Different Properties of SEK1 and MKK7 in Dual Phosphorylation of Stress-induced Activated Protein Kinase SAPK/JNK in Embryonic Stem Cells

Stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), belonging to the mitogen-activated protein kinase family, plays an important role in stress signaling. SAPK/JNK activation requires the phosphorylation of both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7 have been identified as the dual specificity kinases. In this study, we generated mkk7−/− mouse embryonic stem (ES) cells in addition tosek1−/− cells and compared the two kinases in terms of the activation and phosphorylation of JNK. Although SAPK/JNK activation by various stress signals was markedly impaired in both sek1−/− andmkk7−/− ES cells, there were striking differences in the dual phosphorylation profile. The severe impairment observed in mkk7−/− cells was accompanied by a loss of the Thr phosphorylation of JNK without marked reduction in its Tyr-phosphorylated level. On the other hand, Thr phosphorylation of JNK in sek1−/−cells was also attenuated in addition to a decreased level of its Tyr phosphorylation. Analysis in human embryonic kidney 293T cells transfected with a kinase-dead SEK1 or a Thr-Pro-Phe mutant of JNK1 revealed that SEK1-induced Tyr phosphorylation of JNK1 was followed by additional Thr phosphorylation by MKK7. Furthermore, SEK1 but not MKK7 was capable of binding to JNK1 in 293T cells. These results indicate that the Tyr and Thr residues of SAPK/JNK are sequentially phosphorylated by SEK1 and MKK7, respectively, in the stress-stimulated ES cells. Stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), belonging to the mitogen-activated protein kinase family, plays an important role in stress signaling. SAPK/JNK activation requires the phosphorylation of both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7 have been identified as the dual specificity kinases. In this study, we generated mkk7−/− mouse embryonic stem (ES) cells in addition tosek1−/− cells and compared the two kinases in terms of the activation and phosphorylation of JNK. Although SAPK/JNK activation by various stress signals was markedly impaired in both sek1−/− andmkk7−/− ES cells, there were striking differences in the dual phosphorylation profile. The severe impairment observed in mkk7−/− cells was accompanied by a loss of the Thr phosphorylation of JNK without marked reduction in its Tyr-phosphorylated level. On the other hand, Thr phosphorylation of JNK in sek1−/−cells was also attenuated in addition to a decreased level of its Tyr phosphorylation. Analysis in human embryonic kidney 293T cells transfected with a kinase-dead SEK1 or a Thr-Pro-Phe mutant of JNK1 revealed that SEK1-induced Tyr phosphorylation of JNK1 was followed by additional Thr phosphorylation by MKK7. Furthermore, SEK1 but not MKK7 was capable of binding to JNK1 in 293T cells. These results indicate that the Tyr and Thr residues of SAPK/JNK are sequentially phosphorylated by SEK1 and MKK7, respectively, in the stress-stimulated ES cells. stress-activated protein kinase c-Jun N-terminal kinase mitogen-activated protein kinase embryonic day extracellular signal-regulated kinase mitogen-activated protein kinase kinase mitogen-activated protein kinase kinase stress-activated protein kinase/extracellular signal-regulated kinase kinase embryonic stem antibody monoclonal antibody hemagglutinin hygromycin resistance cassette neomycin resistance cassette dominant-negative SEK1 mutant The SAPK/JNK1 is a member of the family of mitogen-activated protein kinase (MAPK). This MAPK is activated not only by many types of cellular stresses, including changes in osmolarity, heat shock, and UV irradiation, but also by serum, lysophosphatidic acid, and inflammatory cytokines (interleukin-1औ and tumor necrosis factor-α). The activated SAPK/JNK phosphorylates transcription factors c-Jun, Jun D, and activating transcription factor-2 to regulate gene expression for the stress response. Activation of SAPK/JNK requires