Biochemical characterization of Cdk2-Speedy/Ringo A2
Tóm tắt
Normal cell cycle progression requires the precise activation and inactivation of cyclin-dependent protein kinases (CDKs), which consist of a CDK and a cyclin subunit. A novel cell cycle regulator called Speedy/Ringo shows no sequence similarity to cyclins, yet can directly bind to and activate CDKs. Speedy/Ringo proteins, which bind to and activate Cdc2 and Cdk2 in vitro, are required for the G2 to M transition during Xenopus oocyte maturation and for normal S-phase entry in cultured human cells. We have characterized the substrate specificity and enzymatic activity of human Cdk2-Speedy/Ringo A2 in order to gain insights into the possible functions of this complex. In contrast to Cdk2-cyclin A, which has a well-defined consensus target site ((S/T)PX(K/R)) that strongly favors substrates containing a lysine at the +3 position of substrates, Cdk2-Speedy/Ringo A2 displayed a broad substrate specificity at this position. Consequently, Cdk2-Ringo/Speedy A2 phosphorylated optimal Cdk2 substrates such as histone H1 and a KSPRK peptide poorly, only ~0.08% as well as Cdk2-cyclin A, but non-canonical Cdk2 substrates such as a KSPRY peptide relatively well, with an efficiency of ~80% compared to Cdk2-cyclin A. Cdk2-Speedy/Ringo A2 also phosphorylated authentic Cdk2 substrates, such as Cdc25 proteins, which contain non-canonical CDK phosphorylation sites, nearly as well as Cdk2-cyclin A. Phosphopeptide mapping indicated that Cdk2-Speedy/Ringo A2 and Cdk2-cyclin A phosphorylate distinct subsets of sites on Cdc25 proteins. Thus, the low activity that Cdk2-Speedy/Ringo A2 displays when assayed on conventional Cdk2 substrates may significantly underestimate the potential physiological importance of Cdk2-Speedy/Ringo A2 in phosphorylating key subsets of Cdk2 substrates. Unlike Cdk2-cyclin A, whose activity depends strongly on activating phosphorylation of Cdk2 on Thr-160, neither the overall catalytic activity nor the substrate recognition by Cdk2-Speedy/Ringo A2 was significantly affected by this phosphorylation. Furthermore, Cdk2-Speedy/Ringo A2 was not a suitable substrate for metazoan CAK (which phosphorylates Cdk2 at Thr-160), supporting the notion that Speedy/Ringo A2 activates Cdk2 in a CAK-independent manner. There are major differences in substrate preferences between CDK-Speedy/Ringo A2 and Cdk2-cyclin complexes. These differences may accommodate the CAK-independent activation of Cdk2 by Speedy/Ringo A2 and they raise the possibility that CDK-Speedy/Ringo A2 complexes could phosphorylate and regulate a subset of non-canonical CDK substrates, such as Cdc25 protein phosphatases, to control cell cycle progression.
Tài liệu tham khảo
Pines J: Cyclins and cyclin-dependent kinases: a biochemical view. Biochem J. 1995, 308: 697-711.
Sherr CJ, Roberts JM: Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995, 9: 1149-1163.
Sherr CJ, Roberts JM: CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999, 13: 1501-1512.
King RW, Deshaies RJ, Peters JM, Kirschner MW: How proteolysis drives the cell cycle. Science. 1996, 274: 1652-1659. 10.1126/science.274.5293.1652.
Morgan DO: The dynamics of cyclin dependent kinase structure. Curr Opin Cell Biol. 1996, 8: 767-772. 10.1016/S0955-0674(96)80076-7.
Morgan DO: Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997, 13: 261-291. 10.1146/annurev.cellbio.13.1.261.
Solomon MJ, Kaldis P: Regulation of CDKs by phosphorylation. Results and Problems in Cell Differentiation. Edited by: Pagano M. 1998, Heidelberg: Springer, 79-109.
Solomon MJ, Glotzer M, Lee TH, Philippe M, Kirschner MW: Cyclin activation of p34cdc2. Cell. 1990, 63: 1013-1024. 10.1016/0092-8674(90)90504-8.
Solomon MJ, Lee T, Kirschner MW: Role of phosphorylation in p34cdc2 activation: identification of an activating kinase. Mol Biol Cell. 1992, 3: 13-27.
Fesquet D, Labbé JC, Derancourt J, Capony JP, Galas S, Girard F, Lorca T, Shuttleworth J, Dorée M, Cavadore JC: 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, 12: 3111-3121.
Poon RY, Yamashita K, Adamczewski JP, 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, 12: 3123-3132.
Solomon MJ, Harper JW, Shuttleworth J: CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15. EMBO J. 1993, 12: 3133-3142.
Solomon MJ: Activation of the various cyclin/cdc2 protein kinases. Curr Opin Cell Biol. 1993, 5: 180-186. 10.1016/0955-0674(93)90100-5.
Kaldis P, Sutton A, Solomon MJ: The Cdk-activating kinase (CAK) from budding yeast. Cell. 1996, 86: 553-564. 10.1016/S0092-8674(00)80129-4.
Espinoza FH, Farrell A, Erdjument-Bromage H, Tempst P, Morgan DO: A cyclin-dependent kinase-activating kinase (CAK) in budding yeast unrelated to vertebrate CAK. Science. 1996, 273: 1714-1717.
Thuret JY, Valay JG, Faye G, Mann C: Civ1 (CAK in vivo), a novel Cdk-activating kinase. Cell. 1996, 86: 565-576. 10.1016/S0092-8674(00)80130-0.
Peeper DS, Parker LL, Ewen ME, Toebes M, Hall FL, Xu M, Zantema A, van der Eb AJ, Piwnica-Worms H: A- and B-type cyclins differentially modulate substrate specificity of cyclin-cdk complexes. EMBO J. 1993, 12: 1947-1954.
Schulman BA, Lindstrom DL, Harlow E: Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. Proc Natl Acad Sci USA. 1998, 95: 10453-10458. 10.1073/pnas.95.18.10453.
Tyers M, Tokiwa G, Futcher B: Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins. EMBO J. 1993, 12: 1955-1968.
Levine K, Huang K, Cross FR: Saccharomyces cerevisiae G1 cyclins differ in their intrinsic functional specificities. Mol Cell Biol. 1996, 16: 6794-6803.
Pines J, Hunter T: Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J Cell Biol. 1991, 115: 1-17. 10.1083/jcb.115.1.1.
Nigg EA: The substrates of the cdc2 kinase. Semin Cell Biol. 1991, 2: 261-270.
Songyang Z, Blechner S, Hoagland N, Hoekstra MF, Piwnica-Worms H, Cantley LC: Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr Biol. 1994, 4: 973-982. 10.1016/S0960-9822(00)00221-9.
Holmes JK, Solomon MJ: A predictive scale for evaluating cyclin-dependent kinase substrates. A comparison of p34cdc2 and p33cdk2. J Biol Chem. 1996, 271: 25240-25246. 10.1074/jbc.271.41.25240.
Lenormand JL, Dellinger RW, Knudsen KE, Subramani S, Donoghue DJ: Speedy: a novel cell cycle regulator of the G2/M transition. EMBO J. 1999, 18: 1869-1877. 10.1093/emboj/18.7.1869.
Ferby I, Blazquez M, Palmer A, Eritja R, Nebreda AR: A novel p34(cdc2)-binding and activating protein that is necessary and sufficient to trigger G(2)/M progression in Xenopus oocytes. Genes Dev. 1999, 13: 2177-2189.
Karaiskou A, Perez LH, Ferby I, Ozon R, Jessus C, Nebreda AR: Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins. J Biol Chem. 2001, 276: 36028-36034. 10.1074/jbc.M104722200.
Porter LA, Dellinger RW, Tynan JA, Barnes EA, Kong M, Lenormand JL, Donoghue DJ: Human Speedy: a novel cell cycle regulator that enhances proliferation through activation of Cdk2. J Cell Biol. 2002, 157: 357-366. 10.1083/jcb.200109045.
Stevenson LM, Deal MS, Hagopian JC, Lew J: Activation mechanism of CDK2: role of cyclin binding versus phosphorylation. Biochemistry. 2002, 41: 8528-8534. 10.1021/bi025812h.
Holmes JK, Solomon MJ: The role of Thr160 phosphorylation of Cdk2 in substrate recognition. Eur J Biochem. 2001, 268: 4647-4652. 10.1046/j.1432-1327.2001.02392.x.
Brown NR, Noble ME, Endicott JA, Johnson LN: The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nat Cell Biol. 1999, 1: 438-443. 10.1038/15674.
Tarricone C, Dhavan R, Peng J, Areces LB, Tsai LH, Musacchio A: Structure and regulation of the CDK5-p25(nck5a) complex. Mol Cell. 2001, 8: 657-669. 10.1016/S1097-2765(01)00343-4.
Brown NR, Noble ME, Lawrie AM, Morris MC, Tunnah P, Divita G, Johnson LN, Endicott JA: Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity. J Biol Chem. 1999, 274: 8746-8756. 10.1074/jbc.274.13.8746.
Cheng A, Xiong W, Ferrell JE, Solomon MJ: Identification and comparative analysis of multiple mammalian Speedy/Ringo proteins. Cell Cycle. 2005, 4: 155-165.
Busino L, Chiesa M, Draetta GF, Donzelli M: Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis. Oncogene. 2004, 23: 2050-2056. 10.1038/sj.onc.1207394.
Cans C, Ducommun B, Baldin V: Proteasome-dependent degradation of human CDC25B phosphatase. Mol Biol Rep. 1999, 26: 53-57. 10.1023/A:1006912105352.
Nilsson I, Hoffmann I: Cell cycle regulation by the Cdc25 phosphatase family. Prog Cell Cycle Res. 2000, 4: 107-114.
Kaldis P, Russo AA, Chou HS, Pavletich NP, Solomon MJ: Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities. Mol Biol Cell. 1998, 9: 2545-2560.
Cheng A, Ross KE, Kaldis P, Solomon MJ: Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases. Genes Dev. 1999, 13: 2946-2957. 10.1101/gad.13.22.2946.
Cheng A, Kaldis P, Solomon MJ: Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms. J Biol Chem. 2000, 275: 34744-34749. 10.1074/jbc.M006210200.
Song H, Hanlon N, Brown NR, Noble ME, Johnson LN, Barford D: Phosphoprotein-protein interactions revealed by the crystal structure of kinase-associated phosphatase in complex with phosphoCDK2. Mol Cell. 2001, 7: 615-626. 10.1016/S1097-2765(01)00208-8.
Chen J, Saha P, Kornbluth S, Dynlacht BD, Dutta A: Cyclin-binding motifs are essential for the function of p21CIP1. Mol Cell Biol. 1996, 16: 4673-4682.
Adams PD, Li X, Sellers WR, Baker KB, Leng X, Harper JW, Taya Y, Kaelin WG: Retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin-cdk complexes. Mol Cell Biol. 1999, 19: 1068-1080.
Zhu L, Enders G, Lees JA, Beijersbergen RL, Bernards R, Harlow E: The pRB-related protein p107 contains two growth suppression domains: independent interactions with E2F and cyclin/cdk complexes. EMBO J. 1995, 14: 1904-1913.
Loog M, Morgan DO: Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates. Nature. 2005, 434: 104-118. 10.1038/nature03329.
Archambault V, Buchler NE, Wilmes GM, Jacobson MD, Cross FR: Two-faced cyclins with eyes on the targets. Cell Cycle. 2005, 4: 125-130.
Kaldis P, Cheng A, Solomon MJ: The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine. J Biol Chem. 2000, 275: 32578-32584. 10.1074/jbc.M003212200.
Hagopian JC, Kirtley MP, Stevenson LM, Gergis RM, Russo AA, Pavletich NP, Parsons SM, Lew J: Kinetic basis for activation of CDK2/cyclin A by phosphorylation. J Biol Chem. 2001, 276: 275-280. 10.1074/jbc.M007337200.
Saha P, Eichbaum Q, Silberman ED, Mayer BJ, Dutta A: p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases. Mol Cell Biol. 1997, 17: 4338-4345.
El-Guindy AS, Miller G: Phosphorylation of Epstein-Barr virus ZEBRA protein at its casein kinase 2 sites mediates its ability to repress activation of a viral lytic cycle late gene by Rta. J Virol. 2004, 78: 7634-7644. 10.1128/JVI.78.14.7634-7644.2004.