title: Expression, purification, and identification of associated proteins of the full-length hCDK12/CyclinK complex.
abstract: The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3'-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a "2A" peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.
authors.affiliation: From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Bartlomiej
authors.initials: B
authors.lastname: Bartkowiak
authors.affiliation: From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 arno@duke.edu.
authors.forename: Arno L
authors.initials: AL
authors.lastname: Greenleaf
mesh_terms: D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D055808:Drug Discovery; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: C113028:CCNK protein, human; D016213:Cyclins; D012313:RNA; D005982:Glutathione Transferase; C447339:CDK12 protein, human; C495894:CDK2 protein, human; C467969:CDK9 protein, human; D051357:Cyclin-Dependent Kinase 2; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases
keywords: Analog-sensitive Kinase; Baculovirus; C-terminal Domain (Carboxyl Tail Domain, CTD); Cyclin-dependent Kinase (CDK); Enzyme Purification; RNA Polymerase II; RNA Processing