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• W2022805818 abstract "c-Jun N-terminal protein kinase (JNK), a member of the mitogen-activated protein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase (JNKK1 and JNKK2), a subfamily of the dual specificity MAP kinase kinase (MEK) family, through phosphorylation on threonine (Thr) 183 and tyrosine (Tyr) 185 residues. The physiological functions of the JNK pathway, however, are not completely understood. A major obstacle is the lack of specific and activated kinase components that can stimulate the JNK pathway in the absence of any stimulus. Here we show that fusion of JNK1 to its upstream activator JNKK2 resulted in its constitutive activation. In HeLa cells, the JNKK2-JNK1 fusion protein showed significant JNK activity, which was comparable with that of JNK1 activated by many stimuli and activators, including EGF, TNF-α, anisomycin, UV irradiation, MEKK1, and small GTP binding proteins Rac1 and Cdc42Hs. Immunoblotting analysis indicated that JNK1 was phosphorylated by JNKK2 in the fusion protein on both Thr183 and Tyr185 residues. Like JNKK2, the JNKK2-JNK1 fusion protein was highly specific for the JNK pathway and did not activate either p38 or ERK2. Transient transfection assays demonstrated that the JNKK2-JNK1 fusion protein was sufficient to stimulate c-Jun transcriptional activity in the absence of any stimulus. Immunofluorescence analysis revealed that the JNKK2-JNK1 fusion protein was predominantly located in the nucleus of transfected HeLa cells. These results indicate that the JNKK2-JNK1 fusion protein is a constitutively active Jun kinase, which will facilitate the investigation of the physiological roles of the JNK pathway. c-Jun N-terminal protein kinase (JNK), a member of the mitogen-activated protein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase (JNKK1 and JNKK2), a subfamily of the dual specificity MAP kinase kinase (MEK) family, through phosphorylation on threonine (Thr) 183 and tyrosine (Tyr) 185 residues. The physiological functions of the JNK pathway, however, are not completely understood. A major obstacle is the lack of specific and activated kinase components that can stimulate the JNK pathway in the absence of any stimulus. Here we show that fusion of JNK1 to its upstream activator JNKK2 resulted in its constitutive activation. In HeLa cells, the JNKK2-JNK1 fusion protein showed significant JNK activity, which was comparable with that of JNK1 activated by many stimuli and activators, including EGF, TNF-α, anisomycin, UV irradiation, MEKK1, and small GTP binding proteins Rac1 and Cdc42Hs. Immunoblotting analysis indicated that JNK1 was phosphorylated by JNKK2 in the fusion protein on both Thr183 and Tyr185 residues. Like JNKK2, the JNKK2-JNK1 fusion protein was highly specific for the JNK pathway and did not activate either p38 or ERK2. Transient transfection assays demonstrated that the JNKK2-JNK1 fusion protein was sufficient to stimulate c-Jun transcriptional activity in the absence of any stimulus. Immunofluorescence analysis revealed that the JNKK2-JNK1 fusion protein was predominantly located in the nucleus of transfected HeLa cells. These results indicate that the JNKK2-JNK1 fusion protein is a constitutively active Jun kinase, which will facilitate the investigation of the physiological roles of the JNK pathway. c-Jun N-terminal protein kinase epidermal growth factor tumor necrosis factor mitogen-activated protein MAP kinase kinase JNK-activating kinase MAP kinase kinase kinase extracellular signal-regulated kinase glutathioneS-transferase phosphate-buffered saline nuclear export sequence JNK1 (also known as stress-activated protein kinase, SAPK), a subfamily of the MAP kinase family, regulates gene expression in response to various extracellular stimuli (1Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2249) Google Scholar). Activation of JNK requires its phosphorylation on both Thr183 and Tyr185 residues (2Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1708) Google Scholar, 3Derijard B. Hibi M. Wu I.H. Barret T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar, 4Kyriakis J.M. Banerjee P. Nikolakaki E. Dai E.A. Rubie T. Ahmad M.F. Avruch