Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Dec 1;90(23):10947–10951. doi: 10.1073/pnas.90.23.10947

Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation.

W Huang 1, A Alessandrini 1, C M Crews 1, R L Erikson 1
PMCID: PMC47898  PMID: 8248196

Abstract

Recombinant Mek1 and Raf-1 proteins produced in Sf9 cells undergo a tight association both in vivo and in vitro, which apparently does not depend on additional factors or the kinase activity of Mek1 or Raf-1. The complex can be disrupted by two polyclonal antibodies raised against Raf-1 peptides. Coinfection with Raf-1 activates Mek1 > 150-fold, and coinfection with Raf-1 and Mek1 activates Erk1 approximately 90-fold. The activation of Mek1 by Raf-1 involves only serine phosphorylation, which is directly proportional to the extent of Mek1 activation. Phosphopeptide maps suggest a single Raf-1 phosphorylation site on mek1.

Full text

PDF
10950

Images in this article

10948Fig. 1
on p.10948
10949Fig. 2
on p.10949
10949Fig. 3
on p.10949
10949Fig. 4
on p.10949
10950Fig. 5
on p.10950

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahn N. G., Campbell J. S., Seger R., Jensen A. L., Graves L. M., Krebs E. G. Metabolic labeling of mitogen-activated protein kinase kinase in A431 cells demonstrates phosphorylation on serine and threonine residues. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5143–5147. doi: 10.1073/pnas.90.11.5143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alessandrini A., Crews C. M., Erikson R. L. Phorbol ester stimulates a protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8200–8204. doi: 10.1073/pnas.89.17.8200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Crews C. M., Alessandrini A. A., Erikson R. L. Mouse Erk-1 gene product is a serine/threonine protein kinase that has the potential to phosphorylate tyrosine. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8845–8849. doi: 10.1073/pnas.88.19.8845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Crews C. M., Alessandrini A., Erikson R. L. Erks: their fifteen minutes has arrived. Cell Growth Differ. 1992 Feb;3(2):135–142. [PubMed] [Google Scholar]
  5. Crews C. M., Alessandrini A., Erikson R. L. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science. 1992 Oct 16;258(5081):478–480. doi: 10.1126/science.1411546. [DOI] [PubMed] [Google Scholar]
  6. Crews C. M., Erikson R. L. Purification of a murine protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product: relationship to the fission yeast byr1 gene product. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8205–8209. doi: 10.1073/pnas.89.17.8205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dent P., Haser W., Haystead T. A., Vincent L. A., Roberts T. M., Sturgill T. W. Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science. 1992 Sep 4;257(5075):1404–1407. doi: 10.1126/science.1326789. [DOI] [PubMed] [Google Scholar]
  8. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  9. Grussenmeyer T., Scheidtmann K. H., Hutchinson M. A., Eckhart W., Walter G. Complexes of polyoma virus medium T antigen and cellular proteins. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7952–7954. doi: 10.1073/pnas.82.23.7952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gómez N., Cohen P. Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature. 1991 Sep 12;353(6340):170–173. doi: 10.1038/353170a0. [DOI] [PubMed] [Google Scholar]
  11. Haystead T. A., Dent P., Wu J., Haystead C. M., Sturgill T. W. Ordered phosphorylation of p42mapk by MAP kinase kinase. FEBS Lett. 1992 Jul 13;306(1):17–22. doi: 10.1016/0014-5793(92)80828-5. [DOI] [PubMed] [Google Scholar]
  12. Howe L. R., Leevers S. J., Gómez N., Nakielny S., Cohen P., Marshall C. J. Activation of the MAP kinase pathway by the protein kinase raf. Cell. 1992 Oct 16;71(2):335–342. doi: 10.1016/0092-8674(92)90361-f. [DOI] [PubMed] [Google Scholar]
  13. Kosako H., Gotoh Y., Matsuda S., Ishikawa M., Nishida E. Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation. EMBO J. 1992 Aug;11(8):2903–2908. doi: 10.1002/j.1460-2075.1992.tb05359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kosako H., Nishida E., Gotoh Y. cDNA cloning of MAP kinase kinase reveals kinase cascade pathways in yeasts to vertebrates. EMBO J. 1993 Feb;12(2):787–794. doi: 10.1002/j.1460-2075.1993.tb05713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kyriakis J. M., App H., Zhang X. F., Banerjee P., Brautigan D. L., Rapp U. R., Avruch J. Raf-1 activates MAP kinase-kinase. Nature. 1992 Jul 30;358(6385):417–421. doi: 10.1038/358417a0. [DOI] [PubMed] [Google Scholar]
  16. Lange-Carter C. A., Pleiman C. M., Gardner A. M., Blumer K. J., Johnson G. L. A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science. 1993 Apr 16;260(5106):315–319. doi: 10.1126/science.8385802. [DOI] [PubMed] [Google Scholar]
  17. Matsuda S., Gotoh Y., Nishida E. Phosphorylation of Xenopus mitogen-activated protein (MAP) kinase kinase by MAP kinase kinase kinase and MAP kinase. J Biol Chem. 1993 Feb 15;268(5):3277–3281. [PubMed] [Google Scholar]
  18. Nishida E., Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993 Apr;18(4):128–131. doi: 10.1016/0968-0004(93)90019-j. [DOI] [PubMed] [Google Scholar]
  19. Payne D. M., Rossomando A. J., Martino P., Erickson A. K., Her J. H., Shabanowitz J., Hunt D. F., Weber M. J., Sturgill T. W. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 1991 Apr;10(4):885–892. doi: 10.1002/j.1460-2075.1991.tb08021.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pelech S. L., Sanghera J. S. MAP kinases: charting the regulatory pathways. Science. 1992 Sep 4;257(5075):1355–1356. doi: 10.1126/science.1382311. [DOI] [PubMed] [Google Scholar]
  21. Rubinfeld B., Munemitsu S., Clark R., Conroy L., Watt K., Crosier W. J., McCormick F., Polakis P. Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1. Cell. 1991 Jun 14;65(6):1033–1042. doi: 10.1016/0092-8674(91)90555-d. [DOI] [PubMed] [Google Scholar]
  22. Schneider C., Newman R. A., Sutherland D. R., Asser U., Greaves M. F. A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol Chem. 1982 Sep 25;257(18):10766–10769. [PubMed] [Google Scholar]
  23. Seger R., Seger D., Lozeman F. J., Ahn N. G., Graves L. M., Campbell J. S., Ericsson L., Harrylock M., Jensen A. M., Krebs E. G. Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases. J Biol Chem. 1992 Dec 25;267(36):25628–25631. [PubMed] [Google Scholar]
  24. Thomas G. MAP kinase by any other name smells just as sweet. Cell. 1992 Jan 10;68(1):3–6. doi: 10.1016/0092-8674(92)90199-m. [DOI] [PubMed] [Google Scholar]
  25. Tsuda L., Inoue Y. H., Yoo M. A., Mizuno M., Hata M., Lim Y. M., Adachi-Yamada T., Ryo H., Masamune Y., Nishida Y. A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila. Cell. 1993 Feb 12;72(3):407–414. doi: 10.1016/0092-8674(93)90117-9. [DOI] [PubMed] [Google Scholar]
  26. Van Aelst L., Barr M., Marcus S., Polverino A., Wigler M. Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6213–6217. doi: 10.1073/pnas.90.13.6213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Williams N. G., Paradis H., Agarwal S., Charest D. L., Pelech S. L., Roberts T. M. Raf-1 and p21v-ras cooperate in the activation of mitogen-activated protein kinase. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5772–5776. doi: 10.1073/pnas.90.12.5772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wu J., Harrison J. K., Vincent L. A., Haystead C., Haystead T. A., Michel H., Hunt D. F., Lynch K. R., Sturgill T. W. Molecular structure of a protein-tyrosine/threonine kinase activating p42 mitogen-activated protein (MAP) kinase: MAP kinase kinase. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):173–177. doi: 10.1073/pnas.90.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES