From the Department of Genetics and Tumor Cell
Biology and § Hartwell Center for Bioinformatics and
Biotechnology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105
Received for publication, July 25, 2002, and in revised form, October 8, 2002
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ABSTRACT |
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The PITSLRE protein kinases, hereafter referred
to as cyclin-dependent kinase 11 (CDK11) due to their
association with cyclin L, are part of large molecular weight protein
complexes that contain RNA polymerase II (RNAP II) as well as numerous
transcription and RNA processing factors. Data presented here
demonstrate that the influence of CDK11p110 on
transcription and splicing does not involve phosphorylation of the RNAP
II carboxyl-terminal domain by CDK11p110. We have isolated
a DRB- and heparin-sensitive protein kinase activity that co-purifies
with CDK11p110 after ion exchange and affinity purification
chromatography. This protein kinase was identified as casein kinase 2 (CK2) by immunoblot and mass spectrometry analyses. In addition to the RNAP II carboxyl-terminal domain, CK2 phosphorylates the
CDK11p110 amino-terminal domain. These data suggest that
CDK11p110 isoforms participate in signaling pathways that
include CK2 and that its function may help to coordinate the regulation
of RNA transcription and processing events. Future experiments will
determine how phosphorylation of CDK11p110 by CK2
specifically affects RNA transcription and/or processing events.
The complex biochemical events of transcription and RNA
processing, resulting in the production of mature RNA transcripts, are
now understood to be highly integrated and co-dependent
processes (1). It is hypothesized that regulation of these events
occurs through the active exchange of associated factors with the RNAP II1 complex (2-4). This
hypothesis is based upon identification of numerous positive and
negative regulatory factors/complexes, influencing both transcription
and RNA processing enzymes, in physical association with RNAP II. Many
of these complexes exert their effects directly or indirectly through
association with the RNAP II CTD. In mammals, the RNAP II CTD is
composed of 52 heptapeptide repeats with the consensus sequence
Tyr-Ser-Pro-Thr-Ser-Pro-Ser (YSPTSPS), which are essential for
viability (5).
The RNAP II CTD is heavily phosphorylated in vivo, and it is
likely that sequential phosphorylation events, as well as
phosphorylation of specific residues by specific protein kinases, help
regulate transcript production. This model appears to fit much of the
data coming from numerous laboratories and was recently proposed as the
most likely means of coordinating the various steps of transcription, RNA processing, and mRNA export (6). Many protein kinases modify the RNAP II CTD. Several of the CTD kinases identified thus far are
from the cyclin-dependent kinase family (CDKs) and include CDK1, CDK7, CDK8, and CDK9. In addition, another regulator of cell
cycle events, casein kinase 2 (CK2), is known to phosphorylate a number
of transcriptional proteins, including the RNAP II CTD and the RAP74
subunit of TFIIF (7, 8).
Data from this laboratory and others
demonstrate that the CDK11p110 (PITSLRE) protein kinases
associate with the cyclin L regulatory protein, bind directly to
various splicing factors, and play a role in pre-mRNA splicing
(9-11).2,3
Moreover, we recently published results indicating a potential role for
CDK11p110 in the regulation of transcription as well (12).
Experiments are described here demonstrating that the
CDK11p110 protein kinases are members of RNAP II-containing
complexes that include CK2. Furthermore, CDK11p110
immunoprecipitation complexes (IP complexes) contain an RNAP II
CTD-directed kinase activity. However, we show that CK2, and not
CDK11p110, is the kinase responsible for this RNAP II CTD
phosphorylation. In addition, we demonstrate that CDK11p110
association with, and amino-terminal phosphorylation by, CK2 in
vivo requires the RD/RE protein interaction domain of
CDK11p110. Thus, the data herein suggest the existence of
CDK11p110 protein complexes in which signaling to the RNA
transcriptional and processing machinery may be influenced by CK2
kinase activity.
Cell Culture and Transfection--
HeLa Tet Off and 293T cells
were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 2% glutamine, and 0.1%
gentamicin. CEM C7 cells were maintained in RPMI supplemented with 10%
heat-inactivated fetal calf serum, 2% glutamine, and 0.1% gentamicin.
Transfections were carried out using FuGENE 6 (Roche Molecular
Biochemicals) according to the manufacturer's instructions with a
reagent/DNA ratio of 3:1.
Antibodies--
The PITSLRE/CDK11 antibodies P2N100, P1C, and
GN1 have been described previously (9, 12, 13). Commercial antibodies used include anti-CK2 Production of Recombinant Protein--
The
GST-CTD32-52 construct was made using a PCR product from
an EST clone. The GST-CTDWT14 construct was a gift from Dr.
J. Corden (14). GST fusion proteins were induced with 0.1 mM isopropyl-1-thio- Mapping of the CK2 Binding Region to CDK11p110 and
the Region of Phosphorylation--
The RD/RE and poly(E) deletion
constructs were made using the QuikChangeTM site-directed mutagenesis
kit (Stratagene) with modifications as described by Wang and Malcolm
(15). CDK11p110 amino-terminal deletion constructs (M91,
N290, N375, and N424) are either described in Ref. 16 or were made
using PCR as previously described in Ref. 16. All constructs
were verified before use by DNA sequence analysis.
Immunoprecipitations and Immunoblots--
Transfected cells were
lysed in 50 mM Hepes (pH 7.9), 150 mM NaCl, 0.1 mM EDTA, 0.5% Tween 20, 10% glycerol, and complete protease inhibitors (Roche Molecular Biochemicals). Incubations with
antibodies for the purpose of immunoprecipitation of protein complexes
were performed in lysis buffer for 2 h at 4 °C. Washes were
performed three times using 1 ml of lysis buffer. Immunoblot analysis
was performed as previously described (9).
Protein Kinase Assays--
For the CTD kinase assays,
immunoprecipitations performed as described above were resuspended in
10 µl of kinase buffer (without ATP). Substrate (1 µg) was
added in a total volume of 10 µl of water, followed by 10 µl of 2×
kinase buffer containing cold and [ Protein Chromatography--
Using a Bio-Rad BioLogic HR
chromatography system, a cellulose phosphate P11 (Whatman) cation
exchange column (5 ml) was loaded with 15 mg of HeLa nuclear extract
protein (3 mg/ml, prepared as described in Ref. 17) and washed with 15 ml of P11 buffer (20 mM Hepes (pH 7.9), 0.1 M
KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride).
Proteins were eluted with a 25-ml linear gradient from 0.1 to 1.0 M KCl P11 buffer, followed by a 10-ml wash of 1.0 M P11 buffer. 1-ml fractions were then collected. The
entire purification procedure, from P11 through the
CDK11p110 antibody affinity purification columns, was
performed as previously described (12). The only exception was that the
0.8 M KCl P11 fractions and the 0.5 M KCl DEAE
fractions were used here.
Mass Spectrometry Analysis of Proteins--
The protein samples
were digested with endoproteinase Lys-C and trypsin according to the
method of Link et al. (18). The resulting peptide mixture
was analyzed by combined capillary liquid chromatography/tandem mass
spectrometry. Mass spectrometry was performed using ThermoFinnigan
LCQ-Deca ion trap mass spectrometry with an electrospray ion source.
Fragment ion (MS2) spectra were subjected to search using
the SEQUEST program of Eng and Yates (ThermoFinnigan).
An RNAP II CTD-directed Kinase Activity Co-immunoprecipitates with
the CDK11p110 Protein Kinase--
We recently published
data demonstrating that CDK11p110 affinity-purified
complexes contain RNAP II (12). Furthermore, we observed that
interference with CDK11p110 activity negatively affected
transcript production in vitro. Given that
CDK11p110 is found in RNAP II complexes, it was reasonable
to determine whether the RNAP II CTD was a substrate for this kinase.
Protein kinase assays, from immunoprecipitations using several
affinity-purified CDK11p110 antibodies in parallel with
known CTD kinases such as CDK7 and CDK1, suggested that the RNAP II CTD
was a good substrate for CDK11p110 (data not shown).
Characterization of this kinase activity revealed that the imperfect
consensus repeats (repeats 32-52) were a much better substrate for
this kinase activity than the perfect repeats (Fig.
1A). To verify that this CTD
kinase activity was due to CDK11p110 and not to a
co-immunoprecipitating kinase, three different kinase-inactive, FLAG-tagged CDK11p110 mutants were transiently transfected
into HeLa cells in parallel with a FLAG-tagged version of the wild-type
CDK11p110 kinase. The point mutations for the kinase
inactive forms of CDK11p110 included K439N, D534N, and
D552N. These mutations were selected based upon previous studies of
serine/threonine kinase structure and function, which demonstrated the
requirement of specific conserved amino acids for enzymatic activity
(19-22). Once the mutant forms of CDK11p110 were
transfected into HeLa cells and given time to express their corresponding proteins, the FLAG epitope was used to immunoprecipitate the exogenous protein and kinase assays performed using the
GST-CTD32-52 substrate. The surprising result was that
there was no change in the CTD-directed kinase activity between the
wild-type and kinase-inactive forms of FLAG-CDK11p110 (Fig.
1B). Two-hybrid and co-immunoprecipitation analyses
indicated that the CDK11p110 isoforms do not form dimers or
other oligomers (data not shown). Also, a reduction in CTD-directed
kinase activity between wild-type and kinase-inactive forms would be
expected if the activity seen with the kinase-dead form of
FLAG-CDK11p110 were due to additional, active endogenous
CDK11p110 kinase in these IP complexes. All of these
results suggested that another kinase was present in the
CDK11p110 IP complexes. This CTD-directed kinase activity
was determined to be DRB-sensitive, with a ~50% decrease in activity
between 10 and 100 µM concentration of DRB (data not
shown). Immunoblot analyses performed to detect cyclin K, CDK1, CDK7,
CDK8, CDK9, ERK1, ERK2, and ERK3 in CDK11p110
immunoprecipitates were all negative (data not shown), indicating that
none of these proteins were responsible for this associated kinase
activity.
CDK11p110 Amino-terminal Domain-directed Kinase
Activity Is Also Present in CDK11 IP Complexes--
A phosphorylated
110-kDa protein band also appeared in these IP kinase
assays. The fact that this band appears even in kinase assays
using FLAG-immunoprecipitated kinase-inactive forms of CDK11p110 indicated that it was not due to
autophosphorylation. IP kinase assays were carried out with several
different CDK11p110 FLAG-tagged deletion constructs
corresponding to various portions of the CDK11p110 protein
to determine whether the CTD-directed kinase activity would
co-immunoprecipitate with and phosphorylate these various fusion
proteins (see Fig. 2C for
construct diagrams). Protein kinase assays were carried out both in the
presence and absence of substrate. The results shown in Fig. 2,
A and B, demonstrate that the CTD kinase activity
co-immunoprecipitates with the amino-terminal domain of
CDK11p110 and phosphorylates a region of
CDK11p110 between amino acid residues 92 and 375.
A CTD Kinase Activity Corresponding to CK2 Co-elutes with Specific
Fractions of CDK11p110 Kinases following P11
Chromatography--
The results suggested that an unknown
DRB-sensitive protein kinase co-immunoprecipitates with
CDK11p110 and that it is this associated protein kinase
activity that is responsible for the observed CTD- and
CDK11p110-directed phosphorylations. To examine this more
carefully, cation exchange chromatography was performed as a first step
toward the purification and identification of the unknown DRB-sensitive
kinase(s). HeLa nuclear extract was loaded onto a P11 phosphocellulose
column, and the proteins were eluted using a linear gradient of
0.1-1.0 M KCl. Aliquots from the collected fractions were
directly assayed by immunoblot for the elution profile of
CDK11p110 as well as RNAP II. The CDK11p110
protein started to elute from the column around 0.39 M KCl,
demonstrated peak elution around 0.89 M KCl, and continued
to elute in the 1.0 M KCl buffer (Fig.
3A). The RNAP II large subunit
also eluted from the P11 column beginning around 0.4 M KCl
and demonstrated peak elution around 0.6 M KCl. Very little
of this protein was eluted by the 1.0 M KCl buffer (data
not shown). To test for the presence of the unknown DRB-sensitive
protein kinase, column fractions were selected representing the entire
range of CDK11p110 elution, and 0.2-ml aliquots were
dialyzed into 0.1 M KCl buffer. CDK11p110
kinase complexes were then immunoprecipitated from the dialyzed samples
using the affinity-purified P2N100 antibody and divided for parallel
immunoblot analyses and protein kinase assays. The results are shown in
Fig. 3B. CDK11p110 was recovered from fraction
42, 50, and 58. The CTD-directed kinase activity was present only in
fractions 50 and 58.
Previously published data led us to believe that the
CDK11p110-associated protein kinase might be CK2. First,
there are three consensus CK2 phosphorylation sites in the imperfect
CTD repeat domain that was phosphorylated but not in the perfect CTD
repeat domain that was not phosphorylated. Second, CK2 had previously been reported to phosphorylate the RNAP II CTD (8). To determine whether CK2 CK2 Specifically Co-purifies with CDK11 following Ion
Exchange and Affinity Chromatography--
Biochemical
purification of CDK11p110-containing complexes was
undertaken to further characterize proteins associated with
CDK11p110. Soluble HeLa cell nuclear extract was subjected
to chromatography using a series of columns as outlined in Fig.
3D. The eluants from these various columns were analyzed to
identify the proteins contained in each fraction. CK2 The Kinase Activity Phosphorylating the RNAP II CTD Is Inhibited by
Heparin--
If CK2 is responsible for the phosphorylation of both the
RNAP II CTD and the CDK11p110 amino-terminal domain in
CDK11p110 IP complexes, then this kinase activity should be
inhibited by heparin. Heparin is known to be a rather potent and
somewhat specific inhibitor of CK2 activity (23), whereas the ability
of DRB to inhibit CK2 has been reported to be variable (24). Kinase
assays were carried out following immunoprecipitation of endogenous
CDK11p110 and TFIIH. GST-CTD32-52 was added to
these kinase reactions along with increasing amounts of heparin. Two
formulations of heparin were tested (see "Materials and Methods"),
and both were found to be effective at inhibiting CK2 kinase activity.
The results shown in Fig. 3E demonstrate that the kinase
activity associated with CDK11P110 and directed against the CTD
was inhibited by heparin. In contrast, TFIIH kinase activity directed
against the CTD was not inhibited. Heparin also inhibited
phosphorylation of endogenous CDK11p110 and the
CDK11p110 amino-terminal domain N375-FLAG, which was
transiently expressed in HeLa cells and subjected to an IP kinase assay
(data not shown).
Expression of Kinase-dead CK2 Mapping the Region of the CDK11 and CK2 Association--
To
further map the association between CDK11p110 and CK2,
specific CDK11p110 amino-terminal deletion constructs were
expressed in cells and examined for the ability to co-immunoprecipitate
CK2. Based upon previous studies by Stamm and colleagues (26), which
indicated that the shuttling of the alternative splicing factor YT521B
is regulated by both RD/RE repeats in its amino terminus and a poly(E) repeat at its carboxyl terminus, these regions were deleted from CDK11p110. Deletion of the RD/RE (i.e. Further characterization of CDK11p110 signaling and
protein complexes and the role they play in transcription and RNA
processing identified CK2 as a member of the CDK11p110
complexes. CK2, a known mediator of cell cycle and transcriptional events (7, 27, 28), phosphorylates the CTD in vitro. In a
recent publication, Gottesfeld and co-workers (11) demonstrated that a
cyclin L-associated kinase activity, attributed to the PITSLRE/CDK11p110 protein kinase, phosphorylated the CTD.
Although human CDK11p110 does interact with human cyclin L
in vivo,2,3 we have shown here that
CDK11p110 is, in fact, not a CTD kinase. This was
demonstrated through the use of several different CDK11p110
kinase-inactive tagged mutants from mammalian cells in the in vitro CTD kinase assays, by examining the sensitivity of the CTD kinase to heparin, and by ectopic expression of kinase-dead CK2 The data presented in this report also demonstrate that CK2
phosphorylates the amino-terminal domain of CDK11p110
in vivo. We have identified the RD/RE repeat region of the
CDK11p110 amino-terminal domain as an essential component
for its association with CK2. In addition, the
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(C-18), anti-ERK3 (D23), anti-TFIIH p62 (Q-19), anti-CDK7 (C-19), and anti-FLAG (Oct-A D8) antibodies from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and anti-FLAG M2
monoclonal antibody and affinity gel from Sigma.
-D-galactopyranoside at
room temperature for 3 h. The cells were first pelleted by centrifugation, resuspended in cold PBS, and then sonicated on ice.
Triton X-100 was added to 1%, and the lysate was spun at 10,000 × g for 20 min at 4 °C. The supernatant was incubated
with glutathione-Sepharose 4B beads overnight, rotating at 4 °C. The beads were washed three times in 100 bed volumes of cold PBS, and the
remaining bound proteins were eluted in 4 bed volumes of 50 mM Tris (pH 8) containing 20 mM glutathione.
The eluted proteins were dialyzed into 40 mM Hepes (pH
7.9), 100 mM KCl, 50 µM ZnSO4,
and 10% glycerol.
-32P]ATP, making
the final volume 30 µl. The kinase reactions were incubated for 10 min at 30 °C. The final protein kinase reaction buffer contained 40 mM Hepes (pH 7.4), 10 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 0.5 mg/ml
acetylated BSA, 10 µM ATP, 3-5 µCi of
[
-32P]ATP, 2 mM benzamidine, 60 mM
-glycerophosphate, 0.1 mM
Na3VO4, and 0.1 mM NaF. For the
heparin- and DRB-containing protein kinase reactions, the drugs were
incubated with the beads for 5 min at room temperature prior to the
addition of substrate and ATP-containing kinase buffer. Heparin was
obtained from Sigma (H-4784; stock solution made in water at 50 mg/ml)
and American Pharmaceutical Partners, Inc. (100 USP units/ml). The
effective concentration range (with no effect on TFIIH) for the Sigma
heparin was 2-10 µg/ml, and the range for the American
Pharmaceutical Partners, Inc. heparin was 0.33-2.5 units/ml
(0.01-0.075 units/30-µl reaction volume). The protein kinase
reaction supernatants were boiled in sample buffer and subjected to
10% SDS-PAGE analysis. The gels were washed extensively in 40%
methanol, 10% acetic acid; dried; and exposed to x-ray film.
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Fig. 1.
A RNAP II CTD-directed kinase activity
co-immunoprecipitates with CDK11p110. A,
Cem C7 cell lysates were immunoprecipitated with either CDK11 or ERK3
polyclonal antibodies. Protein A-agarose beads alone were used as a
negative control. Kinase assays were performed using the washed
immunobead complexes with either imperfect repeat GST-CTD substrate
(GST-CTD32-52) or perfect repeat GST-CTD substrate
(WT14) as described under "Materials and Methods."
B, FLAG-tagged wild type or D534N (catalytically inactive)
forms of CDK11p110 were transiently expressed in HeLa
cells. Immunoprecipitations were performed for FLAG-tagged
CDK11p110 or endogenous CDK11p110 from
transfected or untransfected cells, respectively. Gammabind
Plus-Sepharose beads alone were used as a negative control. The washed
immunobead complexes were split equally for either a kinase assay using
GST-CTD32-52 substrate (left panel)
or for immunoblot analysis using the CDK11p110-specific
antibody P2N100 (right panel).
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Fig. 2.
CTD- and CDK11p110-directed
kinase activity co-immunoprecipitates with the
CDK11p110 amino-terminal domain.
A and B, HeLa cells were transfected with the
following FLAG-tagged constructs. M91 represents CDK11p110
amino acids 92-370; N375 represents CDK11p110 amino acids
1-375; N424 represents CDK11p110 amino acids 1-424; p58
represents the CDK11p110 kinase catalytic domain encoded by
amino acids 339-779; and p110-D552N represents catalytically inactive
full-length CDK11p110. FLAG represents a control
transfection using pcDNA3.1 encoding just the FLAG epitope.
Transfected cell lysates were immunoprecipitated with anti-FLAG
M2-agarose, and the immunoprecipitates were split three ways. Kinase
assays were performed with and without GST-CTD32-52
substrate as described under "Materials and Methods." The third
aliquot was used for immunoblot analysis with a polyclonal anti-FLAG
antibody. C, schematic representation of
CDK11p110 constructs. Kinase assay results are also
indicated on the left. Included on the schematic are the CK2
interaction domain and some of the potential phosphorylation
site(s).
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Fig. 3.
CK2 co-purifies with
CDK11p110. HeLa nuclear extract (15 mg) was subjected
to P11 chromatography, and elution fractions of 1.0 ml were collected
over a linear gradient from 0.1 to 1.0 M KCl. A,
immunoblot analysis of the P11 fractions. Protein aliquots of 30 µl
from the indicated fractions (labeled above the
lanes) were analyzed by immunoblot using the
CDK11p110 P2N100 antibody. The concentration of KCl in
which the protein was eluted from the column is indicated
below the immunoblot panel. B, immunoblot
analysis and kinase assays following P2N100 inmmunoprecipitation.
Selected P11 fractions (200 µl) were dialyzed overnight into 0.1 M P11 buffer and then subjected to immunoprecipitation
using 5 µg of P2N100 antibody and 30 µl of Gammabind
Plus-Sepharose. The resulting bead complexes were divided for parallel
immunoblot analysis and kinase reactions. The upper
panel represents the immunoblot incubated with P2N100
antibody. The far left lane contains
10 µg of HeLa total cell lysate, and the second
from left lane represents a positive
control P2N100 immunoprecipitation from HeLa total cell lysate. The
remaining lanes represent immunoprecipitation from the dialyzed P11
samples, with the identity of the sample shown above the
lanes. The lower panel represents the
corresponding kinase reactions from the immunoprecipitated P11 samples
using 1 µg of GST-CTD32-52 as substrate. C,
the immunoblot described for B was incubated with a
polyclonal antibody to CK2 . HeLa cell lysate was used as a positive
control for CK2, and Gammabind beads plus rabbit IgG were incubated
with HeLa cell lysate as a negative control. The IgG heavy chain
identified by the CK2 polyclonal antibody is indicated to the
left of the panel. D, outline of the
chromatography purification scheme used to purify CDK11p110
protein complexes that contain CK2. E, HeLa cell lysates
were immunoprecipitated with the CDK11p110 P2N100 and TFIIH
p62 subunit antibodies. The immunoprecipitate beads were divided evenly
for immunoblot analysis and kinase reactions. 1 µg of
GST-CTD32-52 was used in each kinase reaction. Heparin was
added to the paired kinase reactions as described under "Materials
and Methods." The components contained in each reaction are indicated
above each lane. The immunoblot analyses
indicated that the IPs for CDK11p110 and TFIIH (CDK7) were
successful (data not shown). F, 293T cells were transfected
with FLAG-CK2
wild type (WT) or kinase-dead
(KD) and AU.1-CK2
expression constructs (as indicated
above the lanes). CDK11 IPs from the transfected
cell lysates and control cells were divided evenly and subjected to
either immunoblot analyses (left panel) or kinase
assays using 1 µg of GST-CTD32-52 as substrate
(right panel). The CK2
immunoblot signal from
the CDK11 IP is not shown because it is the same size as the IgG light
chain signal.
was associated with the CDK11p110
in vivo, the eluted fractions from the P11 column shown in
Fig. 3B were immunoblotted with a human CK2
antibody
(Fig. 3C). As anticipated, CK2
was associated with the
CDK11p110 immunoprecipitated from the HeLa lysate, as well
as the P11 column fractions that eluted between 0.8 and 1.0 M KCl. Conversely, CK2
was not associated with the
CDK11p110 protein in the P11 column fractions that eluted
at salt concentrations less than 0.8 M KCl. CK2
co-immunoprecipitated with CDK11p110 only in those samples
that also demonstrated the CTD directed protein kinase activity.
Finally, both the CK2
and CK2
protein kinase subunits
co-immunoprecipitate with the CDK11p110 protein kinase
isolated from nontransformed human foreskin fibroblast cells (data not shown).
was identified
by combined liquid chromatography/tandem mass spectrometry analysis as
a CDK11p110-co-purifying protein by assignment of
MS2 spectra from nine peptides to the known sequence of
CK2
. It may also be of interest to note that CK2
specifically
co-purifies with a subpopulation of the CDK11p110-RNAP II
complexes that, in a batch elution protocol, elute from the P11 column
at 0.8 M KCl and from the DEAE column at 0.5 M KCl.
Along with CK2
Significantly
Reduces CDK11-associated CTD Kinase Activity--
We tested whether
ectopic expression of kinase dead CK2
would compete with endogenous
CK2
for association with CDK11p110 and thereby reduce
the amount of CTD kinase activity in a CDK11p110 IP. The
human cell line 293T was transfected with combinations of wild type or
D156A kinase-dead (25) CK2
and CK2
. Following expression of the
CK2 proteins, the transfected and control cells, which had not been
transfected, were subjected to CDK11p110 IP
kinase assays using GST-CTD32-52 substrate. The results
shown in Fig. 3F demonstrate that the transfections and IPs
were successful and that expression of kinase-dead CK2
significantly
reduced the amount of CTD phosphorylation associated with
CDK11p110 as compared with wild type. The results also
suggest that expression of CK2
is necessary for increased
association of ectopically expressed CK2
with CDK11p110.
The results further indicate that ectopic CK2
expression alone is
sufficient to increase the amount of CTD kinase activity associated with endogenous CDK11p110. Thus, these results reconfirm
that CK2 association with CDK11p110 is responsible for a
significant amount of the CTD kinase activity in CDK11p110
IP complexes.
RE),
but not the poly(E) (i.e.
E), repeat region resulted in
the loss of CDK11p110 and CK2 association as determined by
co-immunoprecipitation/immunoblot analysis (Fig.
4, left panel). In
addition, when the CK2 protein kinase did not physically associate with
CDK11-N424 in vivo, phosphorylation of the amino-terminal
region of CDK11p110 was not observed (Fig. 4,
right panel). We consistently observed equal or
greater phosphorylation of the CDK11-N424
E protein as compared with
the wild-type CDK11p110. It is possible that the deletion
of the highly charged glutamic acid repeats improved the ability of CK2
to interact with CDK11p110. Thus, the RE domain of
CDK11p110 is at least required for association with CK2.
This RE domain is also required for CDK11p110 interaction
with RNPS1 and for nuclear speckle localization of CDK11p110.4
CDK11p110 and CK2 do not co-immunoprecipitate using
in vitro transcription and translation products. It is
possible that other proteins, factors, or modifications are required
for the stable association of these proteins. However, the
phosphorylation of CDK11p110 by CK2 suggests that these
proteins do interact, at least in an indirect manner.
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Fig. 4.
CK2 cannot associate with a
CDK11p110 deletion mutant lacking the RD/RE
repeat region. A and B, HeLa cells were
transfected with N424 (CDK11p110 amino acids 1-424) wild
type as well as the deletion mutants indicated above each
blot or gel. All constructs are FLAG-tagged at
the carboxyl terminus. Transfected cell lysates were immunoprecipitated
with anti-FLAG M2-agarose. Immunoprecipitates were divided evenly for
immunoblot analysis (left panel) and kinase
reactions (right panel). The immunoblot was
simultaneously incubated with CDK11p110 P2N100 and CK2
polyclonal antibodies. Kinase reactions were performed without any
additional substrate.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. Gottesfeld and co-workers (11) did not use kinase-inactive
CDK11p110 controls, resulting in the discrepant conclusion
that CDK11p110 kinase activity was responsible for CTD
phosphorylation. Rather, as we have shown here, it is the associated
activity of CK2 with CDK11p110 in vivo that is
most likely responsible for CTD-directed kinase activity. It is still
possible that another cyclin L-associated CDK may play a role in CTD
phosphorylation as well.
RE mutation disrupts
the ability of the CDK11p110 kinase to properly localize
within nuclear structures.5
CK2 interacts with multiple signaling pathways, and this protein kinase
is proposed to be an important mediator of cell survival. Increased CK2
activity is associated with increased cellular proliferation and
response to stress, and conversely, loss of CK2 activity is associated
with cell death (see Ref. 29 and references therein). In one case, CK2
is reported to regulate p53 in response to DNA damage. Following UV
irradiation, CK2 purifies in a complex with the FACT transcriptional
elongation factor and phosphorylates serine 392 of p53, thus enhancing
p53 activity (30). Interestingly, both subunits of FACT co-purified
with CDK11p110 and CK2, as identified by both mass
spectrometry and immunoblot analysis (data not shown). The data
reported here further suggest a role for CK2 in regulating the function
of various components of large molecular weight RNA transcription and
processing complexes, including CDK11p110, FACT, and RNAP
II. The association(s) between CK2 and these proteins as well as their
phosphorylation by CK2 may provide further insight into the
mechanism(s) involved in CK2 regulation of RNA transcript production.
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ACKNOWLEDGEMENTS |
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We thank J. Grenet and S. Bothner for excellent technical assistance. We also thank Dr. S. Elledge for providing the cyclin K antibodies and Dr. J. Corden for providing GST-CTD constructs. We thank Dr. Jason Weber for assistance with chromatography, Ashutosh Mishra for assistance with protein chemistry, and Dr. Sandra Pierre for the GST-CTD32-52 protein. The assistance of Dr. C. Naeve and the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital in the production of oligonucleotides and DNA sequencing analysis is also acknowledged.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM44088 (to V. J. K.), CA72572 (to J. M. L.), and P30 CA21765 (to St. Jude Children's Research Hospital) as well as support from the American Lebanese Syrian Associated Charities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Tel.: 901-495-3469; Fax: 901-495-2381; E-mail: vincent.kidd@stjude.org.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M207518200
2 D. Hu, A. Mayeda, J. H. Trembley, J. M. Lahti, and V. J. Kidd, manuscript in preparation.
3 J. H. Trembley, P. Loyer, D. Hu, and V. J. Kidd, unpublished data.
4 J. H. Trembley, R. Gururajan, J. M. Lahti, and V. J. Kidd, unpublished data.
5 J. H. Trembley, R. Gururajan, S. Bothner, J. M. Lahti, and V. J. Kidd, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
RNAP II, RNA
polymerase II;
CDK11, cyclin-dependent kinase 11;
CTD, carboxyl-terminal domain;
DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole;
CK2, casein kinase 2;
IP, immunoprecipitation;
GST, glutathione
S-transferase;
ERK, extracellular signal-regulated
kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Proudfoot, N. J., Furger, A., and Dye, M. J. (2002) Cell 108, 501-512[Medline] [Order article via Infotrieve] |
2. | Hampsey, M., and Reinberg, D. (1999) Curr. Opin. Genet. Dev. 9, 132-139[CrossRef][Medline] [Order article via Infotrieve] |
3. | Otero, G., Fellows, J., Li, Y., de Bizemont, T., Dirac, A. M., Gustafsson, C. M., Erdjument-Bromage, H., Tempst, P., and Svejstrup, J. Q. (1999) Mol. Cell 3, 109-118[Medline] [Order article via Infotrieve] |
4. | Misteli, T., and Spector, D. L. (1999) Mol. Cell 3, 697-705[CrossRef][Medline] [Order article via Infotrieve] |
5. | Dahmus, M. E. (1995) Biochim. Biophys. Acta 1261, 171-182[Medline] [Order article via Infotrieve] |
6. | Orphanides, G., and Reinberg, D. (2002) Cell 108, 439-451[Medline] [Order article via Infotrieve] |
7. | Egyhazi, R., Ossoinak, A., Filhol-Cochet, O., Cochet, C., and Pigon, A. (1999) Mol. Cell. Biochem. 191, 149-159[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Payne, J. M.,
Laybourn, P. J.,
and Dahmus, M. E.
(1989)
J. Biol. Chem.
264,
19621-19629 |
9. |
Loyer, P.,
Trembley, J. H.,
Lahti, J. M.,
and Kidd, V. J.
(1998)
J. Cell Sci.
111,
1495-1506 |
10. | Berke, J. D., Sgambato, V., Zhu, P. P., Lavoie, B., Vincent, M., Krause, M., and Hyman, S. E. (2001) Neuron 32, 277-287[Medline] [Order article via Infotrieve] |
11. |
Dickinson, L. A.,
Edgar, A. J.,
Ehley, J.,
and Gottesfeld, J. M.
(2002)
J. Biol. Chem.
277,
25465-25473 |
12. |
Trembley, J. H., Hu, D.,
Hsu, L. C.,
Yeung, C. Y.,
Slaughter, C.,
Lahti, J. M.,
and Kidd, V. J.
(2002)
J. Biol. Chem.
277,
2589-2596 |
13. |
Xiang, J.,
Lahti, J. M.,
Grenet, J.,
Easton, J.,
and Kidd, V. J.
(1994)
J. Biol. Chem.
269,
15786-15794 |
14. |
Patturajan, M.,
Schulte, R. J.,
Sefton, B. M.,
Berenzney, R.,
Vincent, M.,
Bensaude, O.,
Warren, S. L.,
and Corden, J. L.
(1998)
J. Biol. Chem.
273,
4689-4694 |
15. | Wang, W., and Malcolm, B. A. (1999) Biotechnology 26, 680-681 |
16. |
Tang, D.,
Gururajan, R.,
and Kidd, V. J.
(1998)
J. Biol. Chem.
273,
16601-16607 |
17. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
18. | Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., Garvik, B. M., and Yates, J. R., III (1999) Nat. Biotechnol. 17, 676-682[CrossRef][Medline] [Order article via Infotrieve] |
19. | De Bondt, H. L., Rosenblatt, J., Jancarik, J., Jones, H. D., Morgan, D. O., and Kim, S. H. (1993) Nature 363, 595-602[CrossRef][Medline] [Order article via Infotrieve] |
20. | Jeffrey, P. D., Russo, A. A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, N. P. (1995) Nature 376, 313-320[CrossRef][Medline] [Order article via Infotrieve] |
21. | Taylor, S. S., Knighton, D. R., Zheng, J., Gibbs, C. S., and Zoller, M. J. (1993) Trends Biochem. Sci. 18, 84-89[Medline] [Order article via Infotrieve] |
22. | van den Heuvel, S., and Harlow, E. (1993) Science 262, 2050-2054[Medline] [Order article via Infotrieve] |
23. |
Hathaway, G. M.,
Lubben, T. H.,
and Traugh, J. A.
(1980)
J. Biol. Chem.
255,
8038-8041 |
24. |
Zandomeni, R.,
Zandomeni, M. C.,
Shugar, D.,
and Weinmann, R.
(1986)
J. Biol. Chem.
261,
3414-3419 |
25. | Korn, I., Gutkind, S., Srinivasan, N., Blundell, T. L., Allende, C. C., and Allende, J. E. (1999) Mol. Cell. Biochem. 191, 207-212[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Hartmann, A. M.,
Nayler, O.,
Schwaiger, F. W.,
Obermeier, A.,
and Stamm, S.
(1999)
Mol. Biol. Cell
10,
3909-3926 |
27. | Marshak, D. R., and Russo, G. L. (1994) Cell. Mol. Biol. Res. 40, 513-517[Medline] [Order article via Infotrieve] |
28. |
Pepperkok, R.,
Lorenz, P.,
and Pyerin, W.
(1994)
J. Biol. Chem.
269,
6986-6991 |
29. | Ahmed, K., Gerber, D. A., and Cochet, C. (2002) Trends Cell Biol. 12, 226-230[CrossRef][Medline] [Order article via Infotrieve] |
30. | Keller, D. M., Zeng, X., Wang, Y., Zhang, Q. H., Kapoor, M., Shu, H., Goodman, R., Lozano, G., Zhao, Y., and Lu, H. (2001) Mol. Cell 7, 283-292[Medline] [Order article via Infotrieve] |