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ã Oncogene (2002) 21, 4158 ± 4165 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Physical interaction between pRb and cdk9/cyclinT2 complex Cristiano Simone1,2,5, Luigi Bagella1,3,5, Cristiana Bellan1,3 and Antonio Giordano*,4 1 Department of Pathology, Anatomy and Cell Biology, Thomas Jeerson University, Philadelphia, Pennsylvania, PA 19107 USA; Department of Internal Medicine and Public Medicine, Division of Medical Genetics, University of Bari, Bari 70124 Italy; 3 Institute of Pathologic Anatomy and Histology, University of Siena, Siena 53100 Italy; 4Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, Pennsylvania, PA 19122 USA 2 Cyclin-dependent kinase 9 (cdk9) is a multifunctional kinase with roles in dierent cellular pathways such as transcriptional elongation, dierentiation and apoptosis. Cdk9/cyclin T diers functionally from other cdk/cyclin complexes that regulate cell cycle progression, but maintains structural anity with those complexes. In addition, previous reports have demonstrated that the cdk9 complex is able to phosphorylate p56/pRb in vitro. In this report we show in vitro and in vivo interaction between cdk9/cyclinT2 and the protein product of the retinoblastoma gene (pRb) in human cell lines. The interaction involves the region composed of residues 129 ± 195 of cdk9, cyclinT2 (1 ± 642 aa) and the Cterminal domain of pRb (835 ± 928 aa). We located the minimal region of cdk9 phosphorylation on the Cterminus of pRb, by identifying the residues between 793 and 834. This region contains at least three prolinedirected serines (sp), S795, S807 and S811, which have been reported to be phosphorylated in vivo and which could be targeted by the cdk9 complex. These data suggest that, in logarithmically growing cells, cdk9/ cyclin T2 and pRb are located in a nuclear multiprotein complex probably involved in transduction of cellular signals to the basal transcription machinery and that one of these signals could be the cdk9 phosphorylation of pRb. Oncogene (2002) 21, 4158 ± 4165. doi:10.1038/sj.onc. 1205511 Keywords: cdk9; cyclin T2; pRb; phosphorylation Introduction Cyclin-dependent kinase 9 (cdk9) is a cdc2-related serine/threonine kinase that is isolated using degenerate oligonucleotide primers derived from conserved regions found in cdc2 and cdk2 (Grana et al., 1994). The