the phosphorylation of Tyr and Thr residues located in a Thr-Pro-Tyr motif in the activation loop between VII and VIII of the kinase domain. The phosphorylation is catalyzed by the dual specificity kinases SEK1 (also known as MKK4) and MKK7 (SEK2), which are capable of catalyzing the phosphorylation of both Thr and Tyr residues in vitro (1Davis R., J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar, 2Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar). Targeted gene-disruption experiments in mice demonstrate that both SEK1 and MKK7 are required for embryonic development.Sek1−/− embryos die between embryonic day 10.5 (E10.5) and E12.5 with impaired liver formation (3Yang D. Tournier C. Wysk M. Lu H.-T. Xu J. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3004-3009Crossref PubMed Scopus (259) Google Scholar, 4Ganiatsas S. Kwee L. Fujiwara Y. Perkins A. Ikeda T. Labow M., A. Zon L.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6881-6886Crossref PubMed Scopus (178) Google Scholar, 5Nishina H. Vaz C. Billia P. Nghiem M. Sasaki T. Pompa J.L. Furlonger K. Paige C. Hui C.-C. Fischer K.D. Kishimoto H. Iwatsubo T. Katada T. Woodgett J.R. Penninger J.M. Development. 1999; 126: 505-516Crossref PubMed Google Scholar). Furthermore, we have recently reported that SEK1 is crucial for hepatocyte growth factor-induced activation of SAPK/JNK in developing hepatoblasts of mouse embryos. On the other hand, mkk7−/−embryos die between E11.5 and E12.5 with similar impairment of liver formation and SAPK/JNK activation (6Watanabe T. Nakagawa K. Ohata S. Kitagawa D. Nishitai G. Seo J. Tanemura S. Shimizu N. Kishimoto H. Wada T. Aoki J. Arai H. Iwatsubo T. Mochita M. Watanabe T. Satake M. Ito Y. Matsuyama T. Mak T.W. Penninger J.M. Nishina H. Katada T. Dev. Biol. 2002; 250: 332-347Crossref PubMed Google Scholar). These results clearly show that both SEK1 and MKK7 play indispensable roles in hepatoblast proliferation during mouse embryogenesis. Distinct biochemical properties between SEK1 and MKK7 may be critical for the indispensable roles of the two activators of SAPK/JNK in vivo. In this regard, several in vitro experiments have shown that SAPK/JNK is activated synergistically by SEK1 and MKK7 (7Lawler S. Fleming Y. Goedert M. Cohen P. Curr. Biol. 1998; 8: 1387-1390Abstract Full Text Full Text PDF PubMed Google Scholar, 8Lisnock J. Griffin P. Calaycay J. Franz B. Parsons J. O'Keefe S.J. LoGrasso P. Biochemistry. 2000; 39: 3141-3148Crossref PubMed Scopus (69) Google Scholar, 9Fleming Y. Armstrong C.G. Morrice N. Paterson A. Goedert M. Cohen P. Biochem. J. 2000; 352: 145-154Crossref PubMed Scopus (171) Google Scholar). The synergistic activation may be related to the enzymatic properties of the two MAPKKs: SEK1 prefers the Tyr residue and MKK7 prefers the Thr residue of the MAPK. We have also reported that the synergistic activation of SAPK/JNK in response to stress signals is attenuated with a decreased level of its Tyr phosphorylation insek1−/− mouse ES cells that retain MKK7 at the same level as the wild-type cells (10Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). SAPK/JNK activation by UV irradiation and Fcε-receptor stimulation was also attenuated in themkk7−/− mast cells. Despite the impaired SAPK/JNK activation in the mkk7−/− cells, the expression of SEK1 was strongly up-regulated, and SEK1 protein was phosphorylated upon stimulation (11Sasaki T. Wada T. Kishimoto H. Irie-Sasaki J. Matsumoto G. Goto T. Yao Z. Wakeham A. Mak T.W. Suzuki A. Katada T. Nishina H. Penninger J.M. J. Exp. Med. 2001; 194: 757-768Crossref PubMed Scopus (54) Google Scholar). Thus, both SEK1 and MKK7 seem to be required for the synergistic and functional activation of SAPK/JNK in a variety of mammalian cells. To reveal the molecular mechanism of the synergistic activation of SAPK/JNK by SEK1 and MKK7 in living cells, we generatedmkk7−/− mouse ES cells by gene targeting, in addition to sek1−/− ES cells, and further investigated the contribution of SEK1 and MKK7 to the activation and phosphorylation of the MAPK. Our present results clearly show that both SEK1 and MKK7 are required for the synergistic activation of SAPK/JNK in response to various stimuli in ES cells. Furthermore, we propose a sequential phosphorylation mechanism of SAPK/JNK by the two activators, SEK1 and MKK7, in the stress-stimulated living cells. A 17-kbp DNA fragment of mkk7 gene was isolated from a genomic 129/J mouse library. Targeting vector 1 contained a 585-bp short arm, a 6.7-kbp long arm, three loxP sequences, and a hygromycin resistance cassette (Hyg) in antisense orientation to mkk7 transcription. Targeting vector 2 contained a 709-bp short arm, a 5.2-kbp long arm, and a neomycin resistance cassette (Neo) in antisense orientation tomkk7 transcription. The linearized targeting vector 1 was electroporated into E14K ES cells. ES cell colonies resistant to hygromycin (0.2 mg/ml; Invitrogen) were screened for homologous recombination by PCR (30 s at 94 °C, 30 s at 60 °C, 1 min at 72 °C, for 40 cycles) using primers specific for mkk7genomic sequences and Hyg as described below. Next, themkk7+/hyg ES cells were transfected with Cre recombinase expression vector, and cell colonies sensitive to hygromycin were screened for deletion of the region containing exons 4–13 and Hyg by PCR. The linearized targeting vector 2 was electroporated into mkk7+/del ES cells. Retargeted ES cell colonies resistant to G418 (0.3 mg/ml; Invitrogen) were screened for homologous recombination by PCR using primers specific for mkk7 genomic sequences and Neo as described below. As a result, two mkk7neo/del clones (001 and 002) were independently obtained. Both clones lack MKK7 completely, and they are henceforth referred to as mkk7−/−ES cells in this manuscript. Specific primer sets used were 5′-GCC AAA ACA CGG AGT GCT GG-3′ and 5′-ATG TGA CCA GGC AGG AGT GG-3′ for wild-type (+) allele, 5′-TTA AGG CAA CTG GCA GAG-3′ and 5′-AGC TGA CTC TAG AGC TTG-3′ for hyg allele, 5′-ATC TGC CTG TAG CAT GCC-3′ and 5′-ACT CCA AAC ACC TCC CAC-3′ for del allele, 5′-GGA TGT GGA ATG TGT GCG AG-3′ and 5′-AGC TGG AAC CAC GCG CAA TGT GAG-3′ for neo allele. Recombinant ES cell clones were confirmed by Southern blotting ofXbaI-digested genomic DNA hybridized to a 530-bp 3′-flanking probe. Plasmids that express FLAG-tagged SEK1, MKK7γ2, and HA-tagged JNK1 were constructed as described previously (10Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The cDNA encoding FLAG-tagged SEK1 kinase dominant-negative mutant (dnSEK1) by substituting Lys-129 with Arg (K129R) was cloned into mammalian expression vector pCMV5. The cDNAs encoding SAPK/JNK1 mutants Ala-Pro-Phe (APF), Ala-Pro-Tyr (APY), and Thr-Pro-Phe (TPF), were constructed by Kunkel method using the following three primers: 5′-GGA ACG AGT TTT ATG ATG GCG CCT TTT GTA GTG ACT CGC TAC TAC AGA GCA CC-3′ for APF mutant, 5′-GGA ACG AGT TTT ATG ATG GCG CCT TAT GTA GTG ACT CGC TAC TAC AGA GCA CC-3′ for APY mutant, and 5′-GGA ACG AGT TTT ATG ATG ACG CCT TTT GTA GTG ACT CGC TAC TAC AGA GCA CC-3′ for TPF mutant. Antibodies against SAPK/JNK1 (C-17 and FL), MKK7/MEK7 (T-19), and SEK1/MEK4 (C-20) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-SAPK/JNK (9251), and anti-phospho-SEK1 (9151) Abs were from Cell Signaling Technology. Anti-phospho-Tyr (PY20) and anti-phospho-Thr-Pro (P-Thr-Pro-101) were from and Cell Signaling and were purchased from and and to and were in as described previously (10Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). ES cells were at 2 cells a and transfected 1 day with of DNA using The cells, for 1 were to and for embryonic kidney 293T cells were at 1 cells a and transfected 1 day with of DNA using The cells were and for the of SAPK/JNK and ES cells were at cells and As and stresses, the cells were by for 1 UV and heat for SAPK/JNK were at for 2 using the Santa Cruz Biotechnology, The SAPK/JNK in the was with as an in in the of 60 as described previously H. Fischer K.D. L. A. A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; PubMed Scopus Google Scholar, H. M. Fischer K.D. B. Wakeham A. A. H. A. Mak T.W. Woodgett J.R. Penninger J.M. J. Exp. Med. 1997; PubMed Scopus Google Scholar). To the phosphorylation of Tyr and Thr residues in the Thr-Pro-Tyr motif of ES cells were at cells a and with 2 of a of and 1 The cell were with and protein at for 2 and the were several with the The were by and were to a and with and SAPK/JNK The were by for the of using a the SEK1 and MKK7 were with and and with (C-20) and experiments were at three with of the cell and the results were of the shown are of several To the role of MKK7 in SAPK/JNK we generatedmkk7−/− ES cells using two targeting in the of as the exons 4–13 region gene was from using targeting vector 1 and Cre recombinase was by the of activating phosphorylation in with neomycin resistance cassette using targeting vector 2 As a result, two clones (001 and 002) of mkk7neo/del ES cells that lack MKK7 were independently Thus, the are used as mkk7−/− ES cells in this To the role of MKK7 in activation of mkk7−/−ES cells were various wild-type cells. the of SAPK/JNK in response to a protein of heat for UV irradiation 1 and stress in the two mutant and wild-type ES cells. These markedly SAPK/JNK in wild-type ES cells. was impaired in both mutant cells, the cells contained SAPK/JNK at the same level as wild-type cells Thus, the activation of SAPK/JNK to both SEK1 and MKK7 in ES Thr and Tyr phosphorylation of SAPK/JNK in and mkk7−/−ES cells. ES cells were with 1 of UV irradiation and of and further at for Cell were and SAPK/JNK and SEK1 were with Abs and The Thr phosphorylation and Tyr phosphorylation of with phosphorylated SEK1 were using Abs as described The impairment of SAPK/JNK activation observed in the two mutant cells was further investigated with the of As shown in the activation of SAPK/JNK a at in wild-type ES cells. activation of SAPK/JNK was markedly attenuated in mkk7−/− ES cells without in the of of SAPK/JNK activation in the two mutant cells was also observed in other stress including a and stresses, as and not The results in 2 that SEK1 and MKK7 synergistically in the stimulation of SAPK/JNK in ES cells. To the of the two SEK1 and MKK7 expression were transfected into sek1−/− ES cells SEK1 the impaired SAPK/JNK activation in response to UV irradiation and heat insek1−/− ES cells MKK7 and not the SAPK/JNK activation in sek1−/− ES cells. On the other hand, MKK7 and but not SEK1, the impaired SAPK/JNK activation in response to heat shock, and in mkk7−/− ES cells These results clearly show that SEK1 and MKK7 in the SAPK/JNK activation in ES cells. recently been reported that the phosphorylation of Thr and Tyr residues in the Thr-Pro-Tyr motif of SAPK/JNK is required for the activation of the MAPK and that SEK1 and MKK7 the Tyr and Thr respectively, in vitro (7Lawler S. Fleming Y. Goedert M. Cohen P. Curr. Biol. 1998; 8: 1387-1390Abstract Full Text Full Text PDF PubMed Google Scholar, 8Lisnock J. Griffin P. Calaycay J. Franz B. Parsons J. O'Keefe S.J. LoGrasso P. Biochemistry. 2000; 39: 3141-3148Crossref PubMed Scopus (69) Google Scholar, 9Fleming Y. Armstrong C.G. Morrice N. Paterson A. Goedert M. Cohen P. Biochem. J. 2000; 352: 145-154Crossref PubMed Scopus (171) Google Scholar). we the phosphorylation of SAPK/JNK insek1−/− and mkk7−/−ES cells with wild-type cells. The three cell types were with of and 1 of UV irradiation and and of The SAPK/JNK was and by with In a of the present ES cells the same of SAPK/JNK The of Thr and Tyr phosphorylation the Thr-Pro-Tyr motif of SAPK/JNK in wild-type cells be with anti-phospho-Thr-Pro and and Thr but not Tyr phosphorylation was in mkk7−/− cells A and in which phosphorylation of SEK1 was at the same level as wild-type cells and On the other hand, the Tyr phosphorylation of SAPK/JNK was impaired in sek1−/− cells in with (10Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). the Thr phosphorylation was also markedly attenuated in sek1−/− cells this mutant contained the same of MKK7 as wild-type cells These results indicate that SEK1 phosphorylates the Tyr residue of SAPK/JNK and that MKK7 phosphorylates the Thr residue of Tyr-phosphorylated SAPK/JNK its In other Thr phosphorylation requires the phosphorylation of SAPK/JNK at the Tyr residue by SEK1 in the stress-stimulated ES cells In ES cells, SEK1 and MKK7 clearly to the dual phosphorylation of SAPK/JNK in response to stress and the Thr phosphorylation to the Tyr phosphorylation by we further investigated the of SEK1, which was to be in the Tyr phosphorylation of the we used a human embryonic kidney cell for of of into ES cells. HA-tagged JNK1 was with which kinase in the cells using mammalian In a of the the expression of was Thr and Tyr phosphorylation of in response to 1 of UV irradiation was UV irradiation the Thr and Tyr phosphorylation of and not only Tyr but also Thr phosphorylation was in the cells A and These results clearly show that the Thr phosphorylation of SAPK/JNK requires the Tyr phosphorylation by SEK1 in living cells. Next, we investigated the contribution of the Tyr residue in the Thr-Pro-Tyr motif of phosphorylation of which was to the Thr by MKK7 in the sequential phosphorylation. the we constructed three of mutants and in which the Thr motif was with and The mutants and were in 293T cells by and the phosphorylation and kinase were In a of the the expression of was The Thr and Tyr phosphorylation and its to as be in the wild-type Tyr phosphorylation of the mutant was also its kinase was the Thr phosphorylation not be in the mutant, in which the Tyr residue was by These results clearly show that the Thr phosphorylation of SAPK/JNK requires the phosphorylated Tyr residue in living cells. To the molecular mechanism of the Tyr phosphorylation of SAPK/JNK by SEK1, we the of SAPK/JNK with was with and in 293T cells using mammalian (10Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The transfected cells were with the protein The cell were with and for the phosphorylation of SEK1 using anti-phospho-SEK1 and The expression of was The of and expression expression of and but the of the was in of the experiments was phosphorylated in response to in the and of both phosphorylated and of be with was not The between SEK1 and SAPK/JNK to be with that observed between and SAPK/JNK not These results clearly show that SAPK/JNK with SEK1 with MKK7 and that the be for the Tyr phosphorylation of SAPK/JNK by SEK1 We also the is in ES cells that not the and we not in the ES cells or in mouse including and liver not This may be of expression of the SEK1 may or as a with SAPK/JNK in cells, and there may be a molecular mechanism for SEK1 the phosphorylation of been reported in in vitro experiments that synergistic activation of SAPK/JNK requires the phosphorylation of both Thr and Tyr residues the Thr-Pro-Tyr motif by the two activators, SEK1 and MKK7 (7Lawler S. Fleming Y. Goedert M. Cohen P. Curr. Biol. 1998; 8: 1387-1390Abstract Full Text Full Text PDF PubMed Google Scholar, 8Lisnock J. Griffin P. Calaycay J. Franz B. Parsons J. O'Keefe S.J. LoGrasso P. Biochemistry. 2000; 39: 3141-3148Crossref PubMed Scopus (69) Google Scholar, 9Fleming Y. Armstrong C.G. Morrice N. Paterson A. Goedert M. Cohen P. Biochem. J. 2000; 352: 145-154Crossref PubMed Scopus (171) Google Scholar). Although the two are capable of catalyzing both SEK1 prefers the Tyr phosphorylation and MKK7 prefers the Thr phosphorylation in vitro In a study, we reported that SEK1 is required for synergistic activation of SAPK/JNK in ES cells the activation by various was markedly attenuated in sek1−/− ES cells (10Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This was accompanied with a decreased level of the Tyr phosphorylation. In the present study, we also generatedmkk7−/− ES cells and compared the two mutant ES cells in terms of the activation and phosphorylation of Our present results not only the synergistic activation of SAPK/JNK reported previously but also indicate the properties of SEK1 and MKK7 in the phosphorylation of the MAPK as MKK7 to be a for the synergistic activation and the Thr phosphorylation of The ES cells a in synergistic SAPK/JNK activation in response to a variety of stimuli This be by the of MKK7 and but not by SEK1 Thr phosphorylation of SAPK/JNK observed in wild-type cells was in mkk7−/− ES cells also the properties of MKK7, which Thr phosphorylation of to be SEK1, the Thr phosphorylation was impaired in sek1−/− ES cells, which retain MKK7 expression This was by the additional results as of SEK1 by the expression of its dominant-negative (dnSEK1) Thr phosphorylation of SAPK/JNK in addition to Tyr The SAPK/JNK mutant (TPF), which Tyr not be phosphorylated at the Thr SEK1 SAPK/JNK MKK7 and a of SEK1 and SAPK/JNK without MKK7. Thus, we present a activation mechanism that SEK1-induced Tyr phosphorylation of SAPK/JNK is followed by additional Thr phosphorylation by MKK7 in stress-stimulated ES cells In other MKK7 phosphorylates the Thr of Tyr-phosphorylated On the other hand, SEK1 Tyr phosphorylation of the MAPK in a Thr phosphorylation. Tournier have recently reported that the two MKK7 and SEK1, in various activation of SAPK/JNK using isolated from and dual mice C. C. Davis R.J. Dev. 2001; PubMed Scopus Google Scholar). that MKK7 is important SEK1 in the activation of SAPK/JNK by cytokines in SAPK/JNK activation in response to UV and was in but stimulation of SAPK/JNK was in In SAPK/JNK activation in response to and was cells, but stimulation was observed in sek1−/− cells. Thus, results are from observed in the mutant mkk7−/− ES cells, SAPK/JNK activation by various stimuli was These differences may be by the specificity of cell types ES cells were from embryos and in vitro Thus, the molecular mechanism of SAPK/JNK activation in ES cells may be a in mammalian cells. In cells, other cellular as the of may regulate the protein SEK1, MKK7, and SAPK/JNK to the properties of the in the of kinase of or by cDNA not this not be critical to the two phosphorylated of the Thr-Pro-Tyr motif in SAPK/JNK and to the molecular mechanism of synergistic activation of the MAPK in of cells. other of MAPK family, and have also two is activated by and and is by and Cell. Biol. 1999; PubMed Scopus Google Scholar). the of two is a of mammalian Fleming reported that and have a for the phosphorylation of Tyr residue the motif of MAPK Y. Armstrong C.G. Morrice N. Paterson A. Goedert M. Cohen P. Biochem. J. 2000; 352: 145-154Crossref PubMed Scopus (171) Google Scholar). have also biochemical and may be by two as been observed in in other cells be required for the of the functional of two activators in and have the that SAPK/JNK in as a which into the to and to stimuli Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar, Biochem. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar, Biochem. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar). have shown in that SAPK/JNK to and as and in an Curr. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The activation of SAPK/JNK by the stimuli was at the level of a of at the level of an the response to be In the present study, we have also observed a response in the activation of SAPK/JNK by stress in ES cells Furthermore, as described in the that both SEK1 and MKK7 are required for SAPK/JNK activation and hepatoblast proliferation in developing mice (6Watanabe T. Nakagawa K. Ohata S. Kitagawa D. Nishitai G. Seo J. Tanemura S. Shimizu N. Kishimoto H. Wada T. Aoki J. Arai H. Iwatsubo T. Mochita M. Watanabe T. Satake M. Ito Y. Matsuyama T. Mak T.W. Penninger J.M. Nishina H. Katada T. Dev. Biol. 2002; 250: 332-347Crossref PubMed Google Scholar). This that the MAPK activation also in mammalian cells at an cell level only the two are this MAPK without the two of which SEK1 and the other MKK7. Although the molecular mechanism the two are by various stress signals to be is to that the of the two to SAPK/JNK activation may as a mechanism as previously (10Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).

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