J. Woodgt J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2411) Google Scholar). The MAP kinase kinases that phosphorylate and activate JNK are JNK-activating kinases (JNKK1 and JNKK2, also known as SEK1/MKK4/SAPKK and MKK7, respectively) (5Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1407) Google Scholar, 6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar, 7Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar, 8Tournier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7337-7342Crossref PubMed Scopus (340) Google Scholar, 9Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Wu Z. Wu J. Jacinto E. Karin M. Mol. Cell. Biol. 1997; 17: 7407-7416Crossref PubMed Scopus (95) Google Scholar, 12Lawler S. Cuenda A. Goedert M. Cohen P. FEBS Lett. 1997; 414: 153-158Crossref PubMed Scopus (46) Google Scholar, 13Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 14Yao Z. Diener K. Wang X.S. Zukowski M. Matsumoto G. Zhou G. Mo R. Sasaki T. Nishida H. Hui C.C. Tan T.H. Woodget J.P. Penninger J.M. J. Biol. Chem. 1997; 272: 32378-32383Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), which are members of the dual specificity MAP kinase kinase (MEK) family. The MAP kinase kinase kinases that activate JNKK include MEKKs (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar, 15Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (873) Google Scholar, 16Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar, 17Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar, 18Takekawa M. Posas F. Saito H. EMBO J. 1997; 16: 4973-4982Crossref PubMed Scopus (157) Google Scholar, 19Blank J.L. Gerwins P. Elliott E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 20Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), MLK (21Hirai S. Katoh M. Terada M. Kyriakis J.M. Zon L.I. Rana A. Avruch J. Ohno S. J. Biol. Chem. 1997; 272: 15167-15173Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), TAK1 (22Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1172) Google Scholar), and ASK1 (23Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2006) Google Scholar). This MEKK/JNKK/JNK module appears to be critical in mediating the effects of extracellular stimuli on the activities of several transcription factors, such as c-Jun, ATF-2, and Elk, which are phosphorylated by JNK and control expression of many genes involved in cell growth, differentiation, programmed cell death, and transformation (24Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: F85-104PubMed Google Scholar). The JNK pathway can be activated by a variety of extracellular stimuli such as growth factors, cytokines, tumor promoters, protein synthesis inhibitors, ultraviolet (UV) irradiation, and oncoproteins (1Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2249) Google Scholar, 24Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: F85-104PubMed Google Scholar). Genetic ablation of JNK1 (25Dong C. Yang D.D. Wysk M. Whitmarsh A.J. Davis R.J. Flavell R.A. Science. 1998; 282: 2092-2095Crossref PubMed Scopus (532) Google Scholar), JNK2 (26Sabapathy K. Hu Y. Kallunki T. Schreiber M. David J.P. Jochum W. Wagner E.F. Karin M. Curr. Biol. 1999; 9: 116-125Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 27Yang D.D. Conze D. Whitmarsh A.J. Barrett T. Davis R.J. Rincon M. Flavell R.A. Immunity. 1998; 9: 575-585Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar), JNK3 (28Yang D.D. Kuan C.Y. Whitmarsh A.J. Rincon M. Zheng T.S. Davis R.J. Rakic P. Flavell R.A. Nature. 1997; 389: 865-870Crossref PubMed Scopus (1114) Google Scholar), and SEK1/MKK4/JNKK1 (29Yang D. Tournier C. Wysk M. Lu H.-T. Xu L. Davis R.J. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3004-3009Crossref PubMed Scopus (259) Google Scholar, 30Nishina H. Fischer K.D. Radvanyi L. Shahinian A. Hakem R. Rubie E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (309) Google Scholar) indicates that the JNK pathway is likely required for embryonic development, immune response, and cell survival and death. However, its function in many physiological processes is still poorly understood. Specific chemical inhibitors and constitutively active kinase mutants have successfully been used in exploring the physiological functions of the ERK and p38 MAP kinase pathways (31Cowley S. Paterson H. Kemp P. Marchall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1850) Google Scholar, 32Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Wound G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1258) Google Scholar, 33Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2586) Google Scholar, 34Huang S. Jiang Y. Li Z. Nishida E. Mathias P. Lin S. Ulevitch R.J. Nemerow G.R. Han J. Immunity. 