MOLECULAR AND CELLULAR BIOLOGY, Feb. 2009, p. 650–661
0270-7306/09/$08.00⫹0 doi:10.1128/MCB.00993-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 3
The Human CDK8 Subcomplex Is a Histone Kinase That Requires
Med12 for Activity and Can Function Independently of Mediator䌤
Matthew T. Knuesel,1 Krista D. Meyer,1 Aaron J. Donner,2 Joaquin M. Espinosa,2 and Dylan J. Taatjes1*
Department of Chemistry and Biochemistry1 and Department of Molecular, Cellular, and Developmental Biology,2
University of Colorado, Boulder, Colorado 80309
Received 23 June 2008/Returned for modification 17 July 2008/Accepted 17 November 2008
humans. For instance, a single point mutation in Med12
(R961W) has been linked to a rare syndrome affecting brain
development and mutations within a different region in
Med12—the Q-rich domain in its C terminus—can cause mental retardation in males (46, 48). Furthermore, congenital heart
and neuronal defects can result from mutations within an isoform of Med13 (39), and CDK8 has been directly implicated in
oncogenesis (12, 38).
Clearly, the subunits within the CDK8 subcomplex are important for proper control of developmental programs, and yet
the biochemical mechanisms by which they regulate these processes are not fully defined. For one, it is unclear whether the
CDK8 subcomplex functions only in the context of Mediator or
whether it may operate in part as a separate, independent
entity. Furthermore, no biochemical function has been attributed to Med12 or Med13, which together comprise a major
portion (500 kDa) of the 600-kDa CDK8 subcomplex. Indeed,
nearly all known regulatory functions of the CDK8 subcomplex
have been attributed to its kinase activity (20, 40). For example, phosphorylation of different activators by yeast CDK8
(also called srb10) can alter their activity or cellular stability (8,
42, 54). Yeast CDK8 can also phosphorylate the RNA polymerase II C-terminal domain (Pol II CTD) prior to preinitiation complex assembly to inhibit transcription initiation (18),
whereas human CDK8 appears to switch off transcription by
phosphorylating cyclin H, a critical regulatory subunit within
TFIIH (1). Thus, the kinase activity of CDK8 is a powerful
regulator of gene expression. However, nothing is known about
how CDK8 may be regulated, and few CDK8 substrates have
been identified, particularly in humans.
We describe here the isolation and enzymatic activity of the
CDK8 subcomplex purified both directly from human cells and
also via recombinant expression of human CDK8, cyclin C,
Med12, and Med13 in insect cells. Although our studies indi-
The Mediator complex is a general target of DNA-binding
transcription factors and is required for expression of virtually
all protein-coding genes (35). Four subunits—CDK8, cyclin C,
Med12, and Med13—are known to reversibly associate with
Mediator and modulate its biochemical function. In humans,
CDK8, cyclin C, Med12, and Med13 are believed to associate
as a subcomplex based in part upon biochemical and genetic
experiments in yeast and lower metazoans. In fact, a stable
“CDK8 subcomplex” containing CDK8, cyclin C, Med12, and
Med13 has been isolated from yeast cells expressing TAPtagged cyclin C (5). Genetic experiments in yeasts, Caenorhabditis elegans, and Drosophila melanogaster indicate that CDK8,
cyclin C, Med12, and Med13 are each required for organism
(but not cell) viability and function primarily as a unit (6, 20,
33, 53). That is, similar phenotypes result from mutation of any
single subunit within the CDK8 subcomplex (7). Furthermore,
the expression of CDK8, cyclin C, Med12, and Med13 is regulated differently with respect to other consensus Mediator
subunits, at least in yeast (20). Genetic studies have also revealed that components of the CDK8 subcomplex play critical
roles in development (60). For example, ablation of the CDK8
kinase in mice results in lethality at the preimplantation stage
(57); deletion of Med12 causes severe defects in brain and
neuronal development in C. elegans and zebrafish (21, 47, 56,
61), whereas the loss of either Med12 or Med13 causes identical defects in Drosophila eye and wing development (23, 52).
Significantly, a number of disease-causing mutations in CDK8
subcomplex components are beginning to be uncovered in
* Corresponding author. Mailing address: Department of Chemistry
and Biochemistry, University of Colorado, Campus Box 215, Boulder,
CO 80309. Phone: (303) 492-6269. Fax: (303) 492-5894. E-mail: taatjes
@colorado.edu.
䌤
Published ahead of print on 1 December 2008.
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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.
VOL. 29, 2009
HUMAN CDK8 SUBCOMPLEX
cate the free CDK8 submodule can operate independently, it is
clear that Mediator itself regulates CDK8 activity. Moreover,
mass spectrometry (MS) and biochemical analyses suggest alternate factors work to control CDK8 submodule activity and
stability apart from Mediator in cells. Significantly, we identify
novel substrates for the CDK8 kinase (histone H3, Med13, and
CDK8 itself) that were not anticipated based upon previous
studies in yeast. Furthermore, our studies have uncovered a
key biochemical distinction between Med12 and Med13 that
also appears unique to higher organisms. Together, these results provide insight into CDK8 subcomplex regulation and
suggest the subcomplex itself may regulate gene expression
independently of Mediator in human cells.
MATERIALS AND METHODS
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
stained with SYPRO-Ruby or Coomassie protein stains and quantitated by using
densitometry.
Purification of CDK8-Mediator. Nuclear extract from 100L of HeLa cells was
passed over a phosphocellulose column as described above. After the 0.3 M KCl
wash Mediator was eluted 0.5 M KCl–1 mM MgCl2 HEG and loaded onto a
HiTrap Q column (GE Healthcare). The flow through from the Q column was
incubated with GST-SREBP or GST-VP16 bound GSH resin, washed with 250
cv 0.5 M HEGN, 50 cv 0.15 M KCl HEGN eluted with 30 mM GSH and run on
a linear 15 to 40% glycerol gradient.
Mass spectrometry. Recombinant CDK8 subcomplex or TCA precipitated
endogenous CDK8 subcomplex samples were prepared for analysis by mass
spectrometry as described previously (25). Briefly, the samples were separated by
SDS-PAGE, SYPRO stained, reduced with DTT, alkylated with iodoacetamide,
and digested overnight with trypsin (Promega). Recombinant protein identification was done by using an Agilent LC/MSD Trap XCT fed by an Agilent 1100
series high-pressure liquid chromatograph, while endogenous samples were run
through a Waters nanoACUITY UPLC coupled to a Thermo Finnigan LTQ.
The resulting tandem MS (MS/MS) data was searched by using the MASCOT
search engine against the human IPI database.
Kinase assays. Reactions were carried out with 5 to 50 ng of purified CDK8
subcomplex and 125 to 500 ng of purified substrate in kinase buffer (25 mM Tris
[pH 8.0], 100 mM KCl, 10 to 100 ␮M ATP, 10 mM MgCl2, and 2 mM DTT) with
the addition of 2.5 ␮Ci of [␥-32P]ATP at 30°C for 60 min. Reactions were
separated by SDS-PAGE, silver stained, dried, exposed on a storage PhosphorScreen (GE Healthcare), and imaged with a Typhoon 9400 scanner (GE
Healthcare). For kinase assays involving DNA-PK, 500 ng of linear DNA was
added to activate DNA-PK, and 2 ␮M wortmannin was used to inhibit DNA-PK
function. Purified DNA-PK was obtained from Promega (V5811).
Phosphorylation-specific electrophoretic mobility shift assay. Recombinant
wild-type or kinase dead 4-protein CDK8 subcomplexes were incubated at 30°C
for 60 min in kinase buffer (50 mM Tris [pH 7.9], 100 mM NaCl, 10 mM MgCl2,
and 1 mM DTT) or in phosphatase buffer (supplemented with calf intestinal
phosphatase) at 37°C for 60 min. Proteins were then separated by SDS–5%
PAGE and visualized by silver staining.
Antibodies. Med12 (sc-5374), CDK8 (sc-1521), cyclin C (sc-1061), H3 (sc8654), GST tag (sc-138), TCP-1 ␤ (sc-1378), DNA-PK (sc-1552), and nucleolin
(sc-8031) were obtained from Santa Cruz. H3 phospho-S10 (catalog no. 06-570),
H3 phospho-S28 (catalog no. 07-145), and H3 phospho-T3 (catalog no. 07-424)
were from Upstate. Med13 (A301-278A) and Med23 (A300-425A) were from
Bethyl, Inc. Glu tag (ab1267) and His tag (ab9108) were from Abcam. Pol II
(8WG16), Pol II S2P (MMS-129R), and Pol II S5P (MMS-134R) were from
Covance. Med14 was a laboratory stock antibody.
Purification of core histones. Expression and reconstitution of recombinant
core histones was completed as described previously (34). Purification of Drosophila core histones was completed as described previously (27).
Chromatin assembly. Drosophila core histones were assembled with digested
5S G5E4 DNA into chromatin using the salt dialysis method as described previously (22). Assembled chromatin was visualized by ethidium bromide staining
of phenol-chloroform-extracted micrococcal-nuclease-digested samples.
Cell culture. HCT116 and HEK293 cells were maintained in McCoy’s 5A and
Dulbecco modified Eagle medium (Gibco-Invitrogen), respectively, supplemented with 10% fetal bovine serum and an antibiotic-antimycotic mix.
CDK8 and Med12 knockdowns. Short-hairpin RNAs (shRNAs) targeting either CDK8 or MED12 were generated and expressed using the third-generation
lentiviral delivery system previously described with minor modifications (10, 41).
Briefly, complementary oligonucleotides encoding shRNAs were cloned into the
lentiviral vector pLL3.7neo (a derivative of the original pLL3.7 vector with the
coding region of green fluorescent protein replaced with the coding region of
neomycin phosphotransferase). The shRNA/pLL3.7neo vectors were cotransfected into HEK293FT cells, along with the packaging vectors pMDLg/pRRE,
pRSV-REV, and pMD.G. After 48 h, viral supernatants were harvested and used
to transduce HCT116 cells. HCT116 clones that were neomycin resistant were
grown and screened by immunoblotting for knockdown of the appropriate target.
Cells were lysed in radioimmunoprecipitation assay buffer supplemented with a
cocktail of protease and phosphatase inhibitors.
Electron microscopy. Endogenous CDK8 subcomplex samples were negatively
stained with uranyl acetate, imaged at ⫻29,000 magnification on a Tecnai F20
FEG equipped with a Gatan 4k⫻4k charge-coupled device camera, and segregated into two-dimensional (2D) classes as described previously (36).
S6 fractionation of HeLa nuclear extracts. HeLa nuclear extracts were first
concentrated by ammonium sulfate precipitation (50% [wt/vol]). Concentrated
nuclear extract was passed through a Superose 6 10/300 GL column (GE Health-
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Baculovirus constructs and cell culture. Med13 was isolated from a HeLa
cDNA library and cloned into a modified pAC-GHLT vector (Pharmingen) in
which the glutathione S-transferase (GST) and His tags were removed. Med12
cDNA was obtained from the lab of R. G. Roeder and cloned into a modified
version of the pAC-GHLT vector that contained a C-terminal His tag. Cyclin C
and Glu-tagged CDK8 were gifts from Emma Lees. High-titer viruses of each
gene were coinfected into Sf9 cells at an multiplicity of infection ratio of 1:2:3:3
(K8/CC/12/13) at 27°C for 48 h.
Purification of recombinant CDK8 subcomplexes. Protease inhibitors (1 mM
benzamidine, 1 mM sodium metabisulfite, 1 mM dithiothreitol [DTT], 1.1 ␮g of
aprotinin/ml, and 25 ␮M phenylmethylsulfonyl fluoride) were added to all buffers
immediately before use. Sf9 cells expressing recombinant CDK8 subcomplex
components were lysed by Dounce homogenization in 50 mM Tris (pH 7.6), 150
mM NaCl, and 0.1% NP-40. Extracts were incubated with anti-Glu matrix
(Abcam ab24587), washed with 400 column volumes (cv) 0.5 M KCl HEGN
(0.1% NP-40, 50 mM HEPES [pH 7.6], 0.1 mM EDTA, 10% glycerol) and 80
cv 0.15 M KCl HEGN (0.02% NP-40, 50 mM HEPES [pH 7.6], 0.1 mM EDTA,
10% glycerol) and eluted with 0.15 M KCl HEGN supplemented with 1 mg of
Glu-Glu peptide/ml. Elutions were loaded onto a glycerol gradient containing
300 ␮l of 80% glycerol, 850 ␮l of 40% glycerol, and 1,050 ␮l of 20% glycerol in
0.15 M KCl HEGN and spun at 200,000 ⫻ g for 6 h in a Beckman TLS-55 rotor.
The final glycerol gradient size exclusion served to remove excess free CDK8/
cyclin C from the full complex but did not affect kinase activity.
Purification of the endogenous CDK8 subcomplex. A typical preparation was
started with HeLa nuclear extract from a 500-liter portion of cells; this was
loaded on a Whatman P11 phosphocellulose column at 0.1 M KCl and eluted
with 0.3 M KCl–1 mM MgCl2 HEG, dialyzed to 0.15 M KCl, and passed over a
HiTrap Q column (GE Healthcare). Flowthrough fractions were precipitated by
the addition of 50% (wt/vol) ammonium sulfate and further concentrated by
using Amicon spin filters (Millipore). Residual Mediator was depleted by GSTSREBP affinity chromatography. Superose 6 fractionation isolated 400- to 800kDa complexes. After preclearing with protein A/G resin (GE Healthcare),
concentrated Superose 6 fractions were incubated with anti-CDK8 antibodybound A/G resin, washed with 50 cv 0.5 M KCl HEGN and 10 cv 0.15 M KCl
HEGN and eluted with 50 mM Tris (pH 8.0), 0.75 M ammonium sulfate, 40%
ethylene glycol, and 0.1 mM EDTA. Eluates were desalted into 0.15 M KCl
HEGN with a QuickSpin TE column (Roche) preblocked with bovine serum
albumin; loaded onto a glycerol gradient containing 100 ␮l of 30% glycerol, 850
␮l of 20% glycerol, and 1,150 ␮l of 15% glycerol in 0.15 M KCl HEGN; and spun
as stated above. Endogenous CDK8 subcomplex was retained in the 30% glycerol fraction. (See Fig. 6 for an overview of the purification protocol.)
CDK8 subcomplex stoichiometry experiments. A modified CTD-truncated
GST-tagged Med12 [GST-Med12(aa1-1227)] was incorporated into the recombinant CDK8 kinase-dead complex by using the techniques outlined above.
Having tags on two of the subunits [Glu-CDK8 and GST-Med12(aa1-1227)]
allowed for the multistep affinity purifications summarized in Fig. 2. Purifications
included combinations of the following procedures: (i) Glu purification as described above; (ii) GSH-resin purification; (iii) glycerol gradient sedimentation
(as outlined in above) using 20 to 80% glycerol; and (iv) anti-Med13 immunoprecipitation, followed by elution with 2% Sarkosyl. Since Sarkosyl disrupted
protein-protein interactions, the anti-Med13 purification technique could be
used only as a final step. For each affinity purification step (i.e., steps i to iv),
bound material was washed extensively with 0.3 to 0.5 M KCl HEGN prior to
elution from the resin. After tandem purifications, proteins were separated by
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MOL. CELL. BIOL.
care). Immunoblots of fractions collected were quantitated by using densitometry. Fractions larger than 1.4 MDa were considered to contain CDK8-Mediator,
whereas fractions ranging from 1.4 MDa to 400 kDa were considered as eK8, and
fractions with protein complexes smaller than 400 kDa were considered to
consist of partial subcomplexes.
Quantitative Western analysis of Superose 6-fractionated HeLa nuclear extracts resulted in estimates of ca. 30% of the CDK8 subcomplex components
present in the fractions between 1.4 MDa and 400 kDa—fractions consistent with
the size of the endogenous CDK8 subcomplex. In agreement with this, when
fractionated over a phosphocellulose column, ca. 30% of the total amounts of the
CDK8 subcomplex components probed were retained in the P0.3M fraction (see
Fig. 6A). Since the P0.3M fraction is greatly enriched for the endogenous CDK8
subcomplex, we view this as further evidence that as much as 30% of CDK8
subcomplexes may exist independently of Mediator in human cells. Proportionally more CDK8 (more than 50%) was found in the fractions from 400 kDa to 1.4
MDa, implying that other large complexes containing CDK8 may exist.
RESULTS AND DISCUSSION
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. Phosphorylationspecific 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.
fered a means to systematically examine the kinase activity of
the CDK8 subcomplex.
A 1:1:1:1 stoichiometry for CDK8/cyclin C/Med12/Med13
within the CDK8 subcomplex. Assessing the stoichiometry of
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Recombinant CDK8 subcomplexes. To generate the recombinant CDK8 subcomplex, Sf9 cells were coinfected with hightiter viruses for CDK8, cyclin C, Med12, and Med13. The
CDK8 subcomplex was purified from whole-cell extracts by
immunoprecipitation with an anti-Glu resin, which targeted
the Glu-tagged CDK8 subunit within the subcomplex. Fractionation of the extracts over ion-exchange and gel filtration
columns prior to immunoprecipitation did not measurably improve the purity of the subcomplex (data not shown). A similar
strategy was used to purify the kinase-dead CDK8 subcomplex,
which contained a D173A point mutation in CDK8 that inactivates its kinase function (1). Silver-stained gels of the active
(r 4wt) and kinase-dead (r 4kd) CDK8 subcomplexes are
shown in Fig. 1A. Importantly, each subunit comigrated on a
glycerol gradient, confirming their association in a large complex (data not shown). Immunoblots and electrospray MS/MS
(ESMS) analysis confirmed the identity of each subunit as
Med13, Med12, CDK8, and cyclin C (Fig. 1B and C). Furthermore, a 1:1:1:1 stoichiometry was established for the CDK8
subcomplex based upon densitometry measurements (see below). Thus, it appeared that human CDK8, cyclin C, Med12,
and Med13 could assemble into a stable complex from recombinantly expressed subunits.
Comparison of silver-stained gels of the wild-type (4wt) and
kinase-dead (4kd) CDK8 subcomplexes revealed a shift in
Med13 and/or Med12 mobility, suggesting that these subunits
may be phosphorylated by CDK8. To test this, we treated the
recombinant wild-type (r 4wt) or kinase-dead (r 4kd) complexes with either ATP or phosphatase prior to gel electrophoresis. As shown in Fig. 1D, these experiments indicated the
mobility shifts were due to differential phosphorylation; Western blots (not shown) and ESMS data confirmed that phosphorylation was occurring on Med13 (see below).
To explore a potential biochemical function for Med12 and
Med13, we expressed and purified the CDK8/cyclin C pair
alone and also together with either Med12 or Med13. Each
three-subunit complex (CDK8/cyclin C/Med12 or CDK8/cyclin
C/Med13), as well as the CDK8/cyclin C binary complex, was
stable and could be purified by using standard techniques,
indicating that Med12 and Med13 each independently interact
with the CDK8/cyclin C pair (Fig. 1A and B). Subunits within
these partial subcomplexes also comigrated on a glycerol gradient, confirming their assembly in a complex (data not
shown). Together, these stable, recombinant subcomplexes of-
VOL. 29, 2009
the CDK8 submodule was complicated by several factors. First,
Med13 is phosphorylated, which impacts its migration on a gel
and causes it to overlap with the Med12 protein band. Specifically, Med13 in its unphosphorylated state migrates slightly
below Med12, whereas in intermediate stages it overlaps with
Med12; highly phosphorylated forms of Med13 will migrate
higher than Med12 (Fig. 1D). Because of its multiple phosphorylated states, Med13 is represented by multiple bands,
which hinders an accurate quantitation. Thus, Med12, which
shows no evidence of phosphorylation, represents a more reliable subunit for quantitation. However, because of overlapping Med12/Med13 bands, accurate quantitation of Med12
apart from Med13 was not possible. To remedy this problem,
we expressed a CTD-truncated version of Med12(aa1-1227). This
Med12 truncation does not impact its association with the subcomplex and has no apparent differences from full-length Med12
on impacting the CDK8 submodule kinase activity (data not
shown). With respect to quantitation, this truncated form of
Med12 enables a clear separation between Med12 and Med13 on
a gel to accurately quantify Med12.
With respect to CDK8/cyclin C, we focused on cyclin C,
because, like Med13, CDK8 presents multiple obstacles to
accurate quantitation. First, CDK8, like Med13, is phosphorylated and therefore migrates as several bands. Second, the
CDK8 band overlaps slightly with a nonspecific band that may
represent the heavy chain from the antibody purification (Fig.
2). Fortunately, such issues do not accompany cyclin C. Thus,
to ensure the most accurate results, we focused on Med12 and
cyclin C for the assessment of stoichiometry within the CDK8
submodule. The results, which indicate a 1:1 stoichiometry for
cyclin C and Med12, are summarized in Fig. 2. Densitometry
readings from Coomassie blue-stained gels were also consis-
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tent with a 1:1 stoichiometry between cyclin C and Med12.
Despite the potential uncertainty in quantitation of CDK8 in
these samples (noted above), the molar ratio of cyclin C to
CDK8 ranged from 1:0.73 to 1:1.31 in our experiments, also
suggesting a 1:1 overall stoichiometry. This could also be anticipated based upon predicted CDK8/cyclin C pairing within
the complex.
Despite the problems associated with assessing the molar
ratio of Med13 with densitometry (see above), it is worth
noting that our measurements suggested a 0.5:1 ratio of Med13
relative to cyclin C. We are quite confident the true molar ratio
is 1:1 because of the uncertainty due to its multiply phosphorylated states and also for additional reasons outlined below.
First, in testing alternate purification protocols for the CDK8
subcomplex, we noted that Med13 could be selectively dissociated with 500 mM imidazole, suggesting that Med13 may be
slightly labile within the subcomplex. Second, using titration
gel densitometry techniques (plotting band intensity versus
sample load), we noted that Med13 increased at the same ratio
(i.e., the same slope) as cyclin C, suggesting a 1:1 ratio. Third,
the molecular mass of a 1:1:1:1 subcomplex (600 kDa) is consistent with our electron microscopy (EM) data of the recombinant CDK8 subcomplex (M. T. Kneusel and D. J. Taatjes,
unpublished results), whereas a 950-kDa subcomplex (required
for a 2:2:2:1 ratio) is not. Taken together, these data suggest
Med13 is present in a 1:1 molar ratio with cyclin C and the
stoichiometry of each subunit within the CDK8 subcomplex is
1:1.
Identification of novel substrates for the CDK8 kinase. Although few substrates have been identified for human CDK8,
this kinase clearly plays important roles in gene regulation (1)
and has been linked to oncogenic transformation in colon
cancer (12, 38). Known substrates of CDK8 include TFIIH and
the CTD of the large subunit of Pol II (1, 31). As expected, the
recombinant human subcomplex phosphorylated TFIIH (data
not shown) and the Pol II CTD, whereas the subcomplex
containing a point mutation in CDK8 did not (Fig. 3A, lanes 4
and 5). Phosphorylated bands corresponding in size to CDK8
and Med13/Med12 were also evident with the active CDK8
subcomplex but not in assays with the kinase-dead mutant (Fig.
3A, compare lanes 4 and 5). Subsequent ESMS analysis of
each phosphorylated band confirmed their identities as CDK8
and Med13 (data not shown). Because Med13 and CDK8 were
phosphorylated in the active subcomplex, but not the kinasedead counterpart, we conclude that CDK8, and not a potential contaminating kinase activity, was phosphorylating
these substrates. Phosphorylation of CDK8 and Med13 was
also observed in the context of the entire CDK8-Mediator
complex (Fig. 3A, lane 6), indicating that subunit specificity
is not altered upon subcomplex association with core Mediator. CDK8-Mediator was also observed to phosphorylate
the Pol II CTD, as expected (data not shown). The absence
of additional kinase substrates within Mediator itself suggests that the CDK8 subcomplex occupies a distinct, separable domain within CDK8-Mediator, a finding consistent
with past structural studies (50).
During the course of this work, our lab isolated a derivative
CDK8-Mediator complex that efficiently phosphorylated histone H3 in vitro (36). This result suggested that CDK8 is a
histone H3 kinase, at least when associated with Mediator. To
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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.
HUMAN CDK8 SUBCOMPLEX
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test whether the recombinant CDK8 subcomplex alone would
display similar H3 kinase activity, we performed kinase assays
against purified core histone octamers. The recombinant wildtype CDK8 subcomplex—but not the kinase-dead mutant—
preferentially phosphorylated histone H3 within core histone
octamers (compare Fig. 3B, lanes 5 and 6). Similar results were
obtained with recombinant core histones (data not shown). As
an additional control, we purified human TFIIH (Fig. 3C) to
test in the histone kinase assays. TFIIH is generally required to
initiate transcription and contains a kinase, CDK7, that is
critical for its biochemical function. Both CDK8 and CDK7 are
capable of modifying the Pol II CTD in vitro (Fig. 3D); however, TFIIH was unable to phosphorylate histone octamers in
this assay (Fig. 3B, lane 7), indicating a significant difference in
CDK7 versus CDK8 substrate specificity. Given the emerging
role of H3 phosphorylation in active gene expression (see below), this observation identifies a means by which CDK8 may
positively regulate gene expression in human cells.
To identify the H3 residue(s) modified by the CDK8 subcomplex, we focused on the N-terminal tail of histone H3,
which is a common site for posttranslational modifications. A
series of kinase assays with purified, recombinant H3 were
performed; antibodies against different phosphorylated forms
of H3 were then used to probe for potential site(s) of CDK8
modification. As shown in Fig. 3E, these experiments revealed
that H3S10, but not H3T3 or H3S28, is the major site for
phosphorylation by the CDK8 subcomplex. Notably, whereas
phosphorylation of H3T3 or H3S28 does not correlate with
transcription, H3S10 phosphorylation is strongly tied to transcriptional activation (44). For example, H3S10 phosphorylation helps maintain a transcriptionally active state by inhibiting
methylation of the adjacent H3K9 site, thereby preventing
assembly of repressive HP1 complexes on chromatin (13, 19).
Moreover, H3S10 phosphorylation strongly correlates with activation of genes regulated by such diverse activators as NF-␬B,
RAR␤2, and myc in human cells (3, 28, 59, 64); furthermore,
activation of heat shock genes in Drosophila occurs with a
corresponding increase in H3S10 phosphorylation only at heat
shock loci (43).
Recent work in our laboratory has shown that within the
Mediator complex, CDK8 can efficiently modify H3 within
histone octamers or chromatin templates (36). However, the
activity of the free submodule may vary. Although the data in
Fig. 3 reveals the free CDK8 submodule can phosphorylate
histone octamers, it does not address activity on chromatin.
Therefore, we assembled core histone octamers into chromatin
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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.
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(Fig. 4A) and tested whether the free CDK8 submodule would
similarly modify H3 in this context. As shown in Fig. 4B, the
free CDK8 submodule was not able to modify H3 in a chromatin context, even when doubling the amount of CDK8 submodule used (lane 8) or doubling the amount of chromatin
added (lane 16). Only when incorporated into Mediator could
CDK8 efficiently phosphorylate H3 within chromatin (Fig. 4B,
lanes 5 and 6 and lanes 11 and 12), and H3 phosphorylation
was enhanced if CDK8-Mediator was recruited to chromatin
by an activator (data not shown). These results indicate a
major change in CDK8 function and demonstrate that Mediator itself acts to regulate CDK8 substrate specificity. Because
the majority of H3 is tied up within chromatin in interphase
cells, most CDK8-dependent phosphorylation of H3 likely occurs when associated with Mediator. However, it remains plausible that the free CDK8 subcomplex may cooperate with histone chaperones and/or chromatin disassembly factors to
indirectly modify chromatin templates.
Although some studies have linked CDK8 to transcriptional
activation (9, 14, 32), the observation that CDK8 can mediate
H3S10 phosphorylation provides a molecular mechanism by
which CDK8-dependent activation might occur. Although
other transcriptionally relevant kinases have been linked to
H3S10 modification (e.g., PIM1 and IKK␣), these appear to
regulate only a subset of genes (3, 59, 64). In contrast, CDK8
(as well as core Mediator) appears to be recruited to promoter
and enhancer regions genome-wide (2, 63), suggesting that
H3S10 phosphorylation by CDK8 may be a general phenomenon. In support of this, shRNA knockdown of CDK8 causes
a dramatic reduction in H3 containing the dual modification
H3S10P/K14Ac, as reported previously (36). This tandem H3
modification results from cooperative activity between CDK8
and GCN5L within Mediator. Upon probing H3S10P alone,
we observed that CDK8 knockdown did not significantly reduce the levels of this singly modified H3 in cells (data not
shown). This result likely reflects high H3S10P levels from
Aurora B kinase (a marker for mitosis) and also suggests
CDK8 primarily functions within T/G-Mediator (i.e., together
with GCN5L) when phosphorylating H3 (36).
Med12 activates the CDK8 kinase. Numerous biochemical
roles for the CDK8/cyclin C pair have been identified (albeit
mostly in yeast). No clear function, however, has been determined for Med12 or Med13. Previous biochemical and genetic
data in yeast indicated that ablation of either Med12 or Med13
is phenotypically similar to mutations that inactivated CDK8
(7, 40). However, subtle developmental differences have been
noted upon comparing null mutants in Drosophila: although
CDK8, cyclin C, Med12, or Med13 mutant phenotypes are
largely similar, Med12 or Med13 knockout cells did display
differences in tarsal development compared to cells null for
CDK8 or cyclin C (33). Interestingly, when the three-subunit
partial CDK8 subcomplexes (CDK8/cyclin C/Med12 and
CDK8/cyclin C/Med13 [Fig. 1A]) were tested for kinase function, only the three-subunit complex containing Med12 was
active, whereas the CDK8/cyclin C/Med13 complex showed
only weak activity toward the human CDK8 substrates tested
(Fig. 5). In fact, the three-subunit CDK8/cyclin C/Med12 complex possessed kinase activity comparable to the wild-type,
four-subunit complex for all substrates tested. In addition, it is
worth noting that the CDK8/cyclin C dimer was inactive in
these assays, unless titrated to 10-fold higher concentration
relative to the four-subunit CDK8 subcomplex (Fig. 5, lanes 7
and 15). We also used shRNA to knock down Med12 expression to further test the role of Med12 on CDK8 activity in
human cells. However, this knockdown coordinately decreased
CDK8 protein levels (data not shown) and therefore mimicked
the CDK8 knockdown described above.
The data in Fig. 5 reveal a previously unidentified biochemical function for human Med12 that distinguishes it
from Med13. Future work will be necessary to identify the
mechanism by which Med12 activates the CDK8 kinase. Notably, such a regulatory role for Med12 in yeast is not evident,
as CDK8/cyclin C retains its kinase function independent of
Med12 and Med13 in vitro (40). Whether Med12 is required
for CDK8 activity in lower metazoans is not known; genetic
experiments in worms and flies indicate that Med12- or
Med13-null mutations are indistinguishable during embryonic
development (6, 23, 52, 55, 60). However, Fraser et al. recently
completed a comprehensive RNA interference screen in C.
elegans which identified Med12 as one of only six “hub” genes
that interact with a large number of genes from different signaling pathways (29). Whether this distinction is related to
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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.
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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.
Med12 regulation of CDK8 kinase activity remains to be determined, but this C. elegans screen does suggest that Med12
may perform biochemical functions distinct from Med13 even
in lower metazoans.
Isolation of an endogenous CDK8 subcomplex identifies
potential regulatory cofactors. The purification of a stable,
active human CDK8 subcomplex from recombinant subunits
suggested that a similar subcomplex may exist in human
cells. Although previous work has clearly demonstrated that
CDK8 can reversibly associate with Mediator in human cells
(37, 45), it has not been established whether free, endogenous CDK8 subcomplexes serve any potential regulatory
role independent of Mediator. Given the kinase targets of
the recombinant CDK8 subcomplex identified here (which
include TFIIH, the Pol II CTD, and histone H3), it was
important to establish whether the free CDK8 submodule
may exist as a stable, biochemically active entity in cells.
Therefore, we sought to isolate free CDK8 subcomplexes
FIG. 6. Purification of an endogenous CDK8 subcomplex. (A) Western blot showing CDK8 subcomplex components in the P0.3M fraction.
Each lane contained 10 ␮g of total protein. (B) Purification scheme used
for isolation of the endogenous CDK8 subcomplex.
directly from human cells. Initial fractionation of HeLa nuclear extract over a phosphocellulose column allowed us to
separate a potential CDK8 subcomplex from the majority of
CDK8-Mediator: CDK8-Mediator was enriched in the
P0.5M fraction, whereas CDK8 subcomplex components
were also present in the P0.3M fraction (Fig. 6A). Further
purification from the P0.3M fraction was completed by
tracking kinase activity and protein subunits over additional
ion-exchange, gel filtration, and affinity resins (Fig. 6B).
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This purification scheme resulted in isolation of a complex
containing CDK8, cyclin C, Med12, and Med13 that was free
of core Mediator subunits, as shown by immunoblot assays
(Fig. 7A).
Most convincingly, MS analysis confirmed the presence of all
four CDK8 subcomplex components in this sample (Fig. 7B),
whereas core Mediator subunits were not represented (however, see Fig. 7B). Notably, CDK8-like (CDK8L; also called
CDC2L6 or CDK11), a paralog of CDK8, was observed to
copurify with the endogenous CDK8 subcomplex. Immunoblots of the purified endogenous subcomplex showed no evidence of the CDK8 antibody cross-reacting with CDK8L. For
this reason, and the fact that the CDK8 antibody used in the
final affinity purification step is directed against a region of
CDK8 (amino acids 424 to 464) not conserved within CDK8L,
we conclude that CDK8L most likely associates together with
CDK8 in a single subcomplex. Confirmation of this will ultimately require reagents specific for CDK8L.
MS analysis also identified other submodule-associated factors, including DNA-PK, GCN1L1, and the TRiC chaperonin
complex (Fig. 7B). The presence of DNA-PK and TRiC was
further substantiated with immunoblotting experiments (Fig.
7C; antibodies against CDK8L and GCN1L1 are not available). In addition, CDK8 subcomplex components were observed to coimmunoprecipitate with DNA-PK and the TRiC
subunit TCP-1␤ (Fig. 7D). Given the rigorous purification
protocol, including a final CDK8 antibody binding/elution step
followed by separation based upon size (glycerol gradient
step), it is likely that TRiC, DNA-PK and GCN1L1 are in fact
associated with the CDK8 submodule and do not copurify as
individual entities. However, we cannot rule out the possibility
that TRiC, DNA-PK, or GCN1L1 may bind the CDK8 submodule in a mutually exclusive fashion. Collectively, the data
in Fig. 7 demonstrate that the free CDK8 subcomplex can exist
as a stable entity in human cells. Factors associated with the
endogenous CDK8 subcomplex, however, distinguish it from
the recombinant, four-subunit CDK8 subcomplex and suggest
a means to regulate its activity and stability within cells. Given
the critical role of CDK8 in gene regulation and oncogenesis
(1, 12, 38), it is likely that multiple mechanisms have evolved to
control its biological function.
The biological relevance of DNA-PK, GCN1L1, and TRiC
association with the CDK8 submodule awaits elucidation in
future studies. DNA-PK is well established in DNA repair
mechanisms and is peripherally implicated in transcriptional
regulation (49); much less is known about GCN1L1, although
its yeast ortholog has been shown to bind and regulate the
GCN2 kinase (15, 26). The copurification of the chaperonin
TRiC with the endogenous CDK8 submodule is particularly
intriguing. TRiC does not appear to be a general chaperonin.
Instead, it displays specificity for a subset of regulatory complexes, including elongin BC and SMRT-HDAC3 (11, 16). The
results shown here identify the CDK8 submodule as another
potential TRiC-regulated complex. Notably, TRiC is critical
for the formation of an active human cyclin E/CDK2 complex
(58), and therefore its association with the CDK8 submodule
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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 ␤ 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 ␤ 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.
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may reflect similar roles in the formation of a stable, active
subcomplex. Also noteworthy is the ability of TRiC to modulate aggregation of poly-Q tracts in the Huntington protein (4,
51). Med12 contains a Q-rich region at its C terminus and
TRiC may help regulate interactions from this domain.
That TRiC might regulate CDK8 submodule interactions is
further supported by EM analysis of the endogenous CDK8
submodule. Alignment and 2D classification of single-particle
images revealed the presence of complexes of size and shape
consistent with the free CDK8 submodule, as expected (data
not shown). In addition, 2D averages of single-particle images
clearly showed TRiC complexes whose large, central cavity was
occupied with protein density (Fig. 7E). Mammalian TRiC has
a distinct barrel shape consisting of stacked eight-membered
rings with an outer diameter of ⬃150 Å and a length of 160 Å.
TRiC utilizes ATP hydrolysis to aid in folding cargo proteins
located within its 80 Å-diameter interior cavity. EM analysis
and 3D reconstruction of the recombinant CDK8 subcomplex
(M. T. Knuesel, K. D. Meyer, and D. J. Taatjes, unpublished
results) indicates that its overall dimensions are slightly larger
than the TRiC cavity could accommodate. However, conformational shifts within the CDK8 subcomplex may facilitate its
sequestration within the TRiC central cavity, and/or the CDK8
submodule may exist as a partially assembled entity within
TRiC. However, since both CDK8 and Med13 were found to
coimmunoprecipitate with an antibody against TRiC (Fig. 7D),
this suggests the entire CDK8 subcomplex might associate with
the TRiC chaperonin. Although additional experiments are
necessary to delineate the means by which TRiC might regulate the CDK8 submodule, the EM data suggest that TRiC
may sequester the submodule as a simple means of regulating
its interactions with other proteins such as core Mediator or its
kinase substrates.
The endogenous CDK8 subcomplex displays altered substrate specificity. Following purification of the endogenous
CDK8 subcomplex, we tested its kinase activity against known
human substrates. Notably, the endogenous subcomplex dis-
played similar—but not identical—kinase activity relative to
the recombinant subcomplex. The endogenous sample resembled the recombinant CDK8 subcomplex in its phosphorylation of histone H3, Med13, and CDK8 (Fig. 8A and data not
shown). Although these results may reflect the activity of associated factors—particularly CDK8L—the similarity relative
to the recombinant subcomplex implicates CDK8 in these
modifications. We cannot, however, rule out phosphorylation
by CDK8L in these experiments. CDK8L is a paralog of
CDK8 and may possess similar substrate specificity. In contrast, DNA-PK is unlikely to contribute to the kinase activity
observed in Fig. 8A because DNA-PK requires association
with DNA to become an active kinase (49) and the endogenous
subcomplex is not active in assays containing DNA (i.e., chromatin substrates [Fig. 8A]). Moreover, addition of the
DNA-PK inhibitor wortmannin had no effect on kinase activity
of the endogenous CDK8 subcomplex under the conditions
tested here (Fig. 8B).
Strikingly, the endogenous subcomplex was unable to phosphorylate the Pol II CTD. When normalizing for kinase activity
toward histone H3, the endogenous CDK8 subcomplex phosphorylated the CTD with 2% efficiency compared to the recombinant subcomplex (Fig. 8C). Given this clear difference in
substrate specificity relative to the recombinant subcomplex—
and relative to CDK8-Mediator, which can also phosphorylate
the Pol II CTD—we sought to further test this result in human
cells. Therefore, we examined whether knockdown of CDK8
would impact the cellular levels of phosphorylated Pol II CTD
and observed that CDK8 knockdown did not appreciably impact global CTD phosphorylation patterns (serine 2 or serine 5
[data not shown]). These results are consistent with studies of
yeast CDK8 (srb10), in which CDK8 kinase inhibition did not
notably impact global Pol II CTD phosphorylation (32). Because human (and yeast) cells contain multiple kinases capable
of Pol II CTD phosphorylation, the minimal impact of human
CDK8 knockdown on global CTD phosphorylation was not
unexpected. It remains possible that CDK8-dependent phos-
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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 ␮M. (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.
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phorylation of the Pol II CTD might regulate a subset of
human genes.
Concluding remarks. Given the functionally diverse targets
for the CDK8 kinase, including histone H3, TFIIH, Med13,
and Pol II CTD, precise temporal or context-dependent regulation of CDK8 substrate specificity may be a critical factor
controlling gene expression in humans. We have identified
several means by which CDK8 kinase activity might be regulated in cells. First, the kinase activity of the CDK8 submodule
is dependent upon Med12. Second, incorporation into Mediator regulates CDK8 substrate specificity toward H3S10 within
chromatin. Third, MS analysis of the endogenous CDK8 subcomplex identified CDK8L, the TRiC chaperonin, GCN1L1,
and DNA-PK as potential modifiers of CDK8 activity within
the free subcomplex, and evidence that TRiC might help regulate CDK8 submodule interactions was obtained by EM analysis of endogenous subcomplexes. It is also plausible that phosphorylation or other posttranslational modifications within the
subcomplex itself may impact its kinase activity. Since the
CDK8 subcomplex appears to regulate transcription genomewide (e.g., via regulation of Mediator), a variety of mechanisms
may exist to control CDK8 function.
That the CDK8 subcomplex is stable and biochemically active apart from Mediator in cells suggests the subcomplex may
function autonomously to regulate gene expression. However,
how might the free subcomplex be targeted to its substrates?
Notably, Med12 and CDK8 are known to interact directly with
a number of DNA-binding transcription factors, which would
provide a means for targeting the free CDK8 subcomplex to
specific genomic locations (14, 17, 24, 47, 56, 62). Alternately,
the CDK8 subcomplex may contain chromatin-targeting domains (e.g., cryptic bromodomains or chromodomains) that
remain unidentified. Recruitment may also be mediated
through one of the subcomplex-associated factors, such as
DNA-PK or the TRiC chaperonin, identified in the MS analysis. It is important to note, however, that the Mediator complex is more generally targeted by DNA-binding transcription
factors; consequently, association with Mediator may enable
more widespread targeting of the CDK8 subcomplex to appropriate regulatory sites. Although the majority of CDK8 subcomplexes appear to be Mediator-associated in cells, we estimate that a significant fraction (up to 30%) of CDK8
submodule components may exist independent of Mediator in
HeLa cells (see Materials and Methods and Fig. 9).
It is notable that histone H3, Med13, and CDK8 itself are
not modified by the CDK8 subcomplex in yeast, nor is the yeast
CDK8 subcomplex dependent upon Med12 for activity (18,
40). This is perhaps to be expected given the extensive sequence divergence within Mediator subunits over evolutionary
time. Indeed, compared to the rest of the general transcription
machinery (e.g., TFIID, TFIIH, and Pol II), Mediator has
diverged most significantly (30). This substantial sequence divergence may be reflected in the distinct activity and substrate
specificity of the human CDK8 subcomplex.
ACKNOWLEDGMENTS
We thank Bob Roeder for providing the Med12 cDNA, Emma Lees
for providing cDNAs for CDK8 and cyclin C, and Karolin Luger for
cDNAs encoding core histones. The 5S G5E4 template was a gift from
Jerry Workman.
This study was supported by the NCI P01 CA112181 (D.J.T.) and
R01 CA117907 (J.M.E.) and in part by NIH grants T32 GM07135 and
T32 GM065103 (M.T.K. and K.D.M.).
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