Impaired Synergistic Activation of Stress-activated Protein Kinase SAPK/JNK in Mouse Embryonic Stem Cells Lacking SEK1/MKK4

Stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) family, plays an important role in a stress-induced signaling cascade. SAPK/JNK activation requires the phosphorylation of Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 (MKK4) and MKK7 (SEK2) have been identified as the upstream MAPK kinases. Here we examined the activation and phosphorylation sites of SAPK/JNK and differentiated the contribution of SEK1 and MKK7α1, -γ1, and -γ2 isoforms to the MAPK activation. In SEK1-deficient mouse embryonic stem cells, stress-induced SAPK/JNK activation was markedly impaired, and this defect was accompanied with a decreased level of the Tyr phosphorylation. Analysis in HeLa cells co-transfected with the two MAPK kinases revealed that the Thr and Tyr of SAPK/JNK were independently phosphorylated in response to heat shock by MKK7γ1 and SEK1, respectively. However, MKK7α1 failed to phosphorylate the Thr of SAPK/JNK unless its Tyr residue was phosphorylated by SEK1. In contrast, MKK7γ2 had the ability to phosphorylate both Thr and Tyr residues. In all cases, the dual phosphorylation of the Thr and Tyr residues was essentially required for the full activation of SAPK/JNK. These data provide the first evidence that synergistic activation of SAPK/JNK requires both phosphorylation at the Thr and Tyr residues in living cells and that the preference for the Thr and Tyr phosphorylation was different among the members of MAPK kinases.

[1]  R. Davis,et al.  Signal Transduction by the JNK Group of MAP Kinases , 2000, Cell.

[2]  Y. Kaziro,et al.  Differential Regulation of Mitogen-activated Protein Kinase Kinase 4 (MKK4) and 7 (MKK7) by Signaling from G Protein βγ Subunit in Human Embryonal Kidney 293 Cells* , 1999, The Journal of Biological Chemistry.

[3]  A. Lin,et al.  Identification of c-Jun NH2-terminal Protein Kinase (JNK)-activating Kinase 2 as an Activator of JNK but Not p38* , 1997, The Journal of Biological Chemistry.

[4]  P. Cohen,et al.  Synergistic activation of SAPK1/JNK1 by two MAP kinase kinases in vitro , 1998, Current Biology.

[5]  T. Mak,et al.  Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3 , 1997, Nature.

[6]  L. Zon,et al.  Protein-Damaging Stresses Activate c-Jun N-Terminal Kinase via Inhibition of Its Dephosphorylation: a Novel Pathway Controlled by HSP72 , 1999, Molecular and Cellular Biology.

[7]  E. Nishida,et al.  A novel SAPK/JNK kinase, MKK7, stimulated by TNFα and cellular stresses , 1997, The EMBO journal.

[8]  R. Flavell,et al.  JNK is required for effector T-cell function but not for T-cell activation , 2000, Nature.

[9]  H. Nishina,et al.  Activation of Stress-activated Protein Kinases/c-Jun N-terminal Protein Kinases (SAPKs/JNKs) by a Novel Mitogen-activated Protein Kinase Kinase (MKK7)* , 1997, The Journal of Biological Chemistry.

[10]  P. Griffin,et al.  Activation of JNK3 alpha 1 requires both MKK4 and MKK7: kinetic characterization of in vitro phosphorylated JNK3 alpha 1. , 2000, Biochemistry.

[11]  M. Karin,et al.  Molecular cloning and characterization of human JNKK2, a novel Jun NH2-terminal kinase-specific kinase , 1997, Molecular and cellular biology.

[12]  R. Flavell,et al.  Targeted disruption of the MKK4 gene causes embryonic death, inhibition of c-Jun NH2-terminal kinase activation, and defects in AP-1 transcriptional activity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Davis,et al.  Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[14]  C. Tournier,et al.  The MKK7 Gene Encodes a Group of c-Jun NH2-Terminal Kinase Kinases , 1999, Molecular and Cellular Biology.

[15]  T. Mak,et al.  Impaired CD28-mediated Interleukin 2 Production and Proliferation in Stress Kinase SAPK/ERK1 Kinase (SEK1)/Mitogen-activated Protein Kinase Kinase 4 (MKK4)-deficient T Lymphocytes , 1997, The Journal of experimental medicine.

[16]  P. Cohen,et al.  Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7. , 2000, The Biochemical journal.

[17]  L. Zon,et al.  SEK1 deficiency reveals mitogen-activated protein kinase cascade crossregulation and leads to abnormal hepatogenesis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[18]  S. Noselli,et al.  MKK7 Is A Stress-activated Mitogen-activated Protein Kinase Kinase Functionally Related to hemipterous * , 1997, The Journal of Biological Chemistry.

[19]  Philip R. Cohen,et al.  SKK4, a novel activator of stress‐activated protein kinase‐1 (SAPK1/JNK) , 1997, FEBS letters.

[20]  T. Sasaki,et al.  Defective liver formation and liver cell apoptosis in mice lacking the stress signaling kinase SEK1/MKK4. , 1999, Development.