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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
. 1994 Sep 13;91(19):8960–8963. doi: 10.1073/pnas.91.19.8960

Constitutive activation of Mek1 by mutation of serine phosphorylation sites.

W Huang 1, R L Erikson 1
PMCID: PMC44726  PMID: 8090753

Abstract

A variety of extracellular signals lead to the phosphorylation and activation of mitogen-activated protein kinases (MAP kinases). An activator of MAP kinases, Mek1, phosphorylates MAP kinases at threonine and tyrosine residues and is itself phosphorylated at serine-218 and -222 by the protooncogene product Raf-1. By introducing negatively charged residues that may mimic the effect of phosphorylation at positions 218 and 222, we have activated the capacity of Mek1 to phosphorylate MAP kinase by > 100-fold. The most effective activation by a single substitution resulted from the introduction of aspartate at position 218, whereas the introduction of either aspartate or glutamate at position 222 was ineffective. Expression of the activated Mek1 phosphorylation-site mutants in COS-7 cells led to the activation of MAP kinase in the cells and resulted in an increase in the mass of the transfected COS-7 cell population, suggesting an important role of Mek1 in the transduction of mitogenic signals.

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Selected References

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  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. Alessi D. R., Saito Y., Campbell D. G., Cohen P., Sithanandam G., Rapp U., Ashworth A., Marshall C. J., Cowley S. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J. 1994 Apr 1;13(7):1610–1619. doi: 10.1002/j.1460-2075.1994.tb06424.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson N. G., Maller J. L., Tonks N. K., Sturgill T. W. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature. 1990 Feb 15;343(6259):651–653. doi: 10.1038/343651a0. [DOI] [PubMed] [Google Scholar]
  5. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5889–5892. doi: 10.1073/pnas.90.13.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boulton T. G., Nye S. H., Robbins D. J., Ip N. Y., Radziejewska E., Morgenbesser S. D., DePinho R. A., Panayotatos N., Cobb M. H., Yancopoulos G. D. ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell. 1991 May 17;65(4):663–675. doi: 10.1016/0092-8674(91)90098-j. [DOI] [PubMed] [Google Scholar]
  7. Brott B. K., Alessandrini A., Largaespada D. A., Copeland N. G., Jenkins N. A., Crews C. M., Erikson R. L. MEK2 is a kinase related to MEK1 and is differentially expressed in murine tissues. Cell Growth Differ. 1993 Nov;4(11):921–929. [PubMed] [Google Scholar]
  8. Cowley S., Paterson H., Kemp P., Marshall C. J. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell. 1994 Jun 17;77(6):841–852. doi: 10.1016/0092-8674(94)90133-3. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  13. 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]
  14. Errede B., Levin D. E. A conserved kinase cascade for MAP kinase activation in yeast. Curr Opin Cell Biol. 1993 Apr;5(2):254–260. doi: 10.1016/0955-0674(93)90112-4. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Matsuda S., Kosako H., Takenaka K., Moriyama K., Sakai H., Akiyama T., Gotoh Y., Nishida E. Xenopus MAP kinase activator: identification and function as a key intermediate in the phosphorylation cascade. EMBO J. 1992 Mar;11(3):973–982. doi: 10.1002/j.1460-2075.1992.tb05136.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neiman A. M., Herskowitz I. Reconstitution of a yeast protein kinase cascade in vitro: activation of the yeast MEK homologue STE7 by STE11. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3398–3402. doi: 10.1073/pnas.91.8.3398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rossomando A., Wu J., Weber M. J., Sturgill T. W. The phorbol ester-dependent activator of the mitogen-activated protein kinase p42mapk is a kinase with specificity for the threonine and tyrosine regulatory sites. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5221–5225. doi: 10.1073/pnas.89.12.5221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Sarcevic B., Erikson E., Maller J. L. Purification and characterization of a mitogen-activated protein kinase tyrosine phosphatase from Xenopus eggs. J Biol Chem. 1993 Nov 25;268(33):25075–25083. [PubMed] [Google Scholar]
  25. 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]
  26. Sun H., Charles C. H., Lau L. F., Tonks N. K. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell. 1993 Nov 5;75(3):487–493. doi: 10.1016/0092-8674(93)90383-2. [DOI] [PubMed] [Google Scholar]
  27. Ward Y., Gupta S., Jensen P., Wartmann M., Davis R. J., Kelly K. Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1. Nature. 1994 Feb 17;367(6464):651–654. doi: 10.1038/367651a0. [DOI] [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]
  29. Wu J., Michel H., Rossomando A., Haystead T., Shabanowitz J., Hunt D. F., Sturgill T. W. Renaturation and partial peptide sequencing of mitogen-activated protein kinase (MAP kinase) activator from rabbit skeletal muscle. Biochem J. 1992 Aug 1;285(Pt 3):701–705. doi: 10.1042/bj2850701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zheng C. F., Guan K. L. Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. EMBO J. 1994 Mar 1;13(5):1123–1131. doi: 10.1002/j.1460-2075.1994.tb06361.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zheng C. F., Guan K. L. Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2. J Biol Chem. 1993 May 25;268(15):11435–11439. [PubMed] [Google Scholar]
  32. Zheng C. F., Guan K. L. Dephosphorylation and inactivation of the mitogen-activated protein kinase by a mitogen-induced Thr/Tyr protein phosphatase. J Biol Chem. 1993 Aug 5;268(22):16116–16119. [PubMed] [Google Scholar]

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