1997; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 35Juo P. Kuo C.J. Reynolds S.E. Konz R.F. Raingeaud J. Davis R.J. Biemann H.P. Blenis J. Mol. Cell. Biol. 1997; 17: 24-35Crossref PubMed Scopus (281) Google Scholar, 36Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughllin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3128) Google Scholar, 37Nemoto S. Sheng Z. Lin A. Mol. Cell. Biol. 1998; 18: 3518-3526Crossref PubMed Scopus (209) Google Scholar, 38Nemoto S. Xiang J. Huang S. Lin A. J. Biol. Chem. 1998; 273: 16415-16420Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Such inhibitors and constitutively active mutants, however, have yet to be reported for the JNK pathway. A great deal of effort has been made to generate constitutively active kinase mutants for the JNK pathway but with no success. At the level of MAP kinase kinase kinase, a truncated form of MEKK1, MEKKΔ, has very high activity and can preferentially activate the JNK pathway when expressed at a low level (16Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar). However, it still stimulates the ERK and p38 pathways in transfected cells, even though to a lesser extent (39Xu S. Robbins D.J. Christerson L.B. English J.M. Vanderbilt C.A. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5291-5295Crossref PubMed Scopus (122) Google Scholar). In addition, MEKKΔ also activates the IκB kinase complex, which plays a critical role in NF-κB activation (40Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (354) Google Scholar, 41Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (471) Google Scholar, 42Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar, 43Yin M-J. Christerson L.B. Yamamoto Y. Kwak Y-T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). It has been reported that MEKK4 is a more specific activator for the JNK pathway (20Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), but its human homologue MTK1 also activates p38 (18Takekawa M. Posas F. Saito H. EMBO J. 1997; 16: 4973-4982Crossref PubMed Scopus (157) Google Scholar). It is known that activation of MAP kinase kinases depends on phosphorylation of two conserved Ser/Thr residues between the subkinase domains VII and VIII in the activation-loop (44Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1659) Google Scholar). Replacement of these Ser/Thr residues with acidic amino acids, such as glutamic acid (Glu) or aspartic acid (Asp), has resulted in several constitutively active MAP kinase kinase mutants, including MEK1(ED) (31Cowley S. Paterson H. Kemp P. Marchall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1850) Google Scholar, 32Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Wound G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1258) Google Scholar) and MKK6b(EE) (34Huang S. Jiang Y. Li Z. Nishida E. Mathias P. Lin S. Ulevitch R.J. Nemerow G.R. Han J. Immunity. 1997; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Because these Ser/Thr residues are also conserved in JNKK1 and JNKK2, it was thought that the same strategy might be used to yield a constitutively active JNKK. Surprisingly, the corresponding JNKK mutants, JNKK1(2E) and JNKK2(3E), are catalytically inactive. 2C. Zheng and A. Lin, unpublished results. 2C. Zheng and A. Lin, unpublished results. This suggests that the activation of JNKK may be more complicated than previously thought. To generate constitutively active kinase components for the JNK pathway, we used the approach of enzyme-substrate fusion. The rate of an enzymatic reaction is greatly influenced by the proximity between the enzyme and its substrate. We reasoned that JNK might become constitutively activated if it is physically linked to its upstream activator JNKK. Unlike JNKK1 that activates both JNK and p38 (5Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1407) Google Scholar, 6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar), JNKK2 only stimulates JNK activity (8Tournier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7337-7342Crossref PubMed Scopus (340) Google Scholar, 9Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Wu Z. Wu J. Jacinto E. Karin M. Mol. Cell. Biol. 1997; 17: 7407-7416Crossref PubMed Scopus (95) Google Scholar, 12Lawler S. Cuenda A. Goedert M. Cohen P. FEBS Lett. 1997; 414: 153-158Crossref PubMed Scopus (46) Google Scholar, 13Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 14Yao Z. Diener K. Wang X.S. Zukowski M. Matsumoto G. Zhou G. Mo R. Sasaki T. Nishida H. Hui C.C. Tan T.H. Woodget J.P. Penninger J.M. J. Biol. Chem. 1997; 272: 32378-32383Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 49Yang J. New L. Jiang Y. Han J. Su B. Gene ( Amst. ). 1998; 212: 95-102Crossref PubMed Scopus (29) Google Scholar). In addition, JNKK2 has considerable basal enzymatic activity when overexpressed in cells (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar). Thus, we fused JNK1 to JNKK2 via a short peptide linker and created the JNKK2-JNK1 fusion protein. The JNKK2-JNK1 fusion protein showed profound JNK activity and was able to stimulate c-Jun transcriptional activity in the absence of any stimulus. The constitutively active JNKK2-JNK1 fusion protein will provide a powerful tool for investigating the physiological functions of the JNK pathway. HeLa and human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 mg/ml of streptomycin. To construct the JNKK2-JNK1 fusion construct, pSRα3HA-JNKK2 was first digested with NotI andBglII to release the C-terminal part of JNKK2-(796–1206). A PCR-generated NotI-NcoI fragment encoding the C-terminal part of JNKK2 with a (Gly-Glu)5 linker at its 3′-end and an NcoI-BglII fragment encoding JNK1 were inserted into the NotI-BglII-digested pSRα3HA-JNKK2. A ChameleonTM mutagenesis kit (Stratagene) was used to replace lysine (Lys) 149 with methionine (Met) to create the hemagglutinin (HA)-JNKK2(K149M)-JNK1 fusion protein. To construct pSRα3HA-JNKK2-JNK1 (APY) construct, the JNK1 part in the JNKK2-JNK1 fusion construct was replaced by anNcoI-BglII fragment encoding JNK1 (APY) (9Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The constructs were confirmed by DNA dideoxynucleotide sequencing. Expression vectors of HA-tagged JNKK2, Flag (M2)-tagged JNK1, MEKKΔ, Rac1 V12, Cdc42Hs V12, HA-MEK1(ΔNED), HA-ERK2, HA-MKK6b(EE), M2-p38, GAL4-c-Jun, and GAL4-c-Jun (AA63/73), have been described previously (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar, 9Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 37Nemoto S. Sheng Z. Lin A. Mol. Cell. Biol. 1998; 18: 3518-3526Crossref PubMed Scopus (209) Google Scholar, 38Nemoto S. Xiang J. Huang S. Lin A. J. Biol. Chem. 1998; 273: 16415-16420Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). The reporter gene 5× GAL4-Luc, in which the GAL4 DNA-binding domain was fused to the luciferase gene, has also been described previously (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar). GST-c-Jun-(1–79) and GST-ATF2, GST-JNK1, and GST-p38 were purified on glutathione-agarose, as described (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar, 9Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Histidine-tagged ERK2 was purified on a nickel-chelate column, according to the manufacturer's procedure (Amersham Pharmacia Biotech Inc.). HeLa or 293 cells were transiently transfected with various expression vectors using LipofectAMINE (Life Technologies, Inc., NY), according to the manufacturer's procedure. After 40 h, the cells were treated with different stimuli or left untreated, as indicated in the figure legends. The cells were harvested and the lysates were prepared, as described previously (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar). HA-tagged or M2-tagged protein kinases were immunoprecipitated with specific antibodies for 3 h at 4 °C. The activity of the immune complex was assayed at 30 °C for 30 min in 30 μl of kinase buffer (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar) in the presence of 10 μm ATP/10 μCi [γ-32P]ATP (10 Ci/mmol) with appropriate substrates, as indicated in the figure legends. The reactions were terminated with 4× Laemmli sample buffers. The proteins were resolved by 13% SDS-polyacrylamide gel electrophoresis, followed by autoradiography. The phosphorylated proteins were quantitated by a PhosphorImager (Molecular Dynamics Inc.). For immunoblotting analysis, proteins were resolved by SDS-polyacrylamide gel electrophoresis on 13% gels, blotted onto Immobilon P membranes (Millipore), and subjected to immunoblotting analysis using anti-HA monoclonal antibody (Santa Cruz Biotechnology), anti-M2 monoclonal antibody (Sigma), or anti-phospho-JNK (on both Thr183 and Tyr185) polyclonal antibody (New England Biolabs Inc.), as indicated in the figure legends. The antibody-antigen complexes were visualized by the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech), according to the manufacturer's procedure. Immunofluorescence analysis was performed as described previously (45Lin A. Frost J. Deng T. Smeal T. Al-Alawi N. Kikkaw U. Hunter T. Brenner D. Karin M. Cell. 1992; 70: 777-789Abstract Full Text PDF PubMed Scopus (311) Google Scholar). Briefly, HeLa cells were plated onto coverslips and transfected with various expression vectors. After 36 h, the cells were fixed with ice-cold methanol for 7 min and washed with phosphate-buffered saline (PBS) three times. The coverslips were incubated with anti-HA antibody (1:50) in PBS at 37 °C for 30 min and then washed with PBS. Immune complexes were detected with fluorescein isothiocyanate-conjugated rabbit anti-mouse antibody (1:400, Jackson ImmunoResearch, Inc.) in PBS at room temperature for 1 h. The nuclei were stained with H33258 (1:500) in PBS for 2 min. The coverslips were mounted in Vectashield (Vector Laboratories, Inc.). Fluorescence microscopy was performed with a Zeiss Axioplan microscope. Because the approach of site-directed mutagenesis failed to produce a constitutively active JNKK,2 we explored the possibility of generating a constitutively active JNK through enzyme-substrate fusion. JNK1 was fused in frame with its specific activator JNKK2 (Fig.1 A). A decapeptide linker (Gly-Glu)5, was inserted between the coding sequences of JNKK2 and JNK1 to facilitate the folding (46Robinson M.J. Stippec S.A. Goldsmith E. White M.A. Cobb M.H. Curr. Biol. 1998; 8: 1141-1150Abstract Full Text Full Text PDF PubMed Google Scholar). Cell-free translation ofin vitro generated JNKK2-JNK1 transcripts produced a single polypeptide with an apparent molecular mass of 86 kDa, as expected (Fig. 1 B). Using the same strategy, we also constructed JNKK2(KM)-JNK1, in which the lysine (Lys) 149 in the ATP binding domain of the JNKK2 moiety was replaced by methionine (Met), and JNKK2-JNK1 (APY), in which the Tyr185 residue in the JNK1 moiety was replaced by a nonphosphorylatable alanine (Ala). We tested whether the JNKK2-JNK1 fusion protein has Jun kinase activity and, if so, whether it is a more active Jun kinase than JNK1 stimulated by cotransfection with JNKK2. HeLa cells were transiently transfected with expression vectors encoding HA-JNKK2-JNK1, HA-JNKK2(K149M)-JNK1, HA-JNKK2-JNK1 (APY), M2-JNK1 with or without HA-JNKK2, or empty expression vector. After 40 h, the cells were harvested and the transfected kinases were isolated by immunoprecipitation. The kinase activity was measured by immunocomplex kinase assays with GST-c-Jun-(1–79) as a substrate (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (708) Google Scholar). As reported previously (9Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), coexpression of JNKK2 activated JNK1 modestly (Fig. 2, lanes 2–4). In contrast, the JNKK2-JNK1 fusion protein itself was 30-fold more active than the nonstimulated JNK1 (Fig. 2, lanes 5–7). This activity was not a result of differences in the expression between HA-JNKK2-JNK1 and M2-JNK1/HA-JNKK2, as demonstrated by immunoblotting analysis (Fig. 2). Under the same conditions, the JNKK2(K149M)-JNK1 fusion protein had no detectable Jun kinase activity (Fig. 2,lane 8) nor did the JNKK2-JNK1 (APY) fusion protein (data not shown). These results indicate that the JNKK2-JNK1 fusion protein has profound Jun kinase activity, which likely results from activation of JNK1 by JNKK2 in the fusion protein. Activation of JNK1 requires its phosphorylation on both Thr183 and Tyr185 residues (3Derijard B. Hibi M. Wu I.H. Barret T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar). Because the JNKK2-JNK1 functions as an activated Jun kinase, we examined whether JNK1 is phosphorylated by JNKK2 in the fusion protein. HeLa cells were transiently transfected with expression vectors encoding HA-JNKK2-JNK1, HA-JNKK2(KM)-JNK1, or empty vector. The cells were treated with anisomycin (which is a strong stimulus of JNK) for 15 min or left untreated. The cells were harvested and the extracts were fractionated by SDS-gel electrophoresis, followed by immunoblotting with the anti-JNK antibody (Pharmingen) or the specific anti-phospho-JNK antibody (New England Biolabs, Inc.), which only recognizes the dual-phosphorylated JNK (on both Thr183 and Tyr185). As expected, the anti-phospho-JNK antibody only recognized anisomycin-stimulated, but not nonstimulated, endogenous JNK1 and JNK2 (Fig. 3, left panel,lanes 1 and 2). The anti-phospho-JNK antibody also recognized the transfected HA-JNKK2-JNK1 fusion protein (Fig. 3,left panel, lane 3). Under the same conditions, it failed to detect the inactive HA-JNKK2(KM)-JNK1 mutant (Fig. 3,left panel, lane 4). This was not a result of differences in protein expression, as demonstrated by immunoblotting analysis with anti-JNK antibody (Fig. 3, right panel). Thus, activation of JNK1 in the fusion protein may result from its phosphorylation by JNKK2 on Thr183 and Tyr185residues. To determine whether the JNKK2-JNK1 fusion protein is a constitutively active Jun kinase, we compared its activity with that of JNK1 stimulated by various activators and extracellular stimuli. HeLa cells were transiently transfected with expression vectors encoding HA-JNKK2-JNK1, HA-JNK1 with or without the active forms of MEKK1, Rac1, Cdc42, or empty expression vector. After 40 h, the cells were treated with EGF, TNF-α, anisomycin, UV, or left untreated, as indicated. The activity of HA-JNKK2-JNK1 was equivalent or comparable with that of HA-JNK1 activated by the above activators and stimuli, as measured by immunocomplex kinase assays with GST-c-Jun (1–79) as a substrate (Fig.4 A). Consistently, active forms of Rac1 and Cdc42 did not further stimulate the activity of HA-JNKK2-JNK1 (Fig. 4 B, lanes 4 and5), nor did EGF or TNF-α (Fig. 4 B, lanes 6 and 7). The active form of MEKK1, anisomycin, and UV, on the other hand, slightly stimulated the JNKK2-JNK1 fusion protein (Fig. 4 B, lanes 3, 8, and 9). These results suggest that the JNKK2-JNK1 fusion protein is likely a constitutively active Jun kinase. JNKK2 is a highly specific JNK activator that does not activate p38 or ERK2 (8Tournier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7337-7342Crossref PubMed Scopus (340) Google Scholar, 9Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Wu Z. Wu J. Jacinto E. Karin M. Mol. Cell. Biol. 1997; 17: 7407-7416Crossref PubMed Scopus (95) Google Scholar, 12Lawler S. Cuenda A. Goedert M. Cohen P. FEBS Lett. 1997; 414: 153-158Crossref PubMed Scopus (46) Google Scholar, 13Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 14Yao Z. Diener K. Wang X.S. Zukowski M. Matsumoto G. Zhou G. Mo R. Sasaki T. Nishida H. Hui C.C. Tan T.H. Woodget J.P. Penninger J.M. J. Biol. Chem. 1997; 272: 32378-32383Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 49Yang J. New L. Jiang Y. Han J. Su B. Gene ( Amst. ). 1998; 212: 95-102Crossref PubMed Scopus (29) Google Scholar). To ensure that the JNKK2-JNK1 fusion protein still maintained this high specificity, we determined its effect on the activity of JNK1, p38, and ERK2. In HeLa cells, coexpression of HA-JNKK2-JNK1, but not HA-JNKK2(K149M)-JNK1, stimulated M2-JNK1 activity significantly (Fig.5, lanes 3 and 4). Under the same conditions, the H" @default.
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• W2022805818 title "The JNKK2-JNK1 Fusion Protein Acts As a Constitutively Active c-Jun Kinase That Stimulates c-Jun Transcription Activity" @default.
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