A Novel Human STE20-related Protein Kinase, HGK, That Specifically Activates the c-Jun N-terminal Kinase Signaling Pathway

Abstract

The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors. Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation. TNF-α activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-α-induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK → TAK1 → MKK4, MKK7 → JNK kinase cascade may mediate the TNF-α signaling pathway. The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors. Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation. TNF-α activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-α-induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK → TAK1 → MKK4, MKK7 → JNK kinase cascade may mediate the TNF-α signaling pathway. c-Jun N-terminal kinase mitogen-activated protein kinase extracellular signal-regulated kinase MAPKK, or MEK, MAPK kinase stress-activated protein kinase/ERK kinase germinal center kinase GCK-like kinase kinase homologous to STE20 hematopoietic progenitor kinase-1 MAPK kinase kinase transforming growth factor β-activated kinase MAPK-upstream kinase tumor necrosis factor α polymerase chain reaction polyacrylamide gel electrophoresis hemagglutinin p21-activated kinase glutathioneS-transferase chloramphenicol acetyltransferase polyvinylidene difluoride 4-morpholinepropanesulfonic acid myelin basic protein HPK/GCK-like kinase. The c-June N-terminal kinase (JNK)1 family members belong to the mitogen-activated protein kinase (MAPK) superfamily, which also includes extracellular signal-regulated kinases (ERKs) and the p38 family (reviewed in Ref. 1Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1397) Google Scholar). MAPK superfamily members are serine/threonine-directed kinases that target other kinases, transcription factors, and membrane receptor tails, causing diverse effects such as cell proliferation, transformation, differentiation, and apoptosis. Activation of MAPK members occurs through an assembly of kinase cascades in response to stimuli. The original MAPK cascade (c-Raf → MEK1/2 → ERK1/2) includes activation of the serine/threonine kinase c-Raf, which activates a dual-specificity MAPK kinase (MEK1/MEK2). MAPK/ERK is then activated via tyrosine and threonine phosphorylation of the T-X-Y motif on the phosphorylation loop of the enzyme. This phosphorylation event causes local and global conformational changes in the protein, resulting in an active enzyme structure. The JNK family members were characterized biochemically as two kinases and cloned from human fetal brain and rat brain libraries (2Derijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 3Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar). Presently, the JNK family consists of three genes, JNK1, JNK2, and JNK3, which can be subdivided further into 10 isoforms (4Gupta S. Battett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1183) Google Scholar). JNK1 and JNK2 have four isoforms, while JNK3 has two isoforms; these isoforms arise from alternative splicing. 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The serine/threonine kinase STE20 sequentially activates STE11 and STE7 in the signaling cascade that includes STE11 (MAPKKK), STE7 (MAPKK), and FUS3/KSS1 (MAPK) in the yeast S. cerevisiae pheromone response. STE20 activates this pathway in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors (reviewed in Ref. 31Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (867) Google Scholar). Thus far, p21 (Rac1/Cdc42)-activated kinases (PAKs) and HPK/GCK-related kinases (HPK1, GCK, GLK, and KHS) have been identified as potential upstream kinases for MEKK1. This kinase cascade has been suggested by many laboratories as follows: HPK1, GCK, PAKs → MEKKs → MKK4, MKK7 → JNKs. There is also evidence suggesting that MAPKKKs (e.g.MEKKs and MLK-3) are parallel to, but not downstream of, PAKs and HPK/GCK family kinases (reviewed in Ref. 22Fanger G.R. Gerwins P. Widmann C. Jarpe M.B. Johnson G.L. Curr. Opin. Genet. Dev. 1997; 7: 67-74Crossref PubMed Scopus (299) Google Scholar). the and of a novel human protein kinase, HGK kinase), which is homologous to STE20. of HGK specifically activates the JNK signaling pathway. were to the and of the serine/threonine protein kinase and in a polymerase chain reaction as X.S. Diener K. D. D. Tan T.-H. H. Zukowski M. Yao Z. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). from a human were for in and then to the in the the were for in were isolated and on both from various were from were with an HGK to the of was in by three in were for 293T were by M. Hu 293T were in with fetal and 293T were a of as and transfected the the The of for were as and was to of in A the hemagglutinin was from The the was from a to the of HGK was then to via the the and to was as D. 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 (120) Google Scholar). HGK was cloned into and by two N-terminal was A HGK mutant was by with an by the as R.M. PubMed Scopus Google Scholar). A HGK mutant was by with a acid encoding a dominant-negative MKK7 mutant was Z. Diener K. Wang X.S. Zukowski M. Matsumoto G. Zhou G. Mo R. Sasaki T. Nishina H. Hui C.C. Tan T.-H. Woodgett J. Penninger J.M. J. Biol. Chem. 1997; 272: 32378-32383Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). encoding a dominant-negative MEKK1 mutant was by D. Templeton encoding a dominant-negative mutant was by L.I. Zon and were by J. Woodgett encoding a dominant-negative TAK1 mutant was by K. Matsumoto encoding a dominant-negative mutant was by S. Ohno of and were by J. S. of and were by J. kinase were as C.F. Wang X. C. Templeton D. Tan T.-H. J. Biol. Chem. 1996; 271: 8971-8976Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). were by with and protein in an The were with with and with kinase 10 and then with of the and 10 of in of kinase The kinase reaction was for and with an of The reaction were by The and were M.C.-T. Qu W.R. Wang X. Meyer C.F. Tan T.-H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar). the kinase proteins were a to a polyvinylidene difluoride membrane in 10 and The myelin basic protein were identified and by of the to The membrane was in and then in The proteins on the membrane were then in of for The acid acids were and The were with of The were in of and with and tyrosine and on a The acids were in the for The acid were by the with in were by to novel members of the STE20 family of protein kinases, we a degenerate to cDNA from a human cDNA A novel sequences with homology to the STE20 family of protein kinases was This cDNA was to a human cDNA cDNA of were and the the was on both The nucleotide of the cDNA an open reading frame of 1165 amino acids with a of The amino acid a kinase in the that 11 kinase of the amino acid of the kinase of HGK with other sequences found that the HGK kinase was to HPK1, GLK, GCK, and STE20. the kinase HGK amino acid to the of the HPK1, amino acid to the of the human GLK, amino acid to the of the GCK, amino acid to the of the and amino acid to S. cerevisiae The amino acid of the of HGK with that of kinases is shown in B. and GCK, HGK not the homology of the HGK cDNA an amino acids with two other of HGK that the two to the was to cDNA from human and of the found the to be in the the is the in human and not The of HGK was in a of human by a from the of The HGK to three of and in suggesting a the of HGK protein, a to HGK was and by This specifically a protein of in and cells, as shown by The specificity of the was further in 293T transfected with a HGK cDNA the and the specifically the HGK proteins in the 293T HGK the the HGK and which to and to acid did not the that the HGK kinase activity was not to acid of in and HGK as a serine/threonine protein activity of 293T were transfected with of an HGK, and The were kinase were with an as the of and mutant HGK were by an HGK is a serine/threonine protein kinase. 293T were transfected with of a The were kinase were with an as a electrophoresis in the protein was to was from the and to acid The of threonine and tyrosine are by HGK can activate JNK, 293T were with encoding HGK and an JNK was then from cell and in a protein kinase with as a with HGK in JNK1 activation while transfected with JNK1 activation and of a of HGK, in which in the was to a did not in JNK activation the kinase activity of HGK is for JNK activation. HGK can also activate the p38 kinase or 293T were transfected with HGK with p38 or p38 kinase and were then and their or as substrates. activated p38 and the activated strongly activated p38 or kinase were increased when HGK was in 293T and These suggest that the activation of JNK by HGK is c-Jun is a of JNK and the of HGK can activate the JNK signaling pathway, we the of HGK can in the activation of c-Jun transcriptional activity in vivo. were transfected with an HGK and a chloramphenicol acetyltransferase of the were of HGK JNK1 the transcriptional activity by as with transfected with JNK1 or HGK to the of JNK in 293T cells, of the JNK in the was in to by HGK via the HGK → TAK1 → → JNK kinase of with JNK1 and a mutant of HGK did not the transcriptional activity of This that HGK can activate JNK, which in the transcriptional activity of c-Jun in vivo. MKK4 is an upstream activator of JNK which phosphorylates and activates Recently, another called MKK7, has been cloned and found to specifically activate JNK, but not p38 and HGK activates JNK through MKK4 and MKK7, we with an HGK and a dominant-negative mutant of or a dominant-negative mutant of MKK7 to can HGK-induced JNK activation. The of a dominant-negative of MKK4 or MKK7 inhibited JNK activity by HGK and that HGK upstream of MKK4 The MAPK kinase kinase MEKK1 is a activator of MKK4, which in phosphorylates and activates MEKK1 was shown to function downstream of and M.C.-T. Qu W.R. Wang X. Meyer C.F. Tan T.-H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar, 27Diener D. 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 (120) Google Scholar), two kinases that are homologous to HGK also activates JNK through we the of the mutant MEKK1 on HGK-induced JNK activation. we found the mutant strongly inhibited JNK activation it did not JNK activity These results suggest that HGK downstream kinases for TAK1 was shown to function downstream of W. Zhou G. Hu M.C.-T. Yao Z. Tan T.-H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (163) Google we HGK activates JNK through with HGK the dominant-negative mutant of either TAK1 or and their effects on HGK-induced JNK activation. found that the TAK1 mutant but not the inhibited HGK-induced JNK activation This strongly that TAK1 is a downstream target for HGK activity in the JNK signaling JNK activation is specifically by the dominant-negative TAK1 293T cell were transfected with of of and of of dominant-negative and of dominant-negative was to the of transfected kinase were with an as the of and were by and TNF-α TAK1 and JNK kinase activity K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) and HGK was found to be an upstream kinase of TAK1 and JNK we TNF-α can also HGK kinase the activation of HGK, an encoding the HGK was transfected into 293T The 293T were with and the cell were and to in kinase TNF-α HGK kinase activity increased and of HGK were by with This that TNF-α HGK kinase many other and kinases, HGK kinase in kinase was to we the dominant-negative HGK to further the role of HGK in the JNK activation 293T were transfected with the dominant-negative HGK mutant or the the dominant-negative HGK mutant were then with by JNK kinase found that the dominant-negative HGK or inhibited JNK activation of were by results indicate that HGK is in the TNF-α-induced signaling pathway. A human kinase homologous to yeast STE20, called HGK, was HGK both and homology with members of the STE20 family of kinases, including HPK1, GLK, GCK, and HGK has a in its and a The of HGK not to the motifs found in the family of protein kinases D. A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). HPK1, GLK, GCK, and of HGK in 293T strongly activates the JNK pathway, but has on either the or p38 signaling suggesting that HGK-induced JNK activation is the of HGK not the it to as a dominant-negative the may with other signaling that are for HGK kinase activation. JNK activation was not by the dominant-negative mutant of which is a JNK JNK activation by the homologous kinase, HPK1, can be by this found that JNK activation is also inhibited by a dominant-negative mutant of MEKK1 D. 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 (120) Google Scholar). These suggest that HGK may function of MEKK1 and that protein kinases may through downstream the are kinases that can activate the JNK pathway. MAPKKKs we found that the dominant-negative TAK1 mutant strongly inhibited HGK-induced JNK activation. TAK1 was identified as a kinase that can activate the p38 kinase signaling pathway. found that TAK1 can also activate the JNK pathway K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 20Wang W. Zhou G. Hu M.C.-T. Yao Z. Tan T.-H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). HGK TAK1 as its downstream kinase. isoforms of human HGK were of which has a in the that two The in the of HGK, may mediate its with other homology The that human HGK that is this can activate the JNK pathway that this is not for JNK activation in transfected 293T that the that the was in the while the was in other such as human and not The differential of isoforms of HGK that these isoforms have and may be by The specificity of JNK activation is not well the that the JNK pathway is activated by a of extracellular including UV irradiation, ionizing heat shock, osmotic protein synthesis inhibitors, agents, and proinflammatory it not be that multiple protein kinases can mediate JNK activation. of the specificity of JNK activation is to have upstream kinases, to stimuli. protein kinases have for may have a function in hematopoietic progenitor cells, as its are in hematopoietic and cell M.C.-T. Qu W.R. Wang X. Meyer C.F. Tan T.-H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar, 26Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (200) Google Scholar). This that HGK is in the signaling pathway. also that HGK is in the signaling pathway. are on the of the HGK → TAK1 → MKK4, MKK7 → JNK kinase cascade in and TNF-α signaling for the for the HGK J. R. J. J. S. M.C.-T. K. S. D. J. and L. I. Zon for members of the Tan for the and for and M. for