ARTICLES NATURE CELL BIOLOGY VOLUME 6 | NUMBER 3 | MARCH 2004 215 MKK7 couples stress signalling to G2/M cell-cycle progression and cellular senescence Teiji Wada 1, 2 , Nicholas Joza 1, 2 , Hai-ying M. Cheng 2 , Takehiko Sasaki 2 , Ivona Kozieradzki 2 , Kurt Bachmaier 2 , Toshiaki Katada 3 , Martin Schreiber 4 , Erwin F.Wagner 5 , Hiroshi Nishina 3 and Josef M. Penninger 1,2,6 During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4–5, mkk7 −/− mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7–JNK–c-Jun pathway. These data show that the MKK7–JNK–c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that transduce signals from the cell mem- brane to the nucleus in response to a wide range of stimuli 1−7 . Two inde- pendent stress kinase signalling pathways — p38-MAPK and JNKs (c-Jun N-terminal kinase or SAPKs, stress-activated protein kinases) — partici- pate in many different intracellular signalling pathways that control a spectrum of cellular processes, including cell growth, differentiation, transformation and apoptosis 1−7 . Both JNK-activation kinases — MKK4 and MKK7 — are required for full activation of JNK 8−10 , and genetic inactivation of mkk4 results in embryonic lethality with a liver defect between embryonic day 10.5 (E10.5) and E12.5 (ref. 11). However, nothing is known about the role of MKK7 in embryogenesis 12 . Here we show that genetic inactivation of MKK7 in mice results in defective hepatocyte proliferation and embryonic lethality. Inactivation of MKK7 in embryonic fibroblasts results in impaired proliferation, a G2/M cell-cycle arrest and premature senescence. The G2/M cell-cycle kinase CDC2 was identified as a molecular target for MKK7–JNK sig- nalling. Loss of c-Jun or a expression of c-JunAA mutant in which the JNK phosphorylation sites were inactivates 13,14 also resulted in defec- tive G2/M cell cycle progression and impaired CDC2 expression. 1 IMBA (Institute of Molecular Biotechnology of the Austrian Academy of Sciences), c/o Dr. Bohrgasse 3-5, A-1030 Vienna, Austria. 2 University Health Network, Departments of Medical Biophysics and Immunology, University of Toronto, 620 University Avenue, Ontario M5G 2C1, Toronto, Canada. 3 Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033 Tokyo, Japan. 4 University of Vienna School of Medicine, Dept of Obstetrics and Gynecology Waehringer Guertel 18-20, A-1090 Vienna, Austria. 5 IMP (Research Institute of Molecular Pathology), Dr. Bohrgasse 7, A-1030 Vienna, Austria. 6 Correspondence should be addressed to J.P. (e-mail: josef.penninger@oeaw.ac.at) Published online: 22 February 2004; DOI: 10.1038/ncb1098 Table 1 Embronic lethality in mkk7 –/– mice Litters mkk7 +/+ mkk7 +/– mkk7 –/– Dead (%) Total E9.5 2 4 11 2 0 (0) 17 E10.5 2 5 8 5 0 (0) 18 E11.5 26 55 106 47 15 (31.1) 208 E12.5 16 31 67 33 30 (90.9) 131 E13.5 5 11 17 12 12 (100) 40 E16.5 1 3 4 0 – 7 1W 5 9 21 0 – 39 Embryos were isolated at the indicated time of gestation, analysed for viability and processed for histological staining. Genotypes of embryos were determined by PCR. Viability of embryos was determined by observing heart beating. E = embryonic day; 1W = 1 week after birth. ©2004 Nature Publishing Group