Abstract
Cyclin-dependent kinases have established roles in the regulation of cell cycle, in gene expression and in cell differentiation. Many of these kinases have been considered as drug targets and numerous efforts have been made to develop specific and potent inhibitors against them. The first step in all of these attempts and in many other biochemical analyses is the production of highly purified and reliable kinase, most frequently in a recombinant form. In this chapter we describe our experience in the cloning, expression, and purification of CDKs using CDK7/CycH, CDK8/CycC, and CDK9/CycT1 as an example.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Morgan DO (1997) Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 13:261–291
Andrews B, Measday V (1998) The cyclin family of budding yeast: abundant use of a good idea. Trends Genet 14:66–72
Gopinathan L, Ratnacaram CK, Kaldis P (2011) Established and novel Cdk/cyclin complexes regulating the cell cycle and development. Results Probl Cell Differ 53:365–389
Fisher RP (2005) Secrets of a double agent: CDK7 in cell-cycle control and transcription. J Cell Sci 118:5171–5180
Yankulov KY, Bentley DL (1997) Regulation of CDK7 substrate specificity by MAT1 and TFIIH. EMBO J 16:1638–1646
Pinhero R, Liaw P, Bertens K, Yankulov K (2004) Three cyclin-dependent kinases preferentially phosphorylate different parts of the C-terminal domain of the large subunit of RNA polymerase II. Eur J Biochem 271:1004–1014
Bhaduri S, Pryciak PM (2011) Cyclin-specific docking motifs promote phosphorylation of yeast signaling proteins by G1/S Cdk complexes. Curr Biol 21:1615–1623
Pagliuca FW, Collins MO, Lichawska A, Zegerman P, Choudhary JS, Pines J (2011) Quantitative proteomics reveals the basis for the biochemical specificity of the cell-cycle machinery. Mol Cell 43:406–417
Koivomagi M, Valk E, Venta R, Iofik A, Lepiku M, Morgan DO, Loog M (2011) Dynamics of Cdk1 substrate specificity during the cell cycle. Mol Cell 42:610–623
Kobor MS, Greenblatt J (2002) Regulation of transcription elongation by phosphorylation. Biochim Biophys Acta 1577:261–275
Egly JM, Coin F (2011) A history of TFIIH: two decades of molecular biology on a pivotal transcription/repair factor. DNA Repair (Amst) 10:714–721
Galbraith MD, Donner AJ, Espinosa JM (2010) CDK8: a positive regulator of transcription. Transcription 1:4–12
Price DH (2000) P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol 20:2629–2634
Cho S, Schroeder S, Ott M (2010) CYCLINg through transcription: posttranslational modifications of P-TEFb regulate transcription elongation. Cell Cycle 9:1697–1705
Ping YH, Rana TM (1999) Tat-associated kinase (P-TEFb): a component of transcription preinitiation and elongation complexes. J Biol Chem 274:7399–7404
Yankulov K, Bentley D (1998) Transcriptional control: tat cofactors and transcriptional elongation. Curr Biol 8:R447–R449
Fisher R, Jin P, Chamberlin H, Morgan D (1995) Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase. Cell 83:47–58
Rickert P, Corden JL, Lees E (1999) Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases. Oncogene 18:1093–1102
Peng J, Zhu Y, Milton JT, Price DH (1998) Identification of multiple cyclin subunits of human P-TEFb. Genes Dev 12:755–762
Yankulov K, Yamashita K, Roy R, Egly JM, Bentley DL (1995) The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase. J Biol Chem 270:23922–23925
Matsuoka M, Kato JY, Fisher RP, Morgan DO, Sherr CJ (1994) Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase. Mol Cell Biol 14:7265–7275
Fisher RP, Morgan DO (1994) A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Cell 78:713–724
Larochelle S, Chen J, Knights R, Pandur J, Morcillo P, Erdjument-Bromage H, Tempst P, Suter B, Fisher RP (2001) T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity. EMBO J 20:3749–3759
Ramanathan Y, Rajpara SM, Reza SM, Lees E, Shuman S, Mathews MB, Pe’ery T (2001) Three RNA polymerase II carboxyl-terminal domain kinases display distinct substrate preferences. J Biol Chem 276:10913–10920
Rossignol M, Kolb-Cheynel I, Egly JM (1997) Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH. EMBO J 16:1628–1637
Murhammer DW (2007) Baculovirus and insect cell expression protocols, 2nd edn. Humana Press, Totowa, NJ
Richardson CD (1995) Baculovirus expression protocols. Humana Press, Totowa, NJ
Kikkawa U, Minakuchi R, Takai Y, Nishizuka Y (1983) Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from rat brain. Methods Enzymol 99:288–298
Acknowledgements
We thank Drs. D. Morgan, E. Lees, and D. Price for providing baculoviruses and vectors for the expression of the recombinant kinases. MBP was a gift from Dr. G. Harauz. This study was supported by grants to K. Y. from the Natural Sciences and Engineering Research Council of Canada (NSERC #217548) and the Ontario Genomics Institute (OGI #043567).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Pinhero, R., Yankulov, K. (2016). Expression and Purification of Recombinant CDKs: CDK7, CDK8, and CDK9. In: Orzáez, M., Sancho Medina, M., Pérez-Payá, E. (eds) Cyclin-Dependent Kinase (CDK) Inhibitors. Methods in Molecular Biology, vol 1336. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2926-9_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2926-9_3
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2925-2
Online ISBN: 978-1-4939-2926-9
eBook Packages: Springer Protocols