinase Activity of OSR1—To verify that OSR1 is a serine/threonine protein kinase, recombinant wild type GST-OSR1 and the kinase inactive mutant GST-OSR1KR were expressed in bacteria and assayed with MBP as substrate. Wild type GST-OSR1 phosphorylated both MBP and itself, but GST-OSR1KR showed no detectable activity (Fig. 4A). Phosphoamino acid analysis of autophosphorylated GST-OSR1 revealed primarily phosphothreonine (Fig. 4B). Equal amounts of Myc-tagged OSR1 and OSR1KR expressed in HEK 293 cells were immunoprecipitated with the anti-Myc antibody and assayed with MBP as substrate (data not shown). A phosphorylated band at 58 kDa, representing autophosphorylated Myc-OSR1, and phosphorylated MBP were detected in assays with the wild type OSR1 immunoprecipitate. However, the same bands were also detected in assays with OSR1KR. Similar experiments performed with different epitope tags (3XFLAG) and different cell types (Cos-1 and HeLa) yielded similar results (data not shown). Because OSR1KR had no activity when expressed in bacteria and because MBP is phosphorylated by many abundant protein kinases, we conclude that the phosphorylation comes from contaminating proteins co-purifying with or non-specifically trapped in OSR1 in immune complexes; some of these may be kinases that normally phosphorylate OSR1. As a consequence, MBP was not a suitable substrate to measure OSR1 activity in cell lysates or immunoprecipitates, although it was useful to characterize the activity of the protein expressed in bacteria. The PF1 Domain Is Necessary for OSR1 Kinase Activity—To examine the contribution of the C-terminal region to the kinase activity of OSR1, truncated forms of OSR1 were expressed as GST fusions in bacteria, and the purified proteins were assayed with MBP as substrate. Full-length OSR1, OSR1-(1–433) and OSR1-(1–344) have nearly identical kinase activity toward MBP or themselves. In contrast, OSR1-(1–291), a truncated protein with intact, conserved kinase domain but without the PF1 domain, has no detectable activity toward MBP or itself (Fig. 5). Thus, the PF1 domain is essential for OSR1 kinase activity. Many Ste20p-related kinases contain autoinhibitory domains; removal of the regulatory domains results in a significant increase in kinase activity due to loss of autoinhibition. This has been shown for PAKs, MST1, MST2, TAOs, and SOK1, for example (10Frost J.A. Khokhlatchev A. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28253-28260Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 26Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 32Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (133) Google Scholar, 51Zhao Z.-S. Manser E. Chen Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar, 52Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In the case of OSR1 and other GCK-VI kinases, the PF1 domain may comprise an essential part of the kinase catalytic domain. Because there is no significant difference in the kinase activity of full-length OSR1 and OSR1-(1–433), the PF2 domain is apparently not involved in regulating catalytic activity. Cellular Stimuli That Activate OSR1—To identify possible regulators of OSR1, a number of stimuli were tested on HeLa cells. Autophosphorylation was used to assess activity o" @default.
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• W1995517360 title "Characterization of OSR1, a Member of the Mammalian Ste20p/Germinal Center Kinase Subfamily" @default.
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