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. 1995 Aug 1;309(Pt 3):927–931. doi: 10.1042/bj3090927

The design of peptide-based substrates for the cdc2 protein kinase.

J Srinivasan 1, M Koszelak 1, M Mendelow 1, Y G Kwon 1, D S Lawrence 1
PMCID: PMC1135720  PMID: 7639712

Abstract

The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].

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Selected References

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

  1. Ando S., Tsujimura K., Matsuoka Y., Tokui T., Hisanaga S., Okumura E., Uchiyama M., Kishimoto T., Yasuda H., Inagaki M. Phosphorylation of synthetic vimentin peptides by cdc2 kinase. Biochem Biophys Res Commun. 1993 Sep 15;195(2):837–843. doi: 10.1006/bbrc.1993.2121. [DOI] [PubMed] [Google Scholar]
  2. Beaudette K. N., Lew J., Wang J. H. Substrate specificity characterization of a cdc2-like protein kinase purified from bovine brain. J Biol Chem. 1993 Oct 5;268(28):20825–20830. [PubMed] [Google Scholar]
  3. Bellé R., Derancourt J., Poulhe R., Capony J. P., Ozon R., Mulner-Lorillon O. A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF-1 gamma and EF-1 beta. FEBS Lett. 1989 Sep 11;255(1):101–104. doi: 10.1016/0014-5793(89)81069-5. [DOI] [PubMed] [Google Scholar]
  4. Bischoff J. R., Friedman P. N., Marshak D. R., Prives C., Beach D. Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4766–4770. doi: 10.1073/pnas.87.12.4766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brizuela L., Draetta G., Beach D. Activation of human CDC2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4362–4366. doi: 10.1073/pnas.86.12.4362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chou Y. H., Ngai K. L., Goldman R. The regulation of intermediate filament reorganization in mitosis. p34cdc2 phosphorylates vimentin at a unique N-terminal site. J Biol Chem. 1991 Apr 25;266(12):7325–7328. [PubMed] [Google Scholar]
  7. Cisek L. J., Corden J. L. Phosphorylation of RNA polymerase by the murine homologue of the cell-cycle control protein cdc2. Nature. 1989 Jun 29;339(6227):679–684. doi: 10.1038/339679a0. [DOI] [PubMed] [Google Scholar]
  8. Eggert M., Radomski N., Tripier D., Traub P., Jost E. Identification of phosphorylation sites on murine nuclear lamin C by RP-HPLC and microsequencing. FEBS Lett. 1991 Nov 4;292(1-2):205–209. doi: 10.1016/0014-5793(91)80868-4. [DOI] [PubMed] [Google Scholar]
  9. Elledge S. J., Richman R., Hall F. L., Williams R. T., Lodgson N., Harper J. W. CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2907–2911. doi: 10.1073/pnas.89.7.2907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heald R., McKeon F. Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Cell. 1990 May 18;61(4):579–589. doi: 10.1016/0092-8674(90)90470-y. [DOI] [PubMed] [Google Scholar]
  11. Hoffmann I., Clarke P. R., Marcote M. J., Karsenti E., Draetta G. Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J. 1993 Jan;12(1):53–63. doi: 10.1002/j.1460-2075.1993.tb05631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. House C., Wettenhall R. E., Kemp B. E. The influence of basic residues on the substrate specificity of protein kinase C. J Biol Chem. 1987 Jan 15;262(2):772–777. [PubMed] [Google Scholar]
  13. House C., Wettenhall R. E., Kemp B. E. The influence of basic residues on the substrate specificity of protein kinase C. J Biol Chem. 1987 Jan 15;262(2):772–777. [PubMed] [Google Scholar]
  14. Kamijo M., Yasuda H., Yau P. M., Yamashita M., Nagahama Y., Ohba Y. Preference of human cdc2 kinase for peptide substrate. Pept Res. 1992 Sep-Oct;5(5):281–285. [PubMed] [Google Scholar]
  15. Kemp B. E., Graves D. J., Benjamini E., Krebs E. G. Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem. 1977 Jul 25;252(14):4888–4894. [PubMed] [Google Scholar]
  16. Kemp B. E., Graves D. J., Benjamini E., Krebs E. G. Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem. 1977 Jul 25;252(14):4888–4894. [PubMed] [Google Scholar]
  17. Keryer G., Luo Z., Cavadore J. C., Erlichman J., Bornens M. Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5418–5422. doi: 10.1073/pnas.90.12.5418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kipreos E. T., Wang J. Y. Differential phosphorylation of c-Abl in cell cycle determined by cdc2 kinase and phosphatase activity. Science. 1990 Apr 13;248(4952):217–220. doi: 10.1126/science.2183353. [DOI] [PubMed] [Google Scholar]
  19. Kusubata M., Matsuoka Y., Tsujimura K., Ito H., Ando S., Kamijo M., Yasuda H., Ohba Y., Okumura E., Kishimoto T. cdc2 kinase phosphorylation of desmin at three serine/threonine residues in the amino-terminal head domain. Biochem Biophys Res Commun. 1993 Feb 15;190(3):927–934. doi: 10.1006/bbrc.1993.1138. [DOI] [PubMed] [Google Scholar]
  20. Kwon Y. G., Mendelow M., Lawrence D. S. The active site substrate specificity of protein kinase C. J Biol Chem. 1994 Feb 18;269(7):4839–4844. [PubMed] [Google Scholar]
  21. Kwon Y. G., Mendelow M., Srinivasan J., Lee T. R., Pluskey S., Salerno A., Lawrence D. S. The active site substrate specificity of the cAMP-dependent protein kinase. J Biol Chem. 1993 May 25;268(15):10713–10716. [PubMed] [Google Scholar]
  22. Labbé J. C., Capony J. P., Caput D., Cavadore J. C., Derancourt J., Kaghad M., Lelias J. M., Picard A., Dorée M. MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B. EMBO J. 1989 Oct;8(10):3053–3058. doi: 10.1002/j.1460-2075.1989.tb08456.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Langan T. A., Gautier J., Lohka M., Hollingsworth R., Moreno S., Nurse P., Maller J., Sclafani R. A. Mammalian growth-associated H1 histone kinase: a homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog cells. Mol Cell Biol. 1989 Sep;9(9):3860–3868. doi: 10.1128/mcb.9.9.3860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Matsushime H., Ewen M. E., Strom D. K., Kato J. Y., Hanks S. K., Roussel M. F., Sherr C. J. Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell. 1992 Oct 16;71(2):323–334. doi: 10.1016/0092-8674(92)90360-o. [DOI] [PubMed] [Google Scholar]
  26. McVey D., Brizuela L., Mohr I., Marshak D. R., Gluzman Y., Beach D. Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication. Nature. 1989 Oct 12;341(6242):503–507. doi: 10.1038/341503a0. [DOI] [PubMed] [Google Scholar]
  27. Moreno S., Nurse P. Substrates for p34cdc2: in vivo veritas? Cell. 1990 May 18;61(4):549–551. doi: 10.1016/0092-8674(90)90463-o. [DOI] [PubMed] [Google Scholar]
  28. Norbury C., Nurse P. Animal cell cycles and their control. Annu Rev Biochem. 1992;61:441–470. doi: 10.1146/annurev.bi.61.070192.002301. [DOI] [PubMed] [Google Scholar]
  29. Pearson R. B., Misconi L. Y., Kemp B. E. Smooth muscle myosin kinase requires residues on the COOH-terminal side of the phosphorylation site. Peptide inhibitors. J Biol Chem. 1986 Jan 5;261(1):25–27. [PubMed] [Google Scholar]
  30. Peter M., Nakagawa J., Dorée M., Labbé J. C., Nigg E. A. Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase. Cell. 1990 Mar 9;60(5):791–801. doi: 10.1016/0092-8674(90)90093-t. [DOI] [PubMed] [Google Scholar]
  31. Peter M., Nakagawa J., Dorée M., Labbé J. C., Nigg E. A. In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell. 1990 May 18;61(4):591–602. doi: 10.1016/0092-8674(90)90471-p. [DOI] [PubMed] [Google Scholar]
  32. Pines J. Cyclins and cyclin-dependent kinases: take your partners. Trends Biochem Sci. 1993 Jun;18(6):195–197. doi: 10.1016/0968-0004(93)90185-p. [DOI] [PubMed] [Google Scholar]
  33. Prorok M., Lawrence D. S. Cryopreservation of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase from bovine cardiac muscle. J Biochem Biophys Methods. 1989 May;18(3):167–175. doi: 10.1016/0165-022x(89)90001-8. [DOI] [PubMed] [Google Scholar]
  34. Shenoy S., Choi J. K., Bagrodia S., Copeland T. D., Maller J. L., Shalloway D. Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis. Cell. 1989 Jun 2;57(5):763–774. doi: 10.1016/0092-8674(89)90791-5. [DOI] [PubMed] [Google Scholar]
  35. Smith J. A., Pease L. G. Reverse turns in peptides and proteins. CRC Crit Rev Biochem. 1980;8(4):315–399. doi: 10.3109/10409238009105470. [DOI] [PubMed] [Google Scholar]
  36. Smith L. D., Ecker R. E. The interaction of steroids with Rana pipiens Oocytes in the induction of maturation. Dev Biol. 1971 Jun;25(2):232–247. doi: 10.1016/0012-1606(71)90029-7. [DOI] [PubMed] [Google Scholar]
  37. Songyang Z., Blechner S., Hoagland N., Hoekstra M. F., Piwnica-Worms H., Cantley L. C. Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr Biol. 1994 Nov 1;4(11):973–982. doi: 10.1016/s0960-9822(00)00221-9. [DOI] [PubMed] [Google Scholar]
  38. Till J. H., Annan R. S., Carr S. A., Miller W. T. Use of synthetic peptide libraries and phosphopeptide-selective mass spectrometry to probe protein kinase substrate specificity. J Biol Chem. 1994 Mar 11;269(10):7423–7428. [PubMed] [Google Scholar]
  39. Ward G. E., Kirschner M. W. Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C. Cell. 1990 May 18;61(4):561–577. doi: 10.1016/0092-8674(90)90469-u. [DOI] [PubMed] [Google Scholar]

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