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.

FIG. 1.

Purified recombinant CDK8 subcomplexes. (A) Silver-stained gels of each subcomplex: wild-type CDK8/cyclin C/Med12/Med13 (r 4wt), kinase-dead CDK8/cyclin C/Med12/Med13 (r 4kd), wt CDK8/cyclin C/Med12 (r 3wt 12), wt CDK8/cyclin C/Med13 (r 3wt 13), wt CDK8/cyclin C (r 2 wt), and kd CDK8/cyclin C (r 2kd). (B) Western analysis of the various purified 4-protein CDK8 subcomplexes as well as the three- and two-protein CDK8 partial subcomplexes. This analysis not only shows the presence of CDK8, cyclin C, Med12, and Med13 in the four-protein complexes but also confirms the presence or absence of specific subunits in the partial subcomplexes. (C) MS/MS data obtained by in gel trypsin digestion of the recombinant CDK8 subcomplex (r 4wt). Shown are the percent peptide coverage and the total number of unique peptide matches in the human IPI database. (D) Med13 gel migration is phosphorylation dependent. Phosphorylation-specific electrophoretic mobility shift assay of r 4wt and r 4kd subcomplexes, displaying a shift in Med13 subunit mobility from below Med12 when hypophosphorylated to above Med12 when hyperphosphorylated. Note that this shift requires active CDK8.

FIG. 2.

The stoichiometry of the CDK8 subcomplex is 1:1:1:1. (Top) A table summarizing the various tandem purification techniques used to determine the stoichiometry of the recombinant CDK8 subcomplex. Molar ratios of cyclin C to Med 12 are listed as determined by densitometry. (Bottom) Representative SYPRO-Ruby-stained gel of purified recombinant CDK8 subcomplex consisting of Glu-CDK8, cyclin C, GST-Med12(aa1-1227), and Med13. An asterisk denotes a nonspecific band that interferes with direct quantitation of CDK8.

FIG. 3.

The human CDK8 subcomplex is a histone kinase. (A) Kinase assays with subcomplexes alone (r 4wt; r 4kd) or together with purified GST-Pol II CTD (CTD), as well as a kinase assay with CDK8-Mediator. (B) Kinase assays with r 4wt or r 4kd CDK8 subcomplexes with purified Drosophila core histones (CH) as substrates. Purified TFIIH was also tested as shown. (C) Silver stain of purified TFIIH. (D) Kinase assays comparing the activity of the recombinant CDK8 subcomplex and TFIIH on the Pol II CTD. (E) The human CDK8 subcomplex phosphorylates S10 within histone H3. After incubation with ATP, reaction mixtures containing recombinant core histones (r CH) alone or together with the CDK8 subcomplex were separated by SDS-PAGE and probed with H3 phospho-specific antibodies as shown. The control lane contains Drosophila core histones.

FIG. 4.

CDK8 incorporation into Mediator is required for modification of chromatin templates. (A) Chromatin assembly as assessed by micrococcal nuclease digestion. (B) Kinase assays comparing activity of CDK8-Mediator (Med) or the recombinant CDK8 subcomplex (r 4wt) against core histone octamers or chromatin, as indicated.

FIG. 5.

Med12 activates the CDK8 kinase. (A) Kinase assays with the various subcomplexes and partial subcomplexes: wt or kd CDK8/cyclin C/Med12/Med13 (r 4wt or r 4 kd), wt CDK8/cyclin C/Med12 (r 3wt 12), wt CDK8/cyclin C/Med13 (r 3wt 13), wt or kd CDK8/cyclin C (r 2wt or r 2kd). (B) Kinase assays with subcomplexes described in panel A with addition of GST-Pol II CTD (CTD) or core histone octamers as substrates (Sub). Note that the same autoradiogram exposure is shown for CTD and core histones in the upper panels and a longer exposure for H3 is shown at the bottom.

FIG. 6.

Purification of an endogenous CDK8 subcomplex. (A) Western blot showing CDK8 subcomplex components in the P0.3M fraction. Each lane contained 10 μg of total protein. (B) Purification scheme used for isolation of the endogenous CDK8 subcomplex.

FIG. 7.

MS analysis reveals several factors associated with the endogenous CDK8 subcomplex. (A) Immunoblot analysis of endogenous CDK8 (eK8) showing presence of CDK8, cyclin C, Med12, and Med13, but not other core Mediator subunits. A crude Mediator preparation, consisting of core Mediator and CDK8-Mediator, was used as a positive control. (B) SYPRO-Ruby-stained polyacrylamide gel of the endogenous CDK8 subcomplex (eK8). The identity of each band is indicated and was determined by MS. The number of unique peptide matches is shown in parenthesis. Peptide matches shown each had a Mascot Mowse score greater than 30. Note that 2 core Mediator subunits were detected in the analysis (Med14 and Med24). *, the CDK8L band contained six total nonredundant peptide identifications, with four peptides unique to CDK8L within the human IPI database and two peptides that overlap with the CDK8 sequence. †, The CDK8 band contained two nonredundant peptide matches, with one unique to CDK8 and one that is shared with CDK8L. (C) Immunoblots confirming the MS data that DNA-PK and the TRiC component TCP-1 β are both present in the purified eK8 sample. (D) Coimmunoprecipitation experiments demonstrating the association of the CDK8 subcomplex (as depicted through the Med13 and CDK8 subunits) with the TRiC chaperonin (as probed through the TCP-1 β subunit) or DNA-PK. (E) Representative 2D class averages that are consistent with the structure of the TRiC chaperonin with its interior cavity occupied with cargo protein. Bar, 100 Å.

FIG. 8.

Kinase activity of the endogenous CDK8 subcomplex. (A) Kinase assays showing that the endogenous CDK8 complex (eK8), like the recombinant (r 4wt), phosphorylates free histone octomers but not chromatin templates. (B) DNA-PK does not contribute to the observed kinase activity of eK8. Kinase assays of recombinant DNA-PK (lanes 1 and 2) or eK8 samples (lanes 3 to 6) with substrates as noted. Experiments were completed in the absence or presence of the DNA-PK inhibitor wortmannin (wm) at a concentration of 2 μM. (C) The endogenous CDK8 subcomplex does not phosphorylate the Pol II CTD. Kinase assays of recombinant or endogenous CDK8 subcomplexes with the Pol II CTD as substrate. Samples were normalized for kinase activity on H3 (from panel A). Relative kinase activity toward the Pol II CTD as determined by densitometry is shown as a percentage below the autoradiogram.

FIG. 9.

A significant percentage of CDK8 submodule components in human cells may exist in the eK8 subcomplex. (A) Immunoblot assays for CDK8 subcomplex components after Superose 6 size exclusion chromatography was performed on HeLa nuclear extracts. Titrations of chromatography input extracts (shown at left) were used to normalize quantitations shown in panel B. The fraction number is indicated above the blots, and the elution peak positions of the protein size standards are shown below. (B) Table summarizing densitometry quantitation of immunoblots shown in panel A, as explained in Materials and Methods.