From the Department of Genetics and Tumor Cell
Biology, St. Jude Children's Research Hospital, Memphis, Tennessee
38105 and the § Department of Biochemistry and Molecular
Biology, University of Miami School of Medicine, Miami, Florida
33136
Received for publication, October 1, 2002
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ABSTRACT |
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The PITSLRE protein kinases, hereafter
referred to as CDK11 because of their association with the cyclin L
regulatory partner, belong to large molecular weight protein complexes
that contain RNA polymerase II. These
CDK11p110 complexes have been reported to influence
transcription as well as interact with the general
pre-mRNA-splicing factor RNPS1. Some of these complexes may also
play a role in pre-mRNA splicing. Using a two-hybrid interactive
screen, the splicing protein 9G8 was identified as an in
vivo partner for CDK11p110. The identification of
several splicing-related factors as CDK11p110 interactors
along with the close relationship between transcription and splicing
indicated that CDK11p110 might influence splicing activity
directly. Immunodepletion of CDK11p110 from splicing
extracts greatly reduced the appearance of spliced products using an
in vitro assay system. Moreover, the re-addition of these
CDK11p110 immune complexes to the
CDK11p110-immunodepleted splicing reactions completely
restored splicing activity. Similarly, the addition of purified
CDK11p110 amino-terminal domain protein was sufficient to
inhibit the splicing reaction. Finally, 9G8 is a phosphoprotein
in vivo and is a substrate for CDK11p110
phosphorylation in vitro. These data are among the first
demonstrations showing that a CDK activity is functionally coupled to
the regulation of pre-mRNA-splicing events and further support the
hypothesis that CDK11p110 is in a signaling pathway that
may help to coordinate transcription and RNA-processing events.
The regulation of transcription and RNA-processing events is
complex and, in fact, may be coordinated through the action of several
protein kinases that belong to the larger cell division control family
(i.e. CDKs)1
(1-7). These CDKs are not only regulated during the cell cycle, they
are undoubtedly part of a much larger signal transduction pathway that
is modulated by numerous extracellular and intracellular signals (8). A
model is emerging in which the phosphorylation status of the RNA
polymerase II (RNAP II) carboxyl-terminal domain (CTD) determines which
specific transcription, RNA processing, and polyadenylation factors are
recruited to the CTD (5, 7-9). The CTD is heavily phosphorylated
in vivo, and a number of protein kinases, particularly CDKs,
have been identified that modify this domain in vitro. CDK7,
CDK8, and CDK9 are all known CTD protein kinases that regulate
different aspects of transcription (10-13). Sequential phosphorylation
of specific amino acid residues within the CTD appears to be regulated
by this group of CDKs. These phosphorylation events have been linked to
the changes in the composition of RNAP II complexes that are associated
with transcription from pre-initiation through termination. However, a
functional relationship between a specific CDK and the regulation of
pre-mRNA-splicing events has only been inferred (14, 15).
Based upon the findings reported in this study as well as others (14,
15) demonstrating that the PITSLRE p110 protein kinases
associate with the cyclin L regulatory protein, this protein kinase has
been renamed CDK11p110. In a previous study, we found that
CDK11p110 binds directly to the general splicing factor
RNPS1 (16), and here we show that this protein kinase also interacts
with the splicing factor 9G8 and cyclin L in vivo as well.
We, therefore, hypothesize that CDK11p110 may play an
important role in the regulation of RNA transcript processing in
addition to affecting RNA transcript production (17). Such broad
protein kinase substrate specificity is not too surprising, because
>20 isoforms of CDK11p110 are produced ubiquitously in all
human cells (18). In addition, others have shown that TAT-SF1
U-snRNP complexes containing CDK9 and cyclin T proteins
stimulate splicing in vitro, possibly facilitating the
reciprocal activation of transcription and splicing events through its
ability to function as a dual-function factor (19).
Here we show that the CDK11p110 protein kinases and
associated regulatory proteins such as cyclin L are found in nuclear
complexes containing pre-mRNA splicing factors in vivo
by a number of criteria. These include immunoprecipitation/immunoblot
analyses of the lysate from cells transiently transfected with
tagged cyclin L and tagged 9G8 expression constructs as well as
indirect immunofluorescence studies demonstrating that
CDK11p110 and 9G8 co-localize within portions of the
nucleus. Furthermore, 9G8 is a phosphoprotein in vivo that
can be specifically phosphorylated by the active but not the
kinase-inactive form of CDK11p110 in vitro.
Depletion of CDK11p110 by RNAi diminishes the amount of
phosphorylated 9G8 in vivo as well. Finally, a functional
connection between CDK11p110 and splicing was revealed by a
combination of immunodepletion and dominant negative studies using
in vitro splicing assays. Thus, the data from this and
previous studies show that general splicing factors including RNPS1
(16, 20) and 9G8 interact or co-localize with CDK11p110,
that CDK11p110 protein complexes containing at least some
of these factors are associated with the regulation of cellular
splicing activity, and finally, that 9G8 is a phosphoprotein in
vivo and that it is a specific in vitro substrate
of the CDK11p110 protein kinase.
Yeast Two-hybrid Screening--
The CDK11p110 bait
cDNA was cloned into the pGBT9-DNA-binding domain vector to
generate a hybrid protein containing the sequence for the GAL4
DNA-binding domain in-frame with CDK11p110. This plasmid,
pGBT9/CDK11p110, was then transformed into the yeast strain
HF7C using a Frozen-EZ Yeast Transformation II kit (ZYMO Research). The
yeast cells were then selected on a tryptophan-deficient medium. The
positive yeast transformants were subsequently transformed with a human
B-cell cDNA expression library that contained random cDNAs that
had been fused in-frame to the GAL4 transactivator domain of expression vector pACT2 (16). The transformed yeast cells were then grown on
selective minimal plates (SD medium lacking leucine, tryptophan, and histidine) in the presence of 10 mM
Cell Culture and Transfection--
HeLa Tet-Off cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum, 2% glutamine, and 0.1% gentamicin.
Transfections were performed using PolyFect (Qiagen) according to the
manufacturer's instructions.
Antibodies--
The PITSLRE/CDK11 antibodies P2N100 and
P1C have been described previously (16-18). The polyclonal 9G8
antibody was prepared by continuous immunization of rabbits with the
purified His6-tagged 9G8 protein (Rockland, Inc.).
Commercial antibodies used in these studies included an anti-FLAG M2
monoclonal antibody (Sigma) as well as M2 antibody affinity gel
(Sigma), a c-Myc monoclonal antibody (Clontech), an
Recombinant Protein Production--
The His6-tagged
9G8, CDK11p110 (kinase-active form), and
CDK11p110-D552N (kinase-inactive form) recombinant proteins
were prepared from SF21 insect cells using a baculovirus
expression system. For the purification of CDK11p110 and
CDK11p110-D552N proteins, insect cells overexpressing the
His-tagged proteins were lysed in 50 mM sodium phosphate
buffer (pH 8.0), 500 mM NaCl, 5 mM
Immunoprecipitations and Immunoblots--
HeLa cells or HeLa
cells transfected with the various expression plasmids for 24 h
were lysed in 20 mM Hepes (pH 7.9), 100 mM KCl,
0.2 mM EDTA, 5 mM RNAi for CDK11 p110 and Orthophosphate in Vivo
Labeling Studies--
Orthophosphate labeling was performed on cells
in which endogenous CDK11p110 protein levels were
substantially reduced (i.e. >70%) within a 36-h time frame
without the cells undergoing apoptosis or cell cycle arrest. Apoptosis
and cell cycle of the CDK11p110 RNAi-treated HeLa cells
were examined by annexin V staining and flow cytometry, respectively.
RNA interference conditions were as follows. HeLa cells were treated
with a CDK11p110-specific RNAi using pSUPER plasmid as
described previously (22). The sequence of the CDK11p110
amino-terminal targeting region, 5'-GGGAGATGGCAAGGGAGCA-3', corresponds to nucleotides 572-590 of the human CDK11p110 cDNA
sequence (GenBankTM accession number U04824). After
24 h of CDK11p110 RNAi treatment (the empty pSUPER was
used as a control), the cells were transfected with
pcDNA3/N-FLAG-9G8 for 8 h. 8 h post-FLAG-9G8 transfection, the cells were orthophosphate-labeled for 2-h using 0.5 mCi of 32Pi before harvesting the cells. The
overexpressed FLAG-9G8 protein was then immunoprecipitated with
anti-FLAG M2-agarose beads, separated on a 12% SDS-PAGE, and
autoradiographed. The corresponding gel bands of the labeled 9G8 were
cut from the gel and subjected to Cerenkov counting in a scintillation
analyzer (TRI-CARB 2100TR, Packard Instrument Co.). The various
immunoblots were performed on cell lysates from HeLa cell lysates
harvested 32 h post-RNAi treatment using the indicated antibodies
employed as described throughout this study including anti-P1C specific
for CDK11p110, anti-actin (Santa Cruz Biotechnologies), and
anti-M2 specific for the FLAG tag (Sigma).
Protein Kinase Assays--
The 9G8/CDK11p110 protein
kinase assays were carried out in a protein kinase buffer containing 40 mM Hepes (pH 7.4), 10 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 0.5 mg/ml
acetylated bovine serum albumin, 100 µM ATP, 3-5 µCi
[ In Vitro Splicing Assays--
The 32P-labeled
The Splicing Factor 9G8 Is an in Vivo Partner of
CDK11p110--
The CDK11p110 protein kinase
has previously been shown to be involved in transcriptional elongation
(18), presumably through its association with a number of
transcriptional elongation factors. It has also been demonstrated that
CDK11p110 interacts in vivo with the general
mRNA-splicing factor RNPS1 (16, 20). Other
CDK11p110-interacting proteins were identified by
chromatographic purification and through the complementary use of
two-hybrid yeast systems (16, 18). To explore the possible function of
the CDK11p110 protein kinase in more detail, we used the
wild type CDK11p110 protein kinase as bait for yeast
two-hybrid screening of a B-cell cDNA expression library and
subsequently identified the splicing factor 9G8 as a novel and specific
CDK11p110 interactor. The 9G8 protein belongs to the
Ser-Arg-splicing factor protein family and plays a role in
alternative splicing (26, 27) as well as in promoting the export of
mRNA from the nucleus to the cytoplasm (28). To further verify the
interaction between 9G8 and CDK11p110 in mammalian cells,
we cloned the 9G8 cDNA into a mammalian expression vector with an
amino-terminal FLAG epitope tag and then transfected this construct
into HeLa cells. The anti-FLAG monoclonal antibody M2 was used to
immunoprecipitate the transiently expressed tagged proteins after
36 h, and the products were further analyzed by immunoblotting
using the CDK11p110-specific P2N100 antibody. This
experiment demonstrated that the FLAG-tagged 9G8 protein specifically
interacted with the endogenous CDK11p110 protein kinase,
indicating that 9G8 is an in vivo partner of CDK11p110 (Fig.
1A).
Evidence that CDK11p110, cyclin L, and 9G8 form a ternary
complex in vivo is shown in Fig. 1B. HeLa cells
were transiently transfected with either the expression vector only or
with constructs expressing FLAG-tagged cyclin L and Myc-tagged 9G8
constructs. Cell lysates were then incubated with the M2 FLAG antibody,
and the resulting immunoprecipitates were analyzed by SDS-PAGE and
immunoblotted with the CDK11p110-specific antibody P2N100,
the M2 FLAG antibody (which recognized the transiently expressed cyclin
L protein), or an antibody specific for the Myc tag (which recognized
the transiently expressed 9G8 protein). The results clearly demonstrate
that CDK11p110, cyclin L, and 9G8 immunoprecipitate
together as a single complex in vivo. Finally, when a
membrane containing a CDK11p110 immunoprecipitation from
HeLa cells was immunoblotted with the affinity-purified 9G8 polyclonal
antisera, a doublet that migrated between ~35 and 37 kDa in size was
detected (data not shown). This result is consistent with the
identity of this protein being 9G8 and also indicates that 9G8 was most
probably one of the normal constituents of a portion of the
CDK11p110 immunocomplexes found in HeLa cells.
To delineate the domain(s) of 9G8 required for its interaction with
CDK11p110 in vivo, a number of
CDK11p110 truncation or deletion constructs tagged with the
FLAG epitope were transiently expressed in HeLa cells and
immunoprecipitated with the M2 antibody. This was followed by
immunoblot analysis of the resulting immunoprecipitates using the
affinity-purified 9G8-specific polyclonal antibody. 9G8 interacts with
the amino-terminal portion of CDK11p110. More specifically,
the RE-repeat region (29) of the CDK11p110 protein
kinase was identified as an important region necessary for 9G8
interaction in vivo (Fig. 1C). This result is
consistent with previous analyses of CDK11p110 functional
domains, which indicate that this same region of the amino-terminal
domain is necessary for the majority of the protein-protein interactions between this protein kinase and other proteins (16, 17,
21), whereas the carboxyl-terminal half of the protein encodes the
catalytic domain of the protein kinase that is most homologous to CDK2
(18).
Finally, the immunolocalization of CDK11p110 and 9G8 within
HeLa cells was visualized by indirect immunofluorescence. When indirect immunofluorescence was performed using the previously reported CDK11p110 monoclonal antibody (17) and an affinity-purified
preparation of the newly prepared 9G8 polyclonal antisera, the two
proteins were found to be co-localized within a substantial portion of the nucleus; however, this co-localization did not overlap entirely within the nucleus but only certain speckled regions (Fig.
1D). These immunofluorescence results are consistent
with the co-immunoprecipitation/immunoblot experiments that are shown
in Fig. 1, panels A and B. Previous results from
other laboratories demonstrated that the CDK11p110 protein
kinase also co-localizes with the splicing factor SC-35 using
indirect immunofluorescence (14) and that SC-35 can apparently be
phosphorylated in vitro by a cyclin L-containing
immunocomplex (15). Coupled to our previous results demonstrating an
interaction among CDK11p110, RNPS1 (16) and multiple
transcriptional elongation factors (17), these data suggest that
the CDK11p110 protein kinase can be found in multiple,
distinct complexes containing both transcription and RNA-processing proteins.
CDK11p110 Phosphorylates 9G8 in Vitro and in
Vivo--
Because an in vivo interaction between 9G8 and
CDK11p110 was established, it became important to
investigate the possibility that 9G8 was an in vitro
CDK11p110 protein kinase substrate. Therefore, we purified
wild-type CDK11p110, CDK11p110-D552N (a
catalytically inactive form of the protein kinase), and 9G8 proteins
from baculovirus-infected insect cells expressing the corresponding proteins. The inclusion of the kinase-inactive form of
the CDK11p110 protein kinase in the in vitro
kinase assay was essential for this experiment, since we have
previously shown that the casein kinase 2 protein kinase interacts and
co-purifies with CDK11p110 (21). The inclusion of a
kinase-inactive form of CDK11p110 also eliminates the
possibility that another co-purifying or contaminating protein kinase
is responsible for the in vitro phosphorylation of the 9G8 substrate.
The apparent molecular mass of 9G8 purified from insect cells is
~35 kDa, whereas its theoretical molecular mass is 27 kDa. Following
orthophosphate labeling of both insect and HeLa cells, the 9G8 protein
was immunoprecipitated using the affinity-purified antibody and it was
found to be a
phosphoprotein.2 This
may account for the difference between the apparent and theoretical
molecular weights of 9G8 (this work and Ref. 26). We then determined
whether 9G8 could be phosphorylated by the CDK11p110
protein kinase in vitro. The active wild-type
CDK11p110 protein kinase as well as an inactive form of
CDK11p110 (i.e. CDK11p110-D552N) as
a negative control and appropriate bead controls were all included in
this experiment (Fig. 2A).
This study demonstrated that the purified wild-type
CDK11p110 protein kinase but not the kinase-inactive form
of CDK11p110 (CDK11p110-D552N) is capable of
phosphorylating 9G8 in vitro. To further determine whether
9G8 is indeed a phosphoprotein in vivo, the
His6-tagged form of 9G8 was expressed in insect cells using
a baculovirus vector followed by nickel-nitrilotriacetic acid
affinity column purification of the protein. The purified 9G8 protein
was either treated or left untreated with alkaline phosphatase, and the
gel then visualized by Coomassie Blue staining (Fig. 2B).
The untreated insect cell lysates containing the purified
His6-tagged 9G8 contained two different molecular weight
forms of the 9G8 protein, whereas alkaline phosphatase treatment of the
same lysates containing purified 9G8 resulted in the loss of the slower
migrating form of the protein.
To finally demonstrate that the splicing factor 9G8 is a phosphoprotein
that is phosphorylated by the CDK11p110 protein kinase
in vivo and undoubtedly other kinases as well, we employed
RNAi as the method to substantially reduce the levels of this protein
kinase in cells. Previous work as well as unpublished work3 has demonstrated that
the CDK11p110 protein kinase is essential for cell survival
(16, 17, 21); thus, RNAi (22) was the method chosen due to the greater
possibility so that the levels of CDK11p110 could be
reduced transiently in HeLa cells long enough to perform the
phospholabeling experiments involving 9G8. As shown in Fig. 3, panel A, immunoblotting of
the resulting cell lysate from untreated and
CDK11p110-RNAi-treated cells shows that the endogenous
level of this protein kinase has been reduced by >70% (this
number was obtained after scanning the resulting autoradiograph). These
same HeLa cells were also transfected with a FLAG-9G8 expression
construct, and the expression of the 9G8 protein was confirmed by
immunoblotting with an M2 FLAG-specific antibody (Fig. 3A).
Equivalent loading of proteins in the two lanes was verified by
immunoblotting with an
To conclusively demonstrate that the loss of the endogenous
CDK11p110 protein kinase activity resulted in the
diminished phosphorylation of the 9G8 protein, control and RNAi-treated
HeLa cells were labeled with 32P-labeled inorganic
orthophosphate (Pi) for 2 h and the cell lysates were
immunoprecipitated using the same M2 FLAG-specific antibody from the
first experiment (Fig. 3B). Similar to the diminished level
of the immunoreactive slower migrating FLAG-tagged 9G8-specific protein
band (Fig. 3A), the level of phosphorylation of this same band was reduced to 42% of the level of the identical band in the
control lane in the cellular [32P]orthophosphate-labeling
experiments (Fig. 3B). Additional fine mapping of the
specific residue(s) phosphorylated in 9G8 by CDK11p110 is
currently being explored; however, the large number of potential Ser/Thr phosphorylation sites and tryptic peptides for the 9G8 protein
are creating technical difficulties (e.g. a comparative analysis of two-dimensional tryptic peptide maps) that may take some
time to overcome.
CDK11p110 Complexes Play an Important Role in
Pre-mRNA Splicing--
Considering the experimental evidence
suggesting that the CDK11p110 protein kinase is capable of
forming multiple distinct multiprotein complexes with RNAP II in
vivo that appear to have functions relevant to both transcription
and RNA splicing (16, 18), we decided to assess the possible role of
the CDK11p110 protein kinase in regulating
pre-mRNA-splicing activity. Because 9G8 encodes a known splicing
factor, in vitro splicing reactions were first performed
using a
To establish next whether the interaction between CDK11p110
and 9G8 was functionally relevant, an affinity-purified form of the P2N100 antibody that targets the amino-terminal domain as well as a P1C
monoclonal antibody that targets the carboxyl-terminal domain of
CDK11p110 was used in combination to partially
immunodeplete this protein kinase from HeLa cell NEs. The ability of
these partially CDK11p110-immunodepleted extracts to
facilitate splicing of the same 9G8-dependent
However, a separate and completely distinct approach to this type of
experiment can be now performed using a dominant-negative form of the
CDK11p110 peptide to disrupt the normal interaction of this
protein kinase with other members of this large multiprotein complex.
Previous experiments have shown that the interaction among the
CDK11p110 protein kinase, 9G8 (this study), RNPS1, and
casein kinase 2 (16, 17, 21) requires the amino-terminal domain
of CDK11p110. Therefore, a dominant-negative strategy was
employed to further determine whether interfering with normal
CDK11p110 protein/protein interactions would affect
splicing activity in vitro by interrupting the formation of
the multiprotein complexes necessary for these activities. We
hypothesized that an excess of the same amino-terminal region of the
CDK11p110 protein kinase used to generate the P2N100
antibody, which was used in the immunodepletion studies described
above, might also be able to inhibit splicing activity by competing
with the endogenous full-length CDK11p110 for interaction
with the proteins that are required for splicing activity. Such an
experiment would provide separate and independent confirmation of the
involvement of the CDK11p110 protein kinase complex in the
regulation of in vitro splicing activity, most probably
through the splicing factor 9G8. To test this hypothesis, the
amino-terminal fragment of CDK11p110 corresponding to the
region of CDK11p110 that was used to generate the P2N100
antibody was affinity-purified from Escherichia coli and
used as a competing protein in the in vitro splicing
reaction. As shown in Fig. 5, this purified CDK11p110
peptide fragment was capable of dramatically inhibiting splicing activity (Fig. 5, right panel), consistent with our
hypothesis as well as the data from the immunodepletion studies. Thus,
this type of assay further confirms independently the involvement of the CDK11p110 protein kinase in pre-mRNA-splicing events.
In the this report, we have demonstrated that the
CDK11p110 protein kinase exists in a large multiprotein
complex with a previously identified regulatory subunit, cyclin L (14),
as well as the essential splicing factor 9G8. Of some interest is the
fact that others have suggested that cyclin L and
CDK11p110, possibly because of its association with this
cyclin regulatory partner, may act to regulate splicing factors
(e.g. SC-35) (15). These previous studies (14, 15) have
relied almost entirely upon the co-localization of cyclin L
(i.e. a known regulatory partner for the
CDK11p110 protein kinase) and SC-35.
However, we have shown here that 1) the splicing factor 9G8 interacts
either directly or indirectly physically with the CDK11p110
protein kinase in vivo, 2) that the 9G8-splicing factor is a phosphorylated protein in vivo and that it is phosphorylated
in vitro and in vivo by CDK11p110, 3)
that the specific immunodepletion of CDK11p110 protein
complexes from nuclear lysates results in a substantial loss of
in vitro splicing activity, whereas its re-addition appears to completely restore this in vitro splicing activity, and
finally, 4) that a truncated, dominant-negative form of the
CDK11p110 protein used to generate one of the
CDK11p110 antibodies (i.e. P2N100) and used in
the immunodepletion experiments is also capable of markedly reducing
9G8-dependent in vitro splicing activity. Thus, based upon a number of different types of
experiments, we believe that one of the potentially important cellular
roles of the CDK11p110 protein kinase is to regulate
splicing activity, quite possibly through phosphorylation of the
splicing factor 9G8. Although CDK11p110 appears to
definitely contribute to the in vitro and in vivo phosphorylation status of 9G8, we cannot absolutely rule out the possibility that additional protein kinases may also play an important role in 9G8 function during splicing, particularly because there are so
many putative phosphorylation sites within this protein. Even so, based
upon the work in this study, the CDK11p110 protein kinase
is the first CDK that has been functionally linked directly to the
regulation of RNA-splicing events most probably through its ability to
phosphorylate a splicing factor. In addition, based upon previous
experimental evidence involving the CDK11p110 protein
kinase, we feel that the protein probably has multiple important
functions (16, 17, 21). Others have suggested models of chromatin
regulation in which different proteins are recruited to RNAP II
complexes based upon the phosphorylation status of the CTD to regulate
numerous important cellular functions including transcription, RNA
splicing, RNA polyadenylation, and RNA capping (3, 7, 8, 30-34). At
this time, we can only conclude that one of the major functions of the
CDK11p110 protein kinase complexes containing the
regulatory partner cyclin L appears to be to help coordinate the
regulation of normal splicing and possibly transcription within cells.
Finally, this work establishes yet another CDK with an important role
in RNAP II regulation of transcription and/or RNA-splicing events,
namely CDK11p110, in addition to the functions that are
already attributed to three different CDK family members; namely, CDK7,
CDK8, and CDK9.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amino-1,2,4-triazole. The yeast clones grown on these triple
nutrient-deficient plates were finally subjected to a
-galactosidase
assay, and the resulting positive blue clones were selected for further
DNA sequence analysis.
-actin antibody (I-19, Santa Cruz Biotechnologies), and
c-Myc-agarose beads (Clontech).
-mercaptoethanol, 5 mM imidazole, 1% Nonidet P-40, and
5 mM phenylmethylsulfonyl fluoride. This was followed by
nickel-nitrilotriacetic acid (Qiagen) affinity column purification of
the His6-tagged proteins from insect cell lysates. Proteins
eluted from nickel beads were further purified by DEAE and SP-Sepharose
column with the BioLogic HR purification system (Bio-Rad). The 9G8
protein was purified under denaturing conditions. The insect cells
expressing the His6-tagged 9G8 were lysed in 6 M guanidine-HCl, 100 mM
NaH2PO4, and 10 mM Tris-HCl (pH
8.0). The 9G8 protein eluted from the nickel beads was then re-natured
in a buffer containing 20 mM Hepes (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM
dithiothreitol, and 10% glycerol for 24 h. The re-natured 9G8
protein was subjected to further purification using DEAE and
SP-Sepharose columns with the BioLogic HR purification system
(Bio-Rad). The His6-P2N100 protein used as a
dominant-negative competitor in the in vitro splicing assay
was prepared as described previously (18).
-mercaptoethanol, 0.1%
Nonidet P-40, 10% glycerol, and complete protease inhibitors (Roche
Molecular Biochemicals). The resulting cell lysates were then incubated with either anti-FLAG M2-agarose beads (Sigma) or anti-c-Myc-agarose beads (Clontech) for 4 h at 4 °C. Washes
were performed five times using 1 ml of lysis buffer each time.
Immunoblotting was performed as described previously (16).
-32P]ATP, 2 mM benzamidine, 60 mM
-glycerophosphate, 0.1 mM
Na3VO4, and 0.1 mM NaF using a
final volume of 30 µl with 100 ng of the purified 9G8 protein and 30 ng of either the purified CDK11p110 or
CDK11p110-D552N protein. The reactions were then allowed to
incubate for 20 min at 30 °C and were stopped by the addition of the
SDS-PAGE sample buffer. The phosphorylated proteins were analyzed by
12% SDS-PAGE, and the corresponding gel was then dried and
autoradiographed as described previously (18).
-globin pre-mRNA substrates were prepared by runoff in
vitro transcription using SP6 RNA polymerase and BamHI linearized pSP64-H
E6 plasmid as the template (23, 24). The HeLa
cytoplasmic S100 and HeLa nuclear extracts (NEs) used for the in
vitro splicing reactions were prepared using 15 liters of cultured
HeLa cells obtained from the National Cell Culture Center as performed
by Mayeda and Krainer (25). For the preparation of the
CDK11p110-immunodepleted nuclear extract, 2 mg of the HeLa
nuclear extract was first incubated with 15 µg of the
affinity-purified P2N100 CDK11p110 antibody at 40 °C for
2-4 h and the antibody was prepared as described previously (16, 18).
This was followed by subsequent incubation of the HeLa NE with 15 µg
of the P1C monoclonal antibody and 15 µl of
-bind-Sepharose beads
at 40 °C for 2-4 h. The resulting CDK11p110-immunoprecipitated beads were removed by
centrifugation at 2000 rpm for 5 min. The remaining immunodepleted NE
was then subjected to two additional rounds of CDK11p110
immunodepletion by repeating this procedure. Normal rabbit IgG protein
was used as a control antibody for these immunodepletion experiments.
In vitro splicing reactions were carried out in a final
volume of 25 µl with 20 µg of NE and 20 fmol of
32P-labeled pre-mRNA substrate followed by incubation
at 30 °C for 1-3 h. The spliced RNA products were analyzed by
electrophoresis on a denaturing 5.5% polyacrylamide, 7 M
urea gel (24). The CDK11p110-spliced dominant-negative
protein competition assay was performed by adding 460 ng of the
affinity-purified, truncated CDK11-P2N100 protein that had been
expressed in insect cells to the same 25-µl splicing reaction
containing the same components with the exception of the two CDK11
antibodies. The CDK11-P2N100 protein was allowed to pre-incubate with
the NE for 5 min at 23 °C prior to the addition of the other
reagents. The analysis of the spliced RNA products was performed as
noted above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CDK11p110 interacts with 9G8
in vivo. A, the endogenous
CDK11p110 protein kinase co-immunoprecipitates with cyclin
L (also known as Ania6) subunit and the splicing factor 9G8
in vivo. HeLa cells transfected with FLAG-tagged 9G8 were
immunoprecipitated with M2 FLAG antibody and immunoblotted with M2
anti-FLAG and P2N100 antibodies. Lane 1, lysates from HeLa
cells without transfection; lane 2, lysates from HeLa cells
transfected with the empty expression vector pCDNA3; lane
3, lysates from HeLa cells transfected with pCDNA3/FLAG-9G8.
B, HeLa cells were transiently transfected with
pCDNA3/N-FLAG-cyclin L and pCMV-Myc/9G8 for 24 h. Anti-FLAG M2
beads were incubated with the resulting cell lysates, or the cell
lysates were prepared from cells transfected with the empty expression
vector pCDNA3 (as control) for 4 h at 40 °C. The
immunoprecipitated protein complexes were resolved by 12% SDS-PAGE
followed by immunoblotting with the anti-P2N100, anti-c-Myc, and
anti-M2 FLAG antibodies. C, mapping of the
CDK11p110 protein domain(s) required for interaction with
9G8. The plasmids pUHD10-3 and pcDNA3 containing FLAG-tagged
truncated CDK11p110 fragments were transfected into HeLa
cells followed by immunoprecipitations with the M2 FLAG monoclonal
antibody and then immunoblotted with the 9G8-specific polyclonal
antibody. The truncation mutants that were capable of pulling down 9G8
are marked with a "+", whereas those mutants with which
9G8 did not co-immunoprecipitate are marked with a " ."
D, indirect immunofluorescence analysis of
CDK11p110 and 9G8 co-localization in the nuclei of HeLa
cells. The 9G8 protein was visualized using an affinity-purified rabbit
polyclonal 9G8 antiserum and a goat anti-rabbit Texas Red
secondary antibody, whereas the CDK11p110 and 9G8 proteins
were visualized merged using the mouse P1C monoclonal antibody and a
goat anti-mouse fluorescein isothiocyanate secondary
antibody. The panels are labeled accordingly, and the superimposed
image is indicated as 9G8 CDK11p110.
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Fig. 2.
CDK11p110 phosphorylates 9G8
in vitro. A, in
vitro protein kinase reactions were carried out using recombinant
CDK11p110 purified from insect cells, recombinant
CDK11p110-D552N purified from insect cells, recombinant 9G8
purified from insect cells, CDK11p110 purified from insect
cells with purified 9G8 added as substrate, and
CDK11p110-D552N purified from insect cells with purified
9G8 added as substrate. The reactions were run at 30 °C for 20 min.
B, purified 9G8 protein is phosphorylated. 3 µg of
recombinant 9G8 protein was incubated with 10 units of alkaline
phosphatase (20 units/µl, Roche Molecular Biochemicals) in 50 mM Tris-HCl, and 0.1 mM EDTA (pH 8.5) for
1 h at 37 °C. The alkaline phosphatase (AP)-treated
and untreated 9G8 samples were then subjected to 12% SDS-PAGE analysis
and visualized by staining with Coomassie Blue.
-actin antibody control (Fig. 3A).
Of particular interest was the fact that the steady-state level of the
slower migrating, phosphorylated form of the two 9G8-specific protein
bands was also reduced ~50-60% in the CDK11p110 RNAi
HeLa cells.
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Fig. 3.
CDK11p110 phosphorylates the
splicing factor 9G8 in vivo. A,
immunoblotting of the cell lysates from the control (empty pSUPER) HeLa
( ) and CDK11p110 RNAi-treated HeLa (+) cells with the
various antisera indicated on the left of the
panels. The proper migration of the various proteins is
indicated by the arrows and labeling to the right
of the panel. After 24 h of CDK11p110 RNAi
or control vector treatment, the HeLa cells were transfected with
pcDNA3/N-FLAG-9G8 for 8 h. 50 µg of each cell lysate was
separated by 12% SDS-PAGE and immunoblotted with the indicated
antisera sequentially. B, anti-FLAG M2-agarose beads were
used to immunoprecipitate the [32P]orthophosphate-labeled
9G8-FLAG protein. The HeLa cells were treated with
CDK11p110 RNAi (+) and control pSUPER vector (
) and
transfected with the pcDNA3/N-FLAG-9G8 construct as described in
panel A. 8 h post-FLAG-9G8 transfection, the cells were
[32P]orthophosphate-labeled for 2 h before
harvesting. The overexpressed FLAG-9G8 protein was immunoprecipitated
with the anti-FLAG M2-agarose beads, and the products were separated by
12% SDS-PAGE.
-globin pre-mRNA template, which is known to be
responsive to the addition of the 9G8 protein, as a substrate to verify
that our cDNA isolate encoded a bona fide protein
capable of stimulating in vitro splicing activity in a
9G8-dependent manner as reported by others previously (26, 27). The 9G8 protein purified from insect cells was added to the
splicing-deficient cytoplasmic HeLa S100 cytoplasmic fraction, which is
devoid of splicing activity, in increasing amounts, and the resulting
splicing reactions and their products were then visualized by using
denaturing PAGE analysis (Fig. 4). As
expected, a dose-dependent increase in the production of
the properly spliced
-globin products was observed, confirming the
9G8 dependence of this particular template using in vitro
splicing assays.
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Fig. 4.
Purified 9G8 recombinant protein complements
HeLa cytoplasmic S100 with splicing activity. The HeLa S100
fraction, devoid of endogenous splicing factors, was incubated with the
indicated amount of purified recombinant 9G8 in the presence of the
labeled -globin pre-mRNA template. The in vitro
splicing assay was carried out as described under "Materials and
Methods."
-globin
template was determined by using a standard in vitro splicing assay. As shown in Fig. 5,
left panel, immunodepletion of the CDK11p110
protein kinase complexes from the HeLa cell NE resulted in a dramatic
decrease in the production of the properly spliced
-globin products
that result from the splicing of the template by the exogenously added
9G8 protein. Furthermore, the re-addition of these same
CDK11p110 immune complexes to the in vitro
splicing assays, again using the 9G8-dependent
-globin
template, was able to substantially restore splicing activity. Because
of the rather complex and dynamic nature of CDK11p110
immune complexes, it is impossible at this time to add back individual recombinant proteins that represent each of the proteins that are
present in these CDK11p110 immune complexes. Furthermore, a
number of the CDKp110-interactive proteins from these
immune complexes have not yet been identified. We hope to identify a
sufficient number of the different protein members of these
CDK11p110 immune complexes in the near future to attempt
such an experiment.
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Fig. 5.
CDK11p110 plays a crucial role in
nuclear-splicing activity. A, in vitro
splicing assays were performed using the
CDK11p110-immunodepleted NE. Lane 1, control NE
(rabbit IgG as mock antibody); lane 2, NE partially
immunodepleted of CDK11p110 using the P2N100 and P1C
antibodies as described under "Materials and Methods"; lane
3, in vitro splicing activity after the P2N100/P1C
immunocomplex beads were added back to the P2N100/P1C-immunodepleted
NE. B, CDK11p110 dominant-negative in
vitro splicing assay. The CDK11p110 amino-terminal
protein fragment affinity-purified from E. coli was added to
the NE-splicing reaction mixture as a competitor for
CDK11p110, and the in vitro splicing reactions
were run as described under "Materials and Methods."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We acknowledge and thank Dr. R. Agami (The Netherlands Cancer Institute) for kindly providing the pSUPER plasmid and J. Grenet and S. Bothner for technical assistance as well as Dr. P. Loyer for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM44088 (to V. J. K.) and St. Jude Children's Research Hospital Grant CA21765 as well as funding 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, December 24, 2002, DOI 10.1074/jbc.M210057200
2 D. Hu, A. Mayeda, J. H. Trembley, J. M. Lahti, and V. J. Kidd, unpublished observation.
3 T. Li, A. Inoue, J. M. Lahti, and V. J. Kidd, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; RNAP II, RNA polymerase II; CTD, carboxyl-terminal domain; RNAi, RNA interference; RNPS1, RNA-binding protein with serine-rich domain.
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REFERENCES |
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