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. 1993 Feb;12(2):787–794. doi: 10.1002/j.1460-2075.1993.tb05713.x

cDNA cloning of MAP kinase kinase reveals kinase cascade pathways in yeasts to vertebrates.

H Kosako 1, E Nishida 1, Y Gotoh 1
PMCID: PMC413266  PMID: 8440264

Abstract

A Xenopus 45 kDa protein has been identified as an immediate upstream factor sufficient for full activation of MAP kinase, and is shown to be capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. In this study, we show that purified 45 kDa protein can phosphorylate a kinase-negative mutant of Xenopus MAP kinase on tyrosine and threonine residues, suggesting that the 45 kDa protein functions as a MAP kinase kinase to activate MAP kinase. We then report the cloning and sequencing of a full-length cDNA encoding this 45 kDa MAP kinase kinase, and show that it is highly homologous to four protein kinases in fission and budding yeasts: byr1, wis1, PBS2 and STE7. These yeast kinases are therefore suggested to function as a direct upstream activator for a presumed MAP kinase homolog in each signal transduction pathway involved in the regulation of cell cycle progression or cellular responses to extracellular signals. Finally, we report bacterial expression of recombinant MAP kinase kinase that can be phosphorylated and activated by Xenopus egg extracts.

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Selected References

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  1. Ahn N. G., Seger R., Bratlien R. L., Diltz C. D., Tonks N. K., Krebs E. G. Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase. J Biol Chem. 1991 Mar 5;266(7):4220–4227. [PubMed] [Google Scholar]
  2. Alvarez E., Northwood I. C., Gonzalez F. A., Latour D. A., Seth A., Abate C., Curran T., Davis R. J. Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase. J Biol Chem. 1991 Aug 15;266(23):15277–15285. [PubMed] [Google Scholar]
  3. 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]
  4. Boguslawski G., Polazzi J. O. Complete nucleotide sequence of a gene conferring polymyxin B resistance on yeast: similarity of the predicted polypeptide to protein kinases. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5848–5852. doi: 10.1073/pnas.84.16.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Cobb M. H., Boulton T. G., Robbins D. J. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 1991 Dec;2(12):965–978. doi: 10.1091/mbc.2.12.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Courchesne W. E., Kunisawa R., Thorner J. A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae. Cell. 1989 Sep 22;58(6):1107–1119. doi: 10.1016/0092-8674(89)90509-6. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Elion E. A., Grisafi P. L., Fink G. R. FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation. Cell. 1990 Feb 23;60(4):649–664. doi: 10.1016/0092-8674(90)90668-5. [DOI] [PubMed] [Google Scholar]
  11. Fukui Y., Kaziro Y. Molecular cloning and sequence analysis of a ras gene from Schizosaccharomyces pombe. EMBO J. 1985 Mar;4(3):687–691. doi: 10.1002/j.1460-2075.1985.tb03684.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fukui Y., Kozasa T., Kaziro Y., Takeda T., Yamamoto M. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell. 1986 Jan 31;44(2):329–336. doi: 10.1016/0092-8674(86)90767-1. [DOI] [PubMed] [Google Scholar]
  13. Gotoh Y., Moriyama K., Matsuda S., Okumura E., Kishimoto T., Kawasaki H., Suzuki K., Yahara I., Sakai H., Nishida E. Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF. EMBO J. 1991 Sep;10(9):2661–2668. doi: 10.1002/j.1460-2075.1991.tb07809.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Gotoh Y., Nishida E., Sakai H. Okadaic acid activates microtubule-associated protein kinase in quiescent fibroblastic cells. Eur J Biochem. 1990 Nov 13;193(3):671–674. doi: 10.1111/j.1432-1033.1990.tb19385.x. [DOI] [PubMed] [Google Scholar]
  16. Gotoh Y., Nishida E., Yamashita T., Hoshi M., Kawakami M., Sakai H. Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells. Eur J Biochem. 1990 Nov 13;193(3):661–669. doi: 10.1111/j.1432-1033.1990.tb19384.x. [DOI] [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. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  19. Hattori S., Fukuda M., Yamashita T., Nakamura S., Gotoh Y., Nishida E. Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system. J Biol Chem. 1992 Oct 5;267(28):20346–20351. [PubMed] [Google Scholar]
  20. Hoshi M., Nishida E., Sakai H. Activation of a Ca2+-inhibitable protein kinase that phosphorylates microtubule-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts. J Biol Chem. 1988 Apr 15;263(11):5396–5401. [PubMed] [Google Scholar]
  21. 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]
  22. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  23. Lee K. S., Levin D. E. Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. Mol Cell Biol. 1992 Jan;12(1):172–182. doi: 10.1128/mcb.12.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leevers S. J., Marshall C. J. Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 1992 Feb;11(2):569–574. doi: 10.1002/j.1460-2075.1992.tb05088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Nakielny S., Cohen P., Wu J., Sturgill T. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase. EMBO J. 1992 Jun;11(6):2123–2129. doi: 10.1002/j.1460-2075.1992.tb05271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nishida E., Gotoh Y. Mitogen-activated protein kinase and cytoskeleton in mitogenic signal transduction. Int Rev Cytol. 1992;138:211–238. doi: 10.1016/s0074-7696(08)61589-2. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Pelech S. L., Tombes R. M., Meijer L., Krebs E. G. Activation of myelin basic protein kinases during echinoderm oocyte maturation and egg fertilization. Dev Biol. 1988 Nov;130(1):28–36. doi: 10.1016/0012-1606(88)90410-1. [DOI] [PubMed] [Google Scholar]
  32. Posada J., Cooper J. A. Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science. 1992 Jan 10;255(5041):212–215. doi: 10.1126/science.1313186. [DOI] [PubMed] [Google Scholar]
  33. Posada J., Sanghera J., Pelech S., Aebersold R., Cooper J. A. Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts. Mol Cell Biol. 1991 May;11(5):2517–2528. doi: 10.1128/mcb.11.5.2517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  35. Ray L. B., Sturgill T. W. Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1502–1506. doi: 10.1073/pnas.84.6.1502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rhodes N., Connell L., Errede B. STE11 is a protein kinase required for cell-type-specific transcription and signal transduction in yeast. Genes Dev. 1990 Nov;4(11):1862–1874. doi: 10.1101/gad.4.11.1862. [DOI] [PubMed] [Google Scholar]
  37. Seger R., Ahn N. G., Boulton T. G., Yancopoulos G. D., Panayotatos N., Radziejewska E., Ericsson L., Bratlien R. L., Cobb M. H., Krebs E. G. Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6142–6146. doi: 10.1073/pnas.88.14.6142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shirakabe K., Gotoh Y., Nishida E. A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator. J Biol Chem. 1992 Aug 15;267(23):16685–16690. [PubMed] [Google Scholar]
  39. Sturgill T. W., Ray L. B., Erikson E., Maller J. L. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature. 1988 Aug 25;334(6184):715–718. doi: 10.1038/334715a0. [DOI] [PubMed] [Google Scholar]
  40. Sturgill T. W., Wu J. Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6. Biochim Biophys Acta. 1991 May 17;1092(3):350–357. doi: 10.1016/s0167-4889(97)90012-4. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Thomas S. M., DeMarco M., D'Arcangelo G., Halegoua S., Brugge J. S. Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases. Cell. 1992 Mar 20;68(6):1031–1040. doi: 10.1016/0092-8674(92)90075-n. [DOI] [PubMed] [Google Scholar]
  43. Toda T., Shimanuki M., Yanagida M. Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases. Genes Dev. 1991 Jan;5(1):60–73. doi: 10.1101/gad.5.1.60. [DOI] [PubMed] [Google Scholar]
  44. Torres L., Martín H., García-Saez M. I., Arroyo J., Molina M., Sánchez M., Nombela C. A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants. Mol Microbiol. 1991 Nov;5(11):2845–2854. doi: 10.1111/j.1365-2958.1991.tb01993.x. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Warbrick E., Fantes P. A. The wis1 protein kinase is a dosage-dependent regulator of mitosis in Schizosaccharomyces pombe. EMBO J. 1991 Dec;10(13):4291–4299. doi: 10.1002/j.1460-2075.1991.tb05007.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wood K. W., Sarnecki C., Roberts T. M., Blenis J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992 Mar 20;68(6):1041–1050. doi: 10.1016/0092-8674(92)90076-o. [DOI] [PubMed] [Google Scholar]
  48. Wu J., Rossomando A. J., Her J. H., Del Vecchio R., Weber M. J., Sturgill T. W. Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9508–9512. doi: 10.1073/pnas.88.21.9508. [DOI] [PMC free article] [PubMed] [Google Scholar]

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