CDK11 Complexes Promote Pre-mRNA Splicing*

Dongli HuDagger , Akila Mayeda§, Janeen H. TrembleyDagger , Jill M. LahtiDagger , and Vincent J. KiddDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 alpha -amino-1,2,4-triazole. The yeast clones grown on these triple nutrient-deficient plates were finally subjected to a beta -galactosidase assay, and the resulting positive blue clones were selected for further DNA sequence analysis.

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 alpha -actin antibody (I-19, Santa Cruz Biotechnologies), and c-Myc-agarose beads (Clontech).

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 beta -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).

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 beta -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).

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 [alpha -32P]ATP, 2 mM benzamidine, 60 mM beta -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).

In Vitro Splicing Assays-- The 32P-labeled beta -globin pre-mRNA substrates were prepared by runoff in vitro transcription using SP6 RNA polymerase and BamHI linearized pSP64-Hbeta Delta 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 gamma -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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.


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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.

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 alpha -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.

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 beta -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 beta -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 beta -globin pre-mRNA template. The in vitro splicing assay was carried out as described under "Materials and Methods."

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 beta -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 beta -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 beta -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."

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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