title: The human CDK8 subcomplex is a histone kinase that requires Med12 for activity and can function independently of mediator.
abstract: The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable "CDK8 subcomplex" in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12--but not Med13--to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex--unlike the recombinant submodule--is unable to phosphorylate the Pol II CTD.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Knuesel
authors.forename: Krista D
authors.initials: KD
authors.lastname: Meyer
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Donner
authors.forename: Joaquin M
authors.initials: JM
authors.lastname: Espinosa
authors.forename: Dylan J
authors.initials: DJ
authors.lastname: Taatjes
mesh_terms: D002843:Chromatin / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D056892:Mediator Complex; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism*; D006656:Protamine Kinase / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D002843:Chromatin; D003067:Coenzymes; D016213:Cyclins; C460107:MED12 protein, human; C488666:MED13 protein, human; D056892:Mediator Complex; D046912:Multiprotein Complexes; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D014157:Transcription Factors; D006656:Protamine Kinase; C095686:CDK8 protein, human; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases