Portal de Periódicos da CAPES

Inositol 1,3,4-Trisphosphate 5/6-Kinase Is a Protein Kinase That Phosphorylates the Transcription Factors c-Jun and ATF-2

2001; Elsevier BV; Volume: 276; Issue: 44 Linguagem: Inglês

10.1074/jbc.m106605200

1083-351X

Monita P. Wilson, Yang Sun, Li Cao, Philip W. Majerus,

Insect Resistance and Genetics

Phosphorylation of inositol 1,3,4-trisphosphate by inositol 1,3,4-trisphosphate 5/6-kinase is the first committed step in the formation of higher phosphorylated forms of inositol. We have shown that the eight proteins called the COP9 signalosome complex copurify with calf brain 5/6-kinase. Because the complex has been shown to phosphorylate c-Jun in vitro, we tested both the complex and 5/6-kinase and found that both are able to phosphorylate c-Jun and ATF-2 on serine/threonine residues. These findings establish a link between two major signal transduction systems: the inositol phosphates and the stress response system. Phosphorylation of inositol 1,3,4-trisphosphate by inositol 1,3,4-trisphosphate 5/6-kinase is the first committed step in the formation of higher phosphorylated forms of inositol. We have shown that the eight proteins called the COP9 signalosome complex copurify with calf brain 5/6-kinase. Because the complex has been shown to phosphorylate c-Jun in vitro, we tested both the complex and 5/6-kinase and found that both are able to phosphorylate c-Jun and ATF-2 on serine/threonine residues. These findings establish a link between two major signal transduction systems: the inositol phosphates and the stress response system. inositol 3,4)P3, inositol 1,3,4-trisphosphate 3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate Ins(1,3,4)P3 5/6-kinase 3,4,6)P4, inositol 1,3,4,6- tetrakisphosphate 1,3,4,5,6-pentakisphosphate inositol hexakisphosphate 4,5)P3, inositol 1,4,5-trisphosphate 4,5,6)P4, inositol 3,4,5,6-tetrakisphosphate glutathioneS-transferase Signaling through the inositol phosphate pathway involves a series of kinases and phosphatases that phosphorylate and dephosphorylate the large number of soluble inositol polyphosphates known to exist in eukaryotic cells (1Shears S.B. Pharmacol. Ther. 1991; 49: 79-104Crossref PubMed Scopus (33) Google Scholar). A branch point in this pathway occurs with the production of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3)1, resulting from the hydrolysis of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) by one of the numerous inositol polyphosphate 5-phosphatase isozymes (2Majerus P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Ins(1,3,4)P3 can be dephosphorylated by specific phosphatases, resulting ultimately in the generation of myo-inositol, or it can be phosphorylated further, resulting in the formation of higher phosphorylated forms of inositol. Inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) phosphorylates Ins(1,3,4)P3 to form both inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) and Ins(1,3,4,5)P4 (3Balla T. Guillemette G. Baukal A.J. Catt K.J. J. Biol. Chem. 1987; 262: 9952-9955Abstract Full Text PDF PubMed Google Scholar, 4Shears S.B. Parry J.B. Tang E.K.Y. Irvine R.F. Michell R.H. Kirk C.J. Biochem. J. 1987; 246: 139-147Crossref PubMed Scopus (63) Google Scholar). Ins(1,3,4,6)P4 is the first intermediate in the pathway leading to the formation of the higher phosphorylated inositols including other inositol tetrakisphosphate isomers, inositol 1,3,4,5,6-pentakisphosphate (InsP5), inositol hexakisphosphate (InsP6), and the pyrophosphate forms of inositol (5Safrany S.T. Caffrey J.J. Yang X. Shears S.B. Biol. Chem. 1999; 380: 945-951Crossref PubMed Scopus (33) Google Scholar). Although InsP5 and InsP6 are the most abundant inositol polyphosphates in cells, their functions in vertebrate cells have begun to be elucidated only recently. InsP6 has been reported to inhibit Golgi coatomer K+ channels (6Fleischer B. Xie J. Mayrleitner M. Shears S.B. Palmer D.J. Fleischer S. J. Biol. Chem. 1994; 269: 17826-17832Abstract Full Text PDF PubMed Google Scholar) and to inhibit clathrin cage assembly by binding to the clathrin assembly proteins AP-2 (7Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4442-4447Abstract Full Text PDF PubMed Google Scholar) and AP-3 (8Norris F.A. Ungewickell E. Majerus P.W. J. Biol. Chem. 1995; 270: 214-217Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 9Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Both InsP5 and InsP6 have also been shown to inhibit several serine-threonine protein phosphatases, resulting in stimulation of whole-cell Ca2+ currents in pancreatic cells (10Larsson O. Barker C.J. Sjoholm A. Carlqvist H. Michell R.H. Bertorello A. Nilsson T. Honkanen R.E. Mayr G.W. Zwiller J. Berggren P.-O. Science. 1997; 278: 471-474Crossref PubMed Scopus (122) Google Scholar). Depletion of InsP5 and InsP6 in 293 cells by overexpression of the Salmonella inositol phosphatase SopB results in the inhibition of nuclear mRNA export (11Feng Y. Wente S.R. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 875-879Crossref PubMed Scopus (80) Google Scholar). 5/6-Kinase is conserved from plants to humans and is found even inEntamoeba histolytica (12Field J. Wilson M.P. Mai Z. Majerus P.W. Samuelson J. Mol. Biochem. Parasitol. 2000; 108: 119-123Crossref PubMed Scopus (25) Google Scholar). The human and calf brain enzymes produce more Ins(1,3,4,6)P4 than Ins(1,3,4,5)P4, whereas the ratio of products produced by the plant enzyme is reversed (13Wilson M.P. Majerus P.W. Biochem. Biophys. Res. Commun. 1997; 232: 678-681Crossref PubMed Scopus (55) Google Scholar). In E. histolytica, the enzyme utilizes both Ins(1,3,4)P3 and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as substrates. The two tetrakisphosphate products are produced in equal amounts from Ins(1,3,4)P3, but when Ins(1,4,5)P3 is the substrate only Ins(1,3,4,5)P4 is a product. The activity of the amoebae enzyme is very low compared with that of the human and plant enzymes, which may be because of the fact that the inositol polyphosphates found in E. histolytica are notmyo-derivatives but are neo-derivatives (14Martin J.-B. Laussmann T. Bakker-Grunwald T. Vogel G., K. J. Biol. Chem. 2000; 275: 10134-10140Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In addition to producing two distinct isomers of InsP4 from a single substrate, it has been shown recently that 5/6-kinase can utilize inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) as a substrate for 1-kinase activity to produce InsP5(15Yang X. Shears S.B. Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (60) Google Scholar). Regulation of the activity of 5/6-kinase by cells is not well understood. In adrenal glomerulosa cells, stimulation with angiotensin causes a rise in the level of Ins(1,3,4,6)P4 (16Balla T. Guillemette G. Baukal A.J. Catt K.J. Biochem. Biophys. Res. Commun. 1987; 148: 199-205Crossref PubMed Scopus (22) Google Scholar). A rapid rise in this tetrakisphosphate isomer is also seen after platelet stimulation by thrombin (17King W.G. Downes C.P. Prestwich G.D. Rittenhouse S.E. Biochem. J. 1990; 270: 125-131Crossref PubMed Scopus (13) Google Scholar). In both cases, the levels of the 5/6-kinase substrate Ins(1,3,4)P3 are elevated prior to a rise in Ins(1,3,4,6)P4, and therefore the activity of 5/6-kinase under these conditions may not be changing. We therefore sought to identify several proteins that copurified with calf brain 5/6-kinase, assuming that copurification may reflect an interaction between these proteins within the cell. We identify these proteins as subunits of a large protein complex called the COP9 signalosome. This complex was described originally inArabidopsis seedlings in which a COP (constitutive photomorphogenesis) mutant was identified. The mutant, termedcop9, exhibited light-grown morphology when seedlings were grown in the dark. The protein responsible for the cop9mutation, COP9, was cloned and shown to be a component of a large protein complex, the COP9 signalosome complex (18Wei N. Chamovitz D.A. Deng X.-W. Cell. 1994; 78: 117-124Abstract Full Text PDF PubMed Scopus (301) Google Scholar). The complex consists of eight subunits designated Sgn1 through Sgn8 and is conserved in mammals (19Wei N. Deng X.-W. Trends Genet. 1999; 15: 98-103Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The only known function of the mammalian complex is the in vitro phosphorylation of IκBα, p105, and c-Jun (20Seeger M. Kraft R. Ferrell K. Bech-Otschir D. Dumdey R. Schade R. Gordon C. Naumann M. Dubiel W. FASEB J. 1998; 12: 469-478Crossref PubMed Scopus (315) Google Scholar). Overexpression of the COP9 signalosome subunit Sgn2 in HeLa cells results in an increase in complex assembly and an elevation in the cellular levels of c-Jun, which results in increased AP1 transactivation (21Naumann M. Bech-Otschir D. Huang X. Ferrell K. Dubiel W. J. Biol. Chem. 1999; 274: 35297-35300Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The COP9 signalosome has been shown recently to play a role in the ubiquitin pathway in plants and in fission yeast. The Arabidopsis COP9 signalosome associates with the E3 ubiquitin ligase SCFTIR1 and is required for auxin-responsive protein degradation (22Schwechheimer C. Serino G. Callis J. Crosby W.M. Lyapina S. Deshaies R.J. Gray W.M. Estelle M. Deng X.-W. Science. 2001; 292: 1379-1382Crossref PubMed Scopus (402) Google Scholar). In Schizosaccharomyces pombe, the complex associates with several cullins, ubiquitin ligases that are post-translationally modified by the ubiquitin-like protein NEDD8. COP9 mutants lacking one of the subunits of the complex accumulate NEDD8-modified proteins, indicating that the COP9 complex is required for deneddylation (23Lyapina S. Cope G. Shevchenko A. Serino G. Tsuge T. Zhou C. Wolf D.A. Wei N. Shevchenko A. Deshaies R.J. Science. 2001; 292: 1382-1385Crossref PubMed Scopus (576) Google Scholar). We show here that the calf brain COP9 signalosome complex phosphorylates c-Jun and ATF-2, another transcription factor known to be phosphorylated by stress-activated protein kinases. The calf brain complex contains a small amount of 5/6-kinase, which also phosphorylates c-Jun and ATF-2 in the absence of complex. 5/6-Kinase may represent the as yet unidentified protein kinase activity of the COP9 signalosome complex, which has been referred to as an associated kinase activity (21Naumann M. Bech-Otschir D. Huang X. Ferrell K. Dubiel W. J. Biol. Chem. 1999; 274: 35297-35300Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Phosphorylation of ATF-2 by 5/6-kinase is concentration dependent, enhanced by Mn2+, and unaffected by Ins(3,4,5,6)P4, a potent inhibitor of Ins(1,3,4)P3 phosphorylation and an alternative substrate for 5/6-kinase. Phosphorylation by 5/6-kinase occurs on serine/threonine residues. Fractionation of cytosolic extract from HEK 293 cells stably expressing human 5/6-kinase demonstrates a correlation between the inositol and protein kinase activities of 5/6-kinase. Depletion of 5/6-kinase using a polyclonal antiserum also partially removes the protein kinase activity. Purified, flag-tagged human 5/6-kinase expressed in Sf21 cells phosphorylates ATF-2, indicating that these two activities reside within 5/6-kinase or that the two kinases associate very tightly. In either case, this work establishes a link between the inositol polyphosphate signaling pathway and two of the transcription factors of the mitogen-activated protein kinase pathway. Goat polyclonal antibody against human JAB1, full-length his-tagged ATF-2, rabbit polyclonal antibody against ATF-2, and mouse monoclonal antibody against c-Jun were obtained from Santa Cruz Biotechnology. Recombinant human full-length c-Jun was from Promega. Rabbit polyclonal human 5/6-kinase antibody used for Western blot analysis was generated against the peptide VASLATKASSQ (representing amino acids 404–414 of 5/6-kinase). The rabbit polyclonal antibody used for immunodepletions was prepared against amino acids 124–311 of human 5/6-kinase. Recombinant ATF-2 was expressed in Escherichia coli as a GST fusion protein containing the first 109 amino acids of ATF-2. In vitro protein kinase assays were done as described by Seeger et al. (20Seeger M. Kraft R. Ferrell K. Bech-Otschir D. Dumdey R. Schade R. Gordon C. Naumann M. Dubiel W. FASEB J. 1998; 12: 469-478Crossref PubMed Scopus (315) Google Scholar). Unless otherwise indicated, reactions were done in a volume of 10 µl. Reactions were terminated by the addition of SDS-polyacrylamide gel electrophoresis sample buffer, run on 12% SDS gels, transferred to polyvinylidene difluoride membranes (Millipore), and exposed to x-ray film (Kodak). Ins(1,3,4)P3 5/6-kinase assays were performed as described previously (24Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). All assays were done under first-order rate conditions. [3H]Ins(1,3,4)P3 was prepared by treatment of [3H]Ins(1,3,4,5)P4 (PerkinElmer Life Sciences) with recombinant OCRL (25Zhang X. Jefferson A.B. Authavekiat V. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4853-4856Crossref PubMed Scopus (230) Google Scholar). Purity of the product was determined by high performance liquid chromatography on a 250 × 4.6-mm Adsorbosphere SAX column (Alltech/Applied Science). Recombinant full-length his-tagged ATF-2 (4 µg) and recombinant full-length c-Jun (4 µg) were incubated with 390 ng of calf brain 5/6-kinase in a reaction volume of 75 µl containing 45 µCi of [γ-32P]ATP for 30 min at 37 °C. Samples were run on SDS-polyacrylamide gel electrophoresis, and radiolabeled ATF-2 and c-Jun were excised, electroeluted, and precipitated with 20% TCA. Bovine serum albumin (200 µg/ml) was added as carrier protein. Pellets were washed twice with cold ethanol, three times with cold 50 mmphosphoric acid, a final time with cold ethanol and dried using a speedvac. Samples were hydrolyzed in 5.7 m HCl (Pierce) for 1 h at 100 °C and subjected to phospho amino acid analysis as described by Cooper et al. (26Cooper J.A. Sefton B.M. Hunter T. Methods Enzymol. 1983; 99: 387-431Crossref PubMed Scopus (706) Google Scholar). Stable cell lines overexpressing full-length human 5/6-kinase were prepared using the tetracycline-inducible vector pcDNA 4/TO©(Invitrogen). Cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 300 µg of zeocin/ml, 5 µg of blasticidin/ml, 2 mm glutamine, 10 µg of penicillin/ml, and 100 µg of streptomycin/ml. Expression was induced with 0.1 µg of tetracycline/ml. For partial purification of human 5/6-kinase, 100 150-mm dishes of cells were plated at 60% confluence in the presence of tetracycline. Cells were harvested after 3 days in culture using homogenization buffer containing 20 mm Hepes, pH 7.2, 1 mmEDTA, 1 mm ATP, 10 mm benzamidine, 250 mm sucrose, 200 µg of soybean trypsin inhibitor/ml, 40 µm iodoacetamide, 2 µm pepstatin A, 40 µm bestatin, 1 mm phenylmethylsulfonyl fluoride, 40 µm leupeptin, 1 mmdithiothreitol, 1 mm EGTA, 50 mm NaF, 0.5 mm sodium vanadate, and 5 µg each of calpain inhibitors I and II/ml. The cells were sonicated and spun to remove particulate matter. A total of 360 mg of protein was obtained, which contained 16 µg of 5/6-kinase (as determined by enzymatic activity). Filtered crude extract was loaded onto a 60-ml heparin-agarose column (Sigma) in 20 mm bis-Tris, pH 7.2, 1 mm ATP, 1 mm dithiothreitol, and 1 mm EGTA (buffer A), and the sample was eluted with 0.2 m NaCl in buffer A. Fractions containing 5/6-kinase activity were pooled, and protein was precipitated by the addition of ammonium sulfate to 60% saturation, dialyzed against buffer A containing 3 mmMgCl2, and loaded onto a 1-ml Mono Q column (Amersham Pharmacia Biotech). A 20-ml linear gradient of 0–0.3 mNaCl in buffer A was used for elution. Fractions (1 ml) were aliquoted and stored at −80 °C. Partially purified human 5/6-kinase expressed in 293 cells was used as an enzyme source. Mono Q fraction 14 (10 µl, 2.2 µg of total protein, and 5 ng of 5/6 kinase) was incubated with 10 µg of bovine serum albumin and 6 µl of preimmune or immune rabbit serum in the presence or absence of protein A-Sepharose (43 µl of a 50% slurry) overnight at 4 °C. Samples were spun, and supernatants were assayed for kinase activity. Full-length human 5/6-kinase was expressed in Sf21 cells using the BacPAK™ baculovirus expression system (CLONTECH). Pellets from infected cells were lysed in 10 ml of buffer containing 20 mm Hepes, pH 7.6, 140 mm NaCl, 10% glycerol, 0.1% Nonidet P-40, 0.5 mm dithiothreitol, and Complete™ protease inhibitor mixture tablets (Roche Molecular Biochemicals). Lysate was allowed to bind to anti-flag agarose beads for 1 h, washed with Tris-buffered saline containing 0.5 mm dithiothreitol and 0.1% Nonidet P-40, and then eluted with 2 ml of 0.1 µg of flag peptide/ml Tris-buffered saline containing Complete™ protease inhibitor mixture tablets. The eluate was concentrated and dialyzed against Tris-buffered saline prior to use in assays. During the purification of calf brain Ins(1,3,4)P3 5/6-kinase, several proteins co-chromatographed with the enzyme preparation through a number of steps including affinity elution with InsP6 (24Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The final contaminants, removed using a Mono Q column, were comprised predominantly of eight proteins (Fig.1A) ranging in size from 55 to 20 kDa. To identify these proteins, 10 µg of total protein (Mono Q fraction 13 from Ref. 24Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) was blotted onto a polyvinylidene difluoride membrane for amino-terminal protein sequencing. The sequence obtained for the largest protein (XPLPVQVFNLQGAVEPM) matched at 16/16 residues to human GPS1, identified as a suppressor of a lethalSaccharomyces cerevisiae pheromone pathway mutant (27Spain B.H. Bowdish K.S. Pacal A.R. Staub S.F. Koo D. Chang C.-Y.R. Xie W. Colicelli J. Mol. Cell. Biol. 1996; 16: 6698-6706Crossref PubMed Scopus (102) Google Scholar). GPS1 is a subunit of a large protein complex found in human erythrocytes (20Seeger M. Kraft R. Ferrell K. Bech-Otschir D. Dumdey R. Schade R. Gordon C. Naumann M. Dubiel W. FASEB J. 1998; 12: 469-478Crossref PubMed Scopus (315) Google Scholar) and porcine spleen (28Wei N. Deng X.-W. Photochem. Photobiol. 1998; 68: 237-241Crossref PubMed Scopus (71) Google Scholar, 29Wei N. Tsuge T. Serino G. Dohmae N. Takio K. Matsui M. Deng X.-W. Curr. Biol. 1998; 8: 919-922Abstract Full Text Full Text PDF PubMed Google Scholar). The complex consists of eight subunits, one of which is a protein called JAB1 (Jun activation domain-binding protein 1), which was found in a two-hybrid screen designed to identify proteins that interact with c-Jun (30Claret F.-X Hibi M. Dhut S. Toda T. Karin M. Nature. 1996; 383: 453-457Crossref PubMed Scopus (411) Google Scholar). To confirm that the COP9 signalosome complex co-purified with Ins(1,3,4)P3 5/6-kinase, a Western blot was done using 200 ng of Mono Q fraction 13 blotted with an antibody against human JAB1 (Fig. 1B). The human COP9 signalosome complex has been shown to phosphorylate IκBα, p105, and c-Jun in vitro (20Seeger M. Kraft R. Ferrell K. Bech-Otschir D. Dumdey R. Schade R. Gordon C. Naumann M. Dubiel W. FASEB J. 1998; 12: 469-478Crossref PubMed Scopus (315) Google Scholar). To determine whether the purified calf brain COP9 signalosome complex also functions as a protein kinase, an in vitro kinase reaction was done using full-length c-Jun (40 kDa) as the substrate (Fig.2A). Because the purified calf brain complex contains a small amount of 5/6-kinase, an in vitro protein kinase assay was done using 800 ng of complex mixed with 78 ng of 5/6-kinase. Phosphorylation of c-Jun by the complex was enhanced by the addition of 5/6-kinase. We next assayed 5/6-kinase alone for its ability to phosphorylate c-Jun and found that the purified inositol kinase could function also as a protein kinase. In lanes containing complex, several additional phosphorylated bands are visible. These bands are seen also in reactions containing complex but no c-Jun (data not shown). Although the preparation of purified calf brain 5/6-kinase used in the protein kinase assays seems not to be contaminated with complex on a silver-stained SDS gel (Fig.2B), a Western blot was done with Mono Q fraction 10 (5/6-kinase) and fraction 13 (COP9 signalosome complex) to determine the degree of cross-contamination (Fig. 2C). The COP9 signalosome complex used in the protein kinase reactions contains both JAB1 and 5/6-kinase. By contrast, the 5/6-kinase preparation used contains no JAB1. Therefore, phosphorylation of c-Jun by 5/6-kinase is not caused by contamination by the COP9 signalosome complex. There is a high molecular weight contaminant in the 5/6-kinase preparation visible by silver staining (Fig. 2B). Protein sequencing of this band identified it as clathrin assembly protein 3 (AP-3/A-P180), which also co-purified with inositol polyphosphate 4-phosphatase. Purification of 4-phosphatase also used affinity elution with InsP6, and it was further shown that InsP6inhibits clathrin assembly by AP-3 (8Norris F.A. Ungewickell E. Majerus P.W. J. Biol. Chem. 1995; 270: 214-217Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Phosphorylation of c-Jun occurs in response to activation of the stress-activated protein kinases (31Leppä S. Bohmann D. Oncogene. 1999; 18: 6158-6162Crossref PubMed Scopus (453) Google Scholar), which also results in the phosphorylation of ATF-2 (32Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (476) Google Scholar, 33Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2061) Google Scholar). We therefore tested GST-ATF-2 as a substrate in in vitro kinase assays with 5/6-kinase and COP9 signalosome complex (Fig. 3A). ATF-2 was phosphorylated by both 5/6-kinase and complex. To determine the minimal amount of 5/6-kinase sufficient to phosphorylate ATF-2, serial dilutions of 5/6-kinase were used in kinase reactions with GST-ATF-2. Phosphorylation could be visualized with as little as 0.4 ng of 5/6-kinase (Fig. 3B). Phosphorylation of ATF-2 increases in a linear fashion up to 7.8 ng of 5/6-kinase (shown in Fig.3C). The residues phosphorylated on ATF-2 and c-Jun by 5/6-kinase as determined by phospho amino acid analysis are shown in Fig. 3D. Phosphorylation of ATF-2 occurs primarily on serine, with a small amount visible on threonine (lane 1). Phosphorylation of c-Jun by 5/6 kinase also occurs predominantly on serine residues (lane 2). No tyrosine phosphorylation was observed. Under identical in vitro kinase reaction conditions used for phospho amino acid analysis, a greater amount of radioactivity was incorporated in ATF-2 as compared with c-Jun. Phosphorylation of Ins(1,3,4)P3 by 5/6-kinase has been shown to be inhibited by Ins(3,4,5,6)P4 in rat parotid acinar cells (34Hughes P.J. Hughes A.R. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1989; 264: 19871-19878Abstract Full Text PDF PubMed Google Scholar), rat liver (35Shears S.B. Hughes P.J. Symp. Soc. Exp. Biol. 1990; 44: 181-191PubMed Google Scholar), and porcine brain (36Hughes P.J. Kirk C.J. Michell R.H. Biochim. Biophys. Acta. 1994; 1223: 57-70Crossref PubMed Scopus (7) Google Scholar). Consistent with these reports, calf brain 5/6-kinase is inhibited by Ins(3,4,5,6)P4 with aKi of 30 nm (data not shown). We therefore tested the effects of this InsP4 isomer on the phosphorylation of ATF-2 by 5/6-kinase. In contrast to the inositol kinase activity, the addition of 0.5 µmIns(3,4,5,6)P4 to the protein kinase assay has no effect on phosphorylation of ATF-2 (Fig.4A, lanes 2 and3). No phosphorylation was observed when either 5/6-kinase or ATF-2 were omitted from the reaction (Fig. 4A,lanes 1 and 4, respectively). Phosphorylation by many protein kinases is enhanced by or even dependent on the presence of MnCl2. We therefore tested whether the addition of MnCl2 to the two assays would have any effect. Inositol kinase assays were done in the presence of 6 mm MgCl2 alone and with 6 mmMgCl2 plus 5 mm MnCl2 (Fig.4B). The addition of MnCl2 to the assay results in 37% inhibition of Ins(1,3,4)P3 phosphorylation by 5/6-kinase. By contrast, the addition of 5 mmMnCl2 to the protein kinase assay resulted in enhanced phosphorylation of full-length ATF-2, indicated by the arrowin Fig. 4C. To determine whether human 5/6-kinase can function also as a protein kinase, HEK 293 cells stably transfected with human 5/6-kinase were used as an enzyme source. Soluble extract from 293 cells induced for 3 days with tetracycline was fractionated on a heparin-agarose column followed by a Mono Q column. Fractions from the Mono Q column (1–20) were assayed for phosphorylation of Ins(1,3,4)P3 and GST-AFT-2 (Fig.5A). Fractions 1–8 contained little protein and no 5/6-kinase (data not shown). Shown in thetop panel is a Western blot of fractions 9–20 using an antibody against a carboxyl-terminal peptide of 5/6-kinase. Themiddle panel represents in vitro kinase assays of the same fractions using GST-ATF-2 as a substrate. On thebottom panel, Ins(1,3,4)P3 phosphorylation is plotted for these fractions. There is a strong correlation between inositol kinase activity and phosphorylation of ATF-2. Some phosphorylation of ATF-2 can be visualized in fractions 11 and 12, in which no inositol kinase activity is detected. Because this enzyme preparation is far from pure, it is likely that these fractions contain an additional kinase capable of phosphorylating ATF-2. Immunodepletion of 5/6-kinase was done using Mono Q fraction 14 as an enzyme source and a polyclonal rabbit antiserum generated against a 188-amino acid peptide of human 5/6-kinase as antibody. In vitro kinase assays were done using antibody-treated 5/6-kinase and GST-ATF-2 as a substrate (Fig. 5B, upper). Phosphorylation of ATF-2 by 5/6-kinase was reduced in the absence of protein A-Sepharose (lane 2) and in the presence of protein A-Sepharose (lane 4). Similarly, the addition of immune serum reduced inositol kinase activity by 65% in the absence of protein A-Sepharose and by 93% in the presence of protein A-Sepharose (Fig. 5B, lower). Therefore, partial removal of 5/6-kinase as measured by the phosphorylation of Ins(1,3,4)P3 also reduced the phosphorylation of ATF-2. The addition of protein A-Sepharose to the protein kinase assay consistently reduced the activity slightly. To further establish the link between the two kinase activities of 5/6-kinase, full-length human 5/6-kinase was expressed in Sf21 cells as a flag-tagged fusion protein and purified using an immobilized anti-flag antibody. Silver-stained SDS-polyacrylamide gel electrophoresis of the flag peptide-eluted 5/6-kinase preparation shows a single band (Fig. 6A,lane 1). Western blot analysis using either a flag antibody (Fig. 6A, lane 2) or an antibody against 5/6-kinase (Fig. 6A, lane 3) confirms that the protein band purified from Sf21 cells represents flag-tagged 5/6-kinase. The specific activity of this preparation using Ins(1,3,4)P3 as a substrate is 500,000 min−1/mg of protein, comparable with that obtained for the purified calf brain protein (372,217 min−1/mg of protein). This flag-tagged 5/6-kinase preparation then was used in an in vitro protein kinase assay using full-length ATF-2 as a substrate. As with the calf brain 5/6-kinase, phosphorylation of ATF-2 occurs in a concentration-dependent fashion. ATF-2 phosphorylation by the recombinant fusion protein requires the addition of a much larger amount of enzyme than does phosphorylation by the purified calf brain protein. This could be because of the presence of Nonidet P-40 in the preparation, inappropriate post-translational modification, or the presence of the flag epitope on the protein. The first member of the COP9 signalosome complex was identified originally in a genetic screen of Arabidopsis seedlings. The complex subsequently has been found in many organisms, with the exception of S. cerevisiae (19Wei N. Deng X.-W. Trends Genet. 1999; 15: 98-103Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). We show here that the calf brain COP9 signalosome complex co-purifies with Ins(1,3,4)P3 5/6-kinase, an enzyme conserved from such diverse sources as plants (13Wilson M.P. Majerus P.W. Biochem. Biophys. Res. Commun. 1997; 232: 678-681Crossref PubMed Scopus (55) Google Scholar), E. histolytica (12Field J. Wilson M.P. Mai Z. Majerus P.W. Samuelson J. Mol. Biochem. Parasitol. 2000; 108: 119-123Crossref PubMed Scopus (25) Google Scholar), and humans (24Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Similar to the COP9 signalosome complex, there is no 5/6-kinase homologue in S. cerevisiae. The purification scheme of 5/6-kinase used repeated chromatography on heparin agarose (24Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The cauliflower COP9 complex also binds heparin (37Chamovitz D.A. Wei N. Osterlund M.T. von Arnim A.G. Staub J.M. Matsui M. Deng X.-W. Cell. 1996; 86: 115-121Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Heparin binding alone is unlikely to account for the co-purification, because differential binding in the presence and absence of magnesium as well as affinity elution with InsP6 were used. Even after the final purification step, some 5/6-kinase was detected in the fractions containing complex. It is therefore likely that 5/6-kinase associates with the COP9 signalosome complex. The function of the mammalian COP9 signalosome complex is unknown, although it has been shown to phosphorylate several proteins including c-Jun (20Seeger M. Kraft R. Ferrell K. Bech-Otschir D. Dumdey R. Schade R. Gordon C. Naumann M. Dubiel W. FASEB J. 1998; 12: 469-478Crossref PubMed Scopus (315) Google Scholar). In this report, we show that the complex and 5/6-kinase purified from calf brain phosphorylate c-Jun as well as ATF-2. All preparations of complex used here contain some 5/6-kinase, which may represent the COP9 signalosome-associated kinase activity that to date has not yet been identified. In samples prepared for phospho amino acid analysis, the relative amount of 32P incorporated into ATF-2 was significantly greater than that of c-Jun. This could indicate that ATF-2 is a better in vitro substrate for 5/6-kinase or that there may be multiple sites of phosphorylation on ATF-2. Phosphorylation of ATF-2 by 5/6 kinase occurs predominantly on serine residues, with a minor amount of threonine phosphorylation. There is no evidence of tyrosine phosphorylation of ATF-2 by 5/6-kinase. Phosphorylation of ATF-2 by p38 occurs on threonine residues 69 and 71 (33), which results in increased stability of ATF-2 by protection from ubiquitination and subsequent degradation (38Fuchs S.Y. Tappin I. Ronai Z. J. Biol. Chem. 2000; 275: 12560-12564Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). ATF-2 has also been shown to be phosphorylated on threonine residues 69 and 71 as well as Ser-90 by stress-activated protein kinases, with phosphorylation depending on UV treatment of the cells (32Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (476) Google Scholar). Protein kinase Cα has been reported to phosphorylate ATF-2 on Ser-121 in response to retinoic acid or induction with E1A (39Kawasaki H. Song J. Eckner R. Ugai H. Chiu R. Taira K. Shi Y. Jones N. Yokoyama K.K. Genes Dev. 1998; 12: 233-245Crossref PubMed Scopus (79) Google Scholar). The consequences of phosphorylation of ATF-2 by 5/6-kinase remain to be determined. Phosphorylation of a protein substrate by an inositol kinase has been demonstrated by several lipid kinases. PI3-kinase has been shown to phosphorylate the insulin receptor substrate IRS-1 (40Lam K. Carpenter C.L. Ruderman N.B. Friel J.C. Kelly K.L. J. Biol. Chem. 1994; 269: 20648-20652Abstract Full Text PDF PubMed Google Scholar), the adapter protein p101 and the protein kinase MEK-1 (41Bondev A. Rubio I. Wetzker R. Biol. Chem. 1999; 380: 1337-1340Crossref PubMed Scopus (19) Google Scholar). Autophosphorylation has been reported to occur by PI3-kinase (reviewed in Ref. 46Hunter T. Cell. 1995; 83: 1-4Abstract Full Text PDF PubMed Scopus (264) Google Scholar), type I phosphatidylinositol phosphate 5-kinase isozymes (42Itoh T. Ishihara H. Shibasaki Y. Oka Y. Takenawa T. J. Biol. Chem. 2000; 275: 19389-19394Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and phosphatidylinositol 4-kinase β (43Zhao X.-H. Bondeva T. Balla T. J. Biol. Chem. 2000; 275: 14642-14648Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Phosphorylation of the transcription factors c-Jun and ATF-2 by 5/6-kinase represents the first example of protein phosphorylation by an inositol kinase that utilizes a soluble, as opposed to a lipid, inositol polyphosphate as a substrate. Ins(3,4,5,6)P4 is not only a potent inhibitor of Ins(1,3,4)P3 phosphorylation by 5/6-kinase, but has been reported recently to be a substrate of this enzyme as well (15Yang X. Shears S.B. Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (60) Google Scholar). While inhibiting the inositol kinase activity of 5/6-kinase, this IP4 isomer has no effect on phosphorylation of ATF-2 by 5/6-kinase. We also show that MnCl2 inhibits Ins(1,3,4)P3 phosphorylation by 5/6-kinase but activates ATF-2 phosphorylation. Therefore, the inositol kinase and the protein kinase activities of 5/6-kinase seem to be regulated differently. Differential regulation of protein kinase activity and inositol kinase activity has been reported for PI3-kinase γ. A phosphatidylinositol lipid kinase-negative mutant of PI3-kinase γ retains the ability to autophosphorylate (44Bondeva T. Pirola L. Bulgarelli-Leva G. Rubio I. Wetzker R. Wymann M.P. Science. 1998; 282: 293-296Crossref PubMed Scopus (302) Google Scholar). In addition, increasing concentrations of the βγ subunits of heterotrimeric G proteins increase the lipid kinase activity of PI3-kinase γ, whereas autophosphorylation and phosphorylation of MEK1 are reduced markedly by Gβγ (41Bondev A. Rubio I. Wetzker R. Biol. Chem. 1999; 380: 1337-1340Crossref PubMed Scopus (19) Google Scholar). Human 5/6-kinase expressed in 293 cells and Sf21 cells exhibits both inositol kinase and protein kinase activity. The possibility of a protein kinase responsible for phosphorylation of c-Jun and ATF-2 contaminating the 5/6-kinase preparations used cannot be ruled out completely. This is unlikely, however, because it would require an interaction between the two kinases throughout the calf brain purification in addition to co-immunoprecipitation in the partially purified 293 cell extract. Additionally, the minimal amount of purified calf brain 5/6-kinase sufficient to phosphorylate ATF-2 is 0.4 ng. If a contaminant were responsible for the protein phosphorylation, it would be present in an extremely low concentration. There are no conventional protein kinase domains in 5/6-kinase, such as the ATP binding domain G-X-G-X-X-G (reviewed in Ref. 45Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3857) Google Scholar). This is also the case for PI3-kinase, although a short stretch of amino acids distantly conserved between PI3-kinase and the protein kinase super family is found in the catalytic subdomains VIB and VII of protein kinases (46Hunter T. Cell. 1995; 83: 1-4Abstract Full Text PDF PubMed Scopus (264) Google Scholar). Shown in Fig.7is an alignment of the residues in human PI3-kinase (p110α), which have been reported to be conserved with protein kinases. The two aspartic acid residues (indicated inbold) are conserved amino acids in the protein kinase superfamily. These two residues, along with an arginine (bold), are also conserved in human phosphatidylinositol 4-kinase α and human 5/6-kinase. The first aspartic acid (5/6-kinase amino acid 105), found in subdomain VIB of protein kinases, is proposed to hydrogen-bond with the acceptor amino acid. The second aspartic acid (5/6-kinase amino acid 123), is involved in the chelation of Mg2+. The third conserved amino acid is an arginine found in the inositol lipid kinases and 5/6-kinase but not in protein kinases. Mutation of this arginine (5/6-kinase amino acid 106) has been shown to abolish both inositol lipid and protein kinase activity by PI3-kinase (47Dhand R. Hiles I. Panayotou G. Roche S. Fry M.J. Gout I. Totty N.F. Truong O. Vicendo P. Yonezawa K. Kasuga M. Courtneidge S.A. Waterfield M.D. EMBO J. 1994; 13: 522-533Crossref PubMed Scopus (420) Google Scholar). Mutagenesis of these residues of 5/6-kinase should establish whether they are critical to the inositol and/or protein kinase activities of this enzyme. The only protein kinase to which 5/6-kinase has any sequence similarity is the ε isoform of protein kinase C (24Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Of the three short stretches of sequence similarity between the two proteins, none of the conserved residues have been implicated in the catalytic activity of the protein kinase C isozymes (reviewed in Ref. 48Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1219) Google Scholar). Protein kinase Cε has been shown to be activated by phosphatidylinositol (3,4)P2 and phosphatidylinositol (3,4,5)P3 (49Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar), and thus the regions of identity noted between protein kinase Cε and 5/6-kinase are more likely to be involved in inositol head group binding than in protein kinase activity. Mutagenesis of amino acid residues conserved between 5/6-kinase homologues from different species may shed light upon the structural requirements for inositol versus protein kinase activity. Phosphorylation of the transcription factors c-Jun and ATF-2 by 5/6-kinase implicates this inositol kinase in the stress response pathway. The 5/6-kinase homologue in E. histolytica was identified in a differential display polymerase chain reaction from control versus heat-shocked amoebae, whereby 5/6-kinase mRNA was increased in extracts of heat-shocked parasites (12Field J. Wilson M.P. Mai Z. Majerus P.W. Samuelson J. Mol. Biochem. Parasitol. 2000; 108: 119-123Crossref PubMed Scopus (25) Google Scholar), lending additional support for the role of 5/6-kinase in the response of cells to stress. The trigger(s) responsible for the activation of 5/6-kinase in cells, and the cellular events following this activation remain to be determined. In addition, the role of the inositol polyphosphate products of 5/6-kinase in the stress response pathway have yet to be elucidated. The two functions of 5/6-kinase could represent a divergence of signals resulting from the activation of a single kinase by different stimuli having multiple cellular consequences. We would like to thank Jyotshnabala Kanungo and Marina Kisseleva for helpful discussions and Rosalind Kornfeld and Gregory Longmore for critical reading of the manuscript.