Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic

See more in PubMed

Aimes, R.T., Hemmer, W., and Taylor, S.S. 2000. Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase: Role in catalysis, P-site specificity, and interaction with inhibitors. Biochemistry 39 8325–8332. PubMed

Brown, N.R., Noble, M.E.M., Endicott, J.A., and Johnson, L.N. 1999. The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nat. Cell Biol. 1 438–443. PubMed

Case, D.A., Pearlman, D.A., Caldwell, J.W., Cheatham III, T.E., Ross, W.S., Simmerling, C.L., Darden, T.A., Merz, K.M., Stanton, R.V., Cheng, A.L., et al. 1999. AMBER 6. University of California, San Francisco, CA.

Cheng, A., Ross, K.E., Kaldis, P., and Solomon, M.J. 1999. Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases. Genes Dev. 13 2946–2957. PubMed PMC

Cheng, A., Kaldis, P., and Solomon, M.J. 2000. Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C α and β 2 isoforms. J. Biol. Chem. 275 34744–34749. PubMed

Chow, J.P., Siu, W.Y., Ho, H.T., Ma, K.H., Ho, C.C., and Poon, R.Y.C. 2003. Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints. J. Biol. Chem. 278 40815–40828. PubMed

Cook, A., Lowe, E.D., Chrysina, E.D., Skamnaki, V.T., Oikonomakos, N.G., and Johnson, L.N. 2002. Structural studies on phospho-CDK2/Cyclin A bound to nitrate, a transition state analogue: Implications for the protein kinase mechanism. Biochemistry 41 7301–7311. PubMed

Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz, J.K.M., Ferguson, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W., and Kollman, P.A. 1995. A 2nd generation force-field for simulation of proteins, nucleic-acids and organic-molecules. J. Am. Chem. Soc. 117 5179–5197.

Coulonval, K., Bockstaele, L., Paternot, S., and Roger, P.P. 2003. Phosphorylations of cyclin-dependent kinase 2 revisited using two-dimensional gel electrophoresis. J. Biol. Chem. 278 52052–52060. PubMed

De Bondt, H.L., Rosenblatt, J., Jancarik, J., Jones, H.D., Morgan, D.O., and Kim, S.H. 1993. Crystal structure of cyclin-dependent kinase 2. Nature 363 595–602. PubMed

Endicott, J.A., Noble, M.E.M., and Tucker, J.A. 1999. Cyclin-dependent kinases: Inhibition and substrate recognition. Curr. Opin. Struct. Biol. 9 738–744. PubMed

Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Zakrzewski, V.G., Montgomery, J.A., Stratmann, R.E., Burant, J.C., et al. 1998. Gaussian98. Gaussian, Inc., Pittsburgh, PA.

Gu, Y., Rosenblatt, J., and Morgan, D.O. 1992. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 11 3995–4005. PubMed PMC

Hagopian, J.C., Kirtley, M.P., and Stevenson, L.M. 2001. Kinetic basis for activation of CDK2/Cyclin A by phosphorylation. J. Biol. Chem. 276 275–280. PubMed

Hanks, S. and Quinn, A.M. 1991. Protein kinase catalytic domain sequence database: Identification of conserved features of primary structure and classification of family members. Methods Enzymol. 200 38–62. PubMed

Hemmer, W., McGlone, M., Tsigelny, I., and Taylor, S.S. 1997. Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase. J. Biol. Chem. 272 16946–16954. PubMed

Holmes, J.K. and Solomon, M.J. 1996. A predictive scale for evaluation cyclin-dependent kinase substrates. A comparison of p34cdc2 and p33cdk2. J. Biol. Chem. 271 25240–25246. PubMed

———. 2001. The role of Thr160 phosphorylation of Cdk2 in substrate recognition. Eur. J. Biochem. 268 4647–4652. PubMed

Jeffrey, P.D., Russo, A.A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, N.P. 1995. Mechanism of cdk activation revealed by the structure of a cyclin A–cdk2 complex. Nature 376 313–320. PubMed

Jinno, S., Hung, S.C., and Okayama, H. 1999. Cell cycle start from quiescence controlled by tyrosine phosphorylation of Cdk4. Oncogene 18 565–571. PubMed

Johnson, L.N. and Lewis, R.J. 2001. Structural basis for control by phosphorylation. Chem. Rev. 101 2209–2242. PubMed

Johnson, D.A., Akamine, P., Radzio-Andzelm, E., Madhusudan, and Taylor, S.S. 2001. Dynamics of cAMP-dependent protein kinase. Chem. Rev. 101 2243–2270. PubMed

Knockaert, M., Greengard, P., and Meijer, L. 2002. Pharmacological inhibitors of cyclin-dependent kinases. Trends Pharmacol. Sci. 23 417–425. PubMed

Laaksonen, L. 1992. A graphics program for the analysis and display of molecular dynamics trajectories. J. Mol. Graph. 10 33–34. PubMed

Lew, J. 2003. MAP kinases and CDKs: Kinetic basis for catalytic activation. Biochemistry 42 849–856. PubMed

Morgan, D.O. 1996. The dynamics of cyclin dependent kinase structure. Curr. Opin. Cell Biol. 8 767–772. PubMed

———. 1997. Cyclin-dependent kinases: Engines, clocks, and microprocessors. Annu. Rev. Cell Dev. Biol. 13 261–291. PubMed

Morris, M.C., Gondeau, C., Tainer, J.A., and Divita, G. 2002. Kinetic mechanism of activation of the Cdk2/Cyclin A complex. J. Biol. Chem. 277 23847–23853. PubMed

Otyepka, M., Kříž, Z., and Koča, J. 2002. Dynamics and binding modes of free cdk2 and its two complexes with inhibitors studied by computer simulations. J. Biomol. Struct. Dynam. 20 141–154. PubMed

Pagano, M. 1998. Cell cycle control. New York University School of Medicine, New York.

Philippopoulos, M. and Lim, C. 1995. Molecular dynamics simulation of E. coli ribonuclease H1 in solution: Correlation with NMR and X-ray data and insights into biological function. J. Mol. Biol. 254 771–792. PubMed

Poon, R.Y.C. and Hunter, T. 1995. Dephosphorylation of Cdk2 Thr160 by the cyclin-dependent kinase-interacting phosphatase KAP in the absence of cyclin. Science 270 90–93. PubMed

Rudolph, J., Epstein, D.M., Parker, L., and Eckstein, J. 2001. Specifity of natural and artificial substrates for human CDC25A. Anal. Biochem. 289 43–51. PubMed

Russo, A.A., Jeffrey, P.D., and Pavletich, N.P. 1996. Structural basis of cyclin-dependent kinase 2 activation by phosphorylation. Nat. Struct. Biol. 3 696–700. PubMed

Schulze-Gahmen, U., Brandsen, J., Jones, H.D., Morgan, D., Meijer, L., Veselý, J., and Kim, S.-H. 1995. Multiple modes of ligand recognition: Crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors, olomoucine and isopenthenyladenine. Proteins 22 378–391. PubMed

Sebastian, B., Kakizuka, A., and Hunter, T. 1993. Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15. Proc. Natl. Acad. Sci. 90 3521–3524. PubMed PMC

Spoel, D.V.D., Buuren, A.R.V., Apol, E., Meulenhoff, P.J., Tieleman, P.D., Sijbers, A.L.T.M., Hess, B., Feenstra, K.A., Lindhal, E., Drunen, R.V., et al. 1991–2002. GROMACS. University of Groningen, Groningen, The Netherlands.

Stevenson, L.M., Deal, M.S., Hagopian, J.C., and Lew, J. 2002. Activation mechanism of CDK2: Role of cyclin binding versus phosphorylation. Biochemistry 41 8528–8534. PubMed

Tsigelny, I., Greenberg, J.P., Cox, S., Nichols, W.L., Taylor, S.S., and Ten Eyck, L.F. 1999. 600 ps molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase. Biopolymers 50 513–524. PubMed

Vriend, G. 1997. WHAT IF, 5.0 ed. EMBL, Heidelberg, Germany.

Wang, J.M., Cieplak, P., and Kollman, P.A. 2000. How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J. Comput. Chem. 21 1049–1074.

Watanabe, N., Broome, M., and Hunter, T. 1995. Regulation of the human Wee1Hu Cdk tyrosine 15-kinase during the cell cycle. EMBO J. 14 1878–1891. PubMed PMC

Zukerberg, L.R., Patrick, G.N., Nikolic, M., Humbert, S., Wu, C.L., Lanier, L.M., Gertler, F.B., Vidal, M., Van Etten, R.A., and Tsai, L.H. 2000. Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth. Neuron 26 543–544. PubMed

Two C-terminal ankyrin repeats form the minimal stable unit of the ankyrin repeat protein p18INK4c

Regulatory phosphorylation of cyclin-dependent kinase 2: insights from molecular dynamics simulations

Functional flexibility of human cyclin-dependent kinase-2 and its evolutionary conservation

The mechanism of inhibition of the cyclin-dependent kinase-2 as revealed by the molecular dynamics study on the complex CDK2 with the peptide substrate HHASPRK