Skip to main content
PMC home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1995 Mar;6(3):237–245. doi: 10.1091/mbc.6.3.237

Biochemical and biological analysis of Mek1 phosphorylation site mutants.

W Huang 1, D S Kessler 1, R L Erikson 1
PMCID: PMC301184  PMID: 7612960

Abstract

Recently, we described the constitutive activation of Mek1 by mutation of its two serine phosphorylation sites. We have now characterized the biochemical properties of these Mek1 mutants and performed microinjection experiments to investigate the effect of an activated Mek on oocyte maturation. Single acidic substitution of either serine 218 or 222 activated Mek1 by 10-50 fold. The double acidic substitutions, [Asp218, Asp222] and [Asp218, Glu222], activated Mek1 over 6000-fold. The specific activity of the [Asp218, Asp222] and [Asp218, Glu222] Mek1 mutants, 29 nanomole phosphate per minute per milligram, is similar to that of wild-type Mek1 activated by Raf-1 in vitro. Although the mutants with double acidic substitutions could not be further activated by Raf-1, three of those with single acidic substitution were activated by Raf-1 to the specific activity of activated wild-type Mek1. Injection of the [Asp218, Asp222] Mek1 mutant into Xenopus oocytes activated both MAP kinase and histone H1 kinase and induced germinal vesicle breakdown, an effect that was only partially blocked by inhibition of protein synthesis. These data provide a measure of Mek's potential to influence cell functions and a quantitative basis to assess the biological effects of Mek1 mutants in a variety of circumstances.

Full text

PDF
240

Images in this article

241Image
on p.241
241Image
on p.241

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. 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. Barrett C. B., Erikson E., Maller J. L. A purified S6 kinase kinase from Xenopus eggs activates S6 kinase II and autophosphorylates on serine, threonine, and tyrosine residues. J Biol Chem. 1992 Mar 5;267(7):4408–4415. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Brewster J. L., de Valoir T., Dwyer N. D., Winter E., Gustin M. C. An osmosensing signal transduction pathway in yeast. Science. 1993 Mar 19;259(5102):1760–1763. doi: 10.1126/science.7681220. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Brown A. J., Jones T., Shuttleworth J. Expression and activity of p40MO15, the catalytic subunit of cdk-activating kinase, during Xenopus oogenesis and embryogenesis. Mol Biol Cell. 1994 Aug;5(8):921–932. doi: 10.1091/mbc.5.8.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cairns B. R., Ramer S. W., Kornberg R. D. Order of action of components in the yeast pheromone response pathway revealed with a dominant allele of the STE11 kinase and the multiple phosphorylation of the STE7 kinase. Genes Dev. 1992 Jul;6(7):1305–1318. doi: 10.1101/gad.6.7.1305. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  16. 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]
  17. Dérijard B., Hibi M., Wu I. H., Barrett T., Su B., Deng T., Karin M., Davis R. J. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 1994 Mar 25;76(6):1025–1037. doi: 10.1016/0092-8674(94)90380-8. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Fabian J. R., Daar I. O., Morrison D. K. Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase. Mol Cell Biol. 1993 Nov;13(11):7170–7179. doi: 10.1128/mcb.13.11.7170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fabian J. R., Morrison D. K., Daar I. O. Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes. J Cell Biol. 1993 Aug;122(3):645–652. doi: 10.1083/jcb.122.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fesquet D., Labbé J. C., Derancourt J., Capony J. P., Galas S., Girard F., Lorca T., Shuttleworth J., Dorée M., Cavadore J. C. The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues. EMBO J. 1993 Aug;12(8):3111–3121. doi: 10.1002/j.1460-2075.1993.tb05980.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Fisher R. P., Morgan D. O. A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Cell. 1994 Aug 26;78(4):713–724. doi: 10.1016/0092-8674(94)90535-5. [DOI] [PubMed] [Google Scholar]
  24. Force T., Bonventre J. V., Heidecker G., Rapp U., Avruch J., Kyriakis J. M. Enzymatic characteristics of the c-Raf-1 protein kinase. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1270–1274. doi: 10.1073/pnas.91.4.1270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Galcheva-Gargova Z., Dérijard B., Wu I. H., Davis R. J. An osmosensing signal transduction pathway in mammalian cells. Science. 1994 Aug 5;265(5173):806–808. doi: 10.1126/science.8047888. [DOI] [PubMed] [Google Scholar]
  26. Gonzalez F. A., Raden D. L., Davis R. J. Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases. J Biol Chem. 1991 Nov 25;266(33):22159–22163. [PubMed] [Google Scholar]
  27. Gotoh Y., Nishida E., Matsuda S., Shiina N., Kosako H., Shiokawa K., Akiyama T., Ohta K., Sakai H. In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature. 1991 Jan 17;349(6306):251–254. doi: 10.1038/349251a0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Han J., Lee J. D., Bibbs L., Ulevitch R. J. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science. 1994 Aug 5;265(5173):808–811. doi: 10.1126/science.7914033. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Huang W., Alessandrini A., Crews C. M., Erikson R. L. Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10947–10951. doi: 10.1073/pnas.90.23.10947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Huang W., Erikson R. L. Constitutive activation of Mek1 by mutation of serine phosphorylation sites. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8960–8963. doi: 10.1073/pnas.91.19.8960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kosako H., Gotoh Y., Nishida E. Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation. EMBO J. 1994 May 1;13(9):2131–2138. doi: 10.1002/j.1460-2075.1994.tb06489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Lohka M. J., Hayes M. K., Maller J. L. Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc Natl Acad Sci U S A. 1988 May;85(9):3009–3013. doi: 10.1073/pnas.85.9.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mansour S. J., Matten W. T., Hermann A. S., Candia J. M., Rong S., Fukasawa K., Vande Woude G. F., Ahn N. G. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science. 1994 Aug 12;265(5174):966–970. doi: 10.1126/science.8052857. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Muslin A. J., MacNicol A. M., Williams L. T. Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos. Mol Cell Biol. 1993 Jul;13(7):4197–4202. doi: 10.1128/mcb.13.7.4197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mäkelä T. P., Tassan J. P., Nigg E. A., Frutiger S., Hughes G. J., Weinberg R. A. A cyclin associated with the CDK-activating kinase MO15. Nature. 1994 Sep 15;371(6494):254–257. doi: 10.1038/371254a0. [DOI] [PubMed] [Google Scholar]
  43. Nadin-Davis S. A., Nasim A. A gene which encodes a predicted protein kinase can restore some functions of the ras gene in fission yeast. EMBO J. 1988 Apr;7(4):985–993. doi: 10.1002/j.1460-2075.1988.tb02905.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Poon R. Y., Yamashita K., Adamczewski J. P., Hunt T., Shuttleworth J. The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2. EMBO J. 1993 Aug;12(8):3123–3132. doi: 10.1002/j.1460-2075.1993.tb05981.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Posada J., Yew N., Ahn N. G., Vande Woude G. F., Cooper J. A. Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro. Mol Cell Biol. 1993 Apr;13(4):2546–2553. doi: 10.1128/mcb.13.4.2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Sagata N., Oskarsson M., Copeland T., Brumbaugh J., Vande Woude G. F. Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes. Nature. 1988 Oct 6;335(6190):519–525. doi: 10.1038/335519a0. [DOI] [PubMed] [Google Scholar]
  49. Schorderet-Slatkine S. Action of progesterone and related steroids on oocyte maturation in Xenopus laevis. An in vitro study. Cell Differ. 1972 Aug;1(3):179–189. doi: 10.1016/0045-6039(72)90027-9. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Shibuya E. K., Ruderman J. V. Mos induces the in vitro activation of mitogen-activated protein kinases in lysates of frog oocytes and mammalian somatic cells. Mol Biol Cell. 1993 Aug;4(8):781–790. doi: 10.1091/mbc.4.8.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Solomon M. J., Harper J. W., Shuttleworth J. CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15. EMBO J. 1993 Aug;12(8):3133–3142. doi: 10.1002/j.1460-2075.1993.tb05982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Teague M. A., Chaleff D. T., Errede B. Nucleotide sequence of the yeast regulatory gene STE7 predicts a protein homologous to protein kinases. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7371–7375. doi: 10.1073/pnas.83.19.7371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  55. Wang Y., Xu H. P., Riggs M., Rodgers L., Wigler M. byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype. Mol Cell Biol. 1991 Jul;11(7):3554–3563. doi: 10.1128/mcb.11.7.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Warne P. H., Viciana P. R., Downward J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature. 1993 Jul 22;364(6435):352–355. doi: 10.1038/364352a0. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Yew N., Mellini M. L., Vande Woude G. F. Meiotic initiation by the mos protein in Xenopus. Nature. 1992 Feb 13;355(6361):649–652. doi: 10.1038/355649a0. [DOI] [PubMed] [Google Scholar]
  59. Zhang X. F., Settleman J., Kyriakis J. M., Takeuchi-Suzuki E., Elledge S. J., Marshall M. S., Bruder J. T., Rapp U. R., Avruch J. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature. 1993 Jul 22;364(6435):308–313. doi: 10.1038/364308a0. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. 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]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES