Nature 358, 417 - 421 (30 July 1992); doi:10.1038/358417a0

Raf-1 activates MAP kinase-kinase

John M. Kyriakis, Harald App^*, Xian-feng Zhang, Papia Banerjee, David L. Brautigan^†, Ulf R. Rapp^* & Joseph Avruch

Diabetes Unit, Medical Services, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, and Department of Medicine, Harvard Medical School, Boston,Massachusetts 02114, USA
^* Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA
^† Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA

THE normal cellular homologue of the acutely transforming oncogene v-ra/is c-raf-l, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli¹. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases ErkI and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K)^2–7. Here we report that MAPK-K as well as Erkl and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells³ can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M _r 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-l protooncogene product to be identified.

References

1.	Rapp, U. R. Oncogene 6, 495−500 (1991). | PubMed | ChemPort |
2.	Ahn, N. G., Weiel, J. E., Chan, C. P. & Krebs, E. G. J. biol. Chem. 265, 11487−11494 (1990). | PubMed | ChemPort |
3.	Kyriakis, J. M., Brautigan, D. L., Ingelbritsen, T. S. & Avruch, J. J. biol. Chem. 266, 10043−10046 (1991). | PubMed | ChemPort |
4.	Anderson, N. G., Maller, J. L., Tonks, N. K. & Sturgill, T. W. Nature 343, 651−653 (1990). | Article | PubMed | ISI | ChemPort |
5.	Ahn, N. G., et al. J. biol. Chem. 266, 4220−4227 (1991). | PubMed | ISI | ChemPort |
6.	Gomez, N. & Cohen, P. Nature 353, 170−173 (1991). | Article | PubMed | ISI | ChemPort |
7.	Allemain, G. L., Her, J.-H., Wu, J., Sturgill, T. W. & Weber, M. J. Molec. cell. Biol. 12, 2222−2229 (1992). | PubMed |
8.	Rapp, U. R., Reynolds, F. H. & Stephenson, J. R. J. Virol. 45, 914−924 (1983). | PubMed | ISI | ChemPort |
9.	Matsuda, S. et al. EMBO J. 11, 973−982 (1992). | PubMed | ISI | ChemPort |
10.	Pulverer, B. J., et al. Nature 353, 670−674 (1991). | Article | PubMed | ISI | ChemPort |
11.	Sturgill, T. W., Ray, L. B., Erikson, E. & Maller, J. L. Nature 334, 715−718 (1988). | Article | PubMed | ISI | ChemPort |
12.	Dent, P. et al. Nature 348, 302−308 (1990). | Article | PubMed | ISI | ChemPort |
13.	Anderson, N. G. et al. Biochem. J. 277, 573−576 (1991). | PubMed | ChemPort |
14.	Lee, R.-M., Cobb, M. H. & Blackshear, P. J. J. biol. Chem. 267, 1088−1092 (1992). | PubMed | ISI | ChemPort |
15.	Downward, J. et al. Nature 346, 719−723 (1990). | Article | PubMed | ISI | ChemPort |
16.	Burgering, B. M. et al. EMBO J. 10, 1103−1109 (1991). | PubMed | ISI | ChemPort |
17.	Satoh, T., Nakafuku, M., Miyajima, A. & Kaziro, Y. Proc natn. Acad. Sci. U.S.A. 88, 3314−3318 (1991). | ChemPort |
18.	Leevers, S. J. & Marshall, C. J. EMBO J. 11, 569−574 (1992). | PubMed | ISI | ChemPort |
19.	Bruder, J. T., Heidecker, G. & Rapp, U. R. Genes Dev. 6, 545−556 (1992). | PubMed | ISI | ChemPort |
20.	Troppmair, J. et al. Oncogene (in the press).
21.	Grove, J. R., Price, D. J., Goodman, H. M. & Avruch, J. Science 238, 530−533 (1987). | PubMed | ChemPort |
22.	Hsi, E. D. et al. J. Biol. Chem. 264, 10836−10842 (1989). | PubMed | ISI | ChemPort |
23.	Kyriakis, J. M. & Avruch, J. J. biol. Chem. 265, 17355−17363 (1990). | PubMed | ISI | ChemPort |
24.	Takeda, M. et al. J. biol. Chem. 258, 10455−10463 (1988).
25.	Jones, S. W., Erikson, R. L., Ingebritsen, V. M. & Ingebritsen, T. S. J. biol. Chem. 264, 7747−7753 (1989). | PubMed | ChemPort |
26.	Kyriakis, J. M. & Avruch, J. Biochem. Biophys. Acta 1054, 73−82 (1990). | PubMed | ChemPort |