Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Jul;16(7):3472–3479. doi: 10.1128/mcb.16.7.3472

Identification of an autoinhibitory region in the activation loop of the Mos protein kinase.

S C Robertson 1, D J Donoghue 1
PMCID: PMC231342  PMID: 8668163

Abstract

The Mos protein is a serine/threonine protein kinase which acts to regulate progression through meiosis in vertebrate oocytes. Although Mos function is dependent on its ability to act as a protein kinase, little is known about the factors which regulate Mos kinase activity. To understand the mechanism by which Mos kinase activity is regulated, we have used molecular modeling to construct a three-dimensional model of Mos based on the crystallographic coordinates of cyclic AMP-dependent kinase (PKA). This model identified a loop in Mos which is positioned near the active site and appears capable of blocking substrate access to the active site. Mutagenesis was used to construct altered forms of the Mos protein with deletions of parts or all of the loop. In vitro kinase assays showed that Mos proteins with the loop removed had up to a fourfold increase in kinase activity compared with the wild-type protein, indicating that the loop acts in an autoinhibitory manner for Mos kinase activity. Point mutations were also made on individual residues of the loop which were determined from the molecular model to be capable of reaching the active site. Determination of the kinase activities of these mutants showed that individual mutations in the loop region are capable of either increasing or decreasing kinase activity with regard to the wild-type protein. These data suggest that the loop identified in Mos acts as an autoinhibitor of kinase activity.

Full Text

The Full Text of this article is available as a PDF (535.3 KB).

Selected References

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

  1. 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]
  2. Chou P. Y., Fasman G. D. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry. 1974 Jan 15;13(2):211–222. doi: 10.1021/bi00699a001. [DOI] [PubMed] [Google Scholar]
  3. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  4. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. De Bondt H. L., Rosenblatt J., Jancarik J., Jones H. D., Morgan D. O., Kim S. H. Crystal structure of cyclin-dependent kinase 2. Nature. 1993 Jun 17;363(6430):595–602. doi: 10.1038/363595a0. [DOI] [PubMed] [Google Scholar]
  7. Freeman R. S., Ballantyne S. M., Donoghue D. J. Meiotic induction by Xenopus cyclin B is accelerated by coexpression with mosXe. Mol Cell Biol. 1991 Mar;11(3):1713–1717. doi: 10.1128/mcb.11.3.1713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Freeman R. S., Kanki J. P., Ballantyne S. M., Pickham K. M., Donoghue D. J. Effects of the v-mos oncogene on Xenopus development: meiotic induction in oocytes and mitotic arrest in cleaving embryos. J Cell Biol. 1990 Aug;111(2):533–541. doi: 10.1083/jcb.111.2.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Freeman R. S., Meyer A. N., Li J., Donoghue D. J. Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor. J Cell Biol. 1992 Feb;116(3):725–735. doi: 10.1083/jcb.116.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Freeman R. S., Pickham K. M., Kanki J. P., Lee B. A., Pena S. V., Donoghue D. J. Xenopus homolog of the mos protooncogene transforms mammalian fibroblasts and induces maturation of Xenopus oocytes. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5805–5809. doi: 10.1073/pnas.86.15.5805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haccard O., Lewellyn A., Hartley R. S., Erikson E., Maller J. L. Induction of Xenopus oocyte meiotic maturation by MAP kinase. Dev Biol. 1995 Apr;168(2):677–682. doi: 10.1006/dbio.1995.1112. [DOI] [PubMed] [Google Scholar]
  12. Haccard O., Sarcevic B., Lewellyn A., Hartley R., Roy L., Izumi T., Erikson E., Maller J. L. Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase. Science. 1993 Nov 19;262(5137):1262–1265. doi: 10.1126/science.8235656. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Hannink M., Donoghue D. J. Lysine residue 121 in the proposed ATP-binding site of the v-mos protein is required for transformation. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7894–7898. doi: 10.1073/pnas.82.23.7894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Herzog N. K., Nash M., Ramagli L. S., Arlinghaus R. B. v-mos protein produced by in vitro translation has protein kinase activity. J Virol. 1990 Jun;64(6):3093–3096. doi: 10.1128/jvi.64.6.3093-3096.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hu S. H., Parker M. W., Lei J. Y., Wilce M. C., Benian G. M., Kemp B. E. Insights into autoregulation from the crystal structure of twitchin kinase. Nature. 1994 Jun 16;369(6481):581–584. doi: 10.1038/369581a0. [DOI] [PubMed] [Google Scholar]
  17. Huang W., Kessler D. S., Erikson R. L. Biochemical and biological analysis of Mek1 phosphorylation site mutants. Mol Biol Cell. 1995 Mar;6(3):237–245. doi: 10.1091/mbc.6.3.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hubbard S. R., Wei L., Ellis L., Hendrickson W. A. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature. 1994 Dec 22;372(6508):746–754. doi: 10.1038/372746a0. [DOI] [PubMed] [Google Scholar]
  19. Ito M., Guerriero V., Jr, Chen X. M., Hartshorne D. J. Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis. Biochemistry. 1991 Apr 9;30(14):3498–3503. doi: 10.1021/bi00228a021. [DOI] [PubMed] [Google Scholar]
  20. Jeffrey P. D., Russo A. A., Polyak K., Gibbs E., Hurwitz J., Massagué J., Pavletich N. P. Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature. 1995 Jul 27;376(6538):313–320. doi: 10.1038/376313a0. [DOI] [PubMed] [Google Scholar]
  21. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  22. Knighton D. R., Zheng J. H., Ten Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. doi: 10.1126/science.1862343. [DOI] [PubMed] [Google Scholar]
  23. Kosako H., Gotoh Y., Nishida E. Mitogen-activated protein kinase kinase is required for the mos-induced metaphase arrest. J Biol Chem. 1994 Nov 11;269(45):28354–28358. [PubMed] [Google Scholar]
  24. 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]
  25. Masui Y., Clarke H. J. Oocyte maturation. Int Rev Cytol. 1979;57:185–282. doi: 10.1016/s0074-7696(08)61464-3. [DOI] [PubMed] [Google Scholar]
  26. Masui Y., Markert C. L. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J Exp Zool. 1971 Jun;177(2):129–145. doi: 10.1002/jez.1401770202. [DOI] [PubMed] [Google Scholar]
  27. Maxwell S. A., Arlinghaus R. B. Serine kinase activity associated with Maloney murine sarcoma virus-124-encoded p37mos. Virology. 1985 May;143(1):321–333. doi: 10.1016/0042-6822(85)90119-9. [DOI] [PubMed] [Google Scholar]
  28. Nebreda A. R., Hunt T. The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs. EMBO J. 1993 May;12(5):1979–1986. doi: 10.1002/j.1460-2075.1993.tb05847.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
  30. Papkoff J., Nigg E. A., Hunter T. The transforming protein of Moloney murine sarcoma virus is a soluble cytoplasmic protein. Cell. 1983 May;33(1):161–172. doi: 10.1016/0092-8674(83)90345-8. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Puls A., Proikas-Cezanne T., Marquardt B., Propst F., Stabel S. Kinase activities of c-Mos and v-Mos proteins: a single amino acid exchange is responsible for constitutive activation of the 124 v-Mos kinase. Oncogene. 1995 Feb 16;10(4):623–630. [PubMed] [Google Scholar]
  33. Sagata N., Daar I., Oskarsson M., Showalter S. D., Vande Woude G. F. The product of the mos proto-oncogene as a candidate "initiator" for oocyte maturation. Science. 1989 Aug 11;245(4918):643–646. doi: 10.1126/science.2474853. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Sagata N., Watanabe N., Vande Woude G. F., Ikawa Y. The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature. 1989 Nov 30;342(6249):512–518. doi: 10.1038/342512a0. [DOI] [PubMed] [Google Scholar]
  36. Soderling T. R. Protein kinases. Regulation by autoinhibitory domains. J Biol Chem. 1990 Feb 5;265(4):1823–1826. [PubMed] [Google Scholar]
  37. Taylor S. S., Radzio-Andzelm E. Three protein kinase structures define a common motif. Structure. 1994 May 15;2(5):345–355. doi: 10.1016/s0969-2126(00)00036-8. [DOI] [PubMed] [Google Scholar]
  38. Taylor S. S., Zheng J., Radzio-Andzelm E., Knighton D. R., Ten Eyck L. F., Sowadski J. M., Herberg F. W., Yonemoto W. M. cAMP-dependent protein kinase defines a family of enzymes. Philos Trans R Soc Lond B Biol Sci. 1993 Jun 29;340(1293):315–324. doi: 10.1098/rstb.1993.0073. [DOI] [PubMed] [Google Scholar]
  39. Van Beveren C., van Straaten F., Galleshaw J. A., Verma I. M. Nucleotide sequence of the genome of a murine sarcoma virus. Cell. 1981 Nov;27(1 Pt 2):97–108. doi: 10.1016/0092-8674(81)90364-0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Yew N., Oskarsson M., Daar I., Blair D. G., Vande Woude G. F. mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities. Mol Cell Biol. 1991 Feb;11(2):604–610. doi: 10.1128/mcb.11.2.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. van der Hoorn F. A., Hulsebos E., Berns A. J., Bloemers H. P. Molecularly cloned c-mos(rat) is biologically active. EMBO J. 1982;1(11):1313–1317. doi: 10.1002/j.1460-2075.1982.tb01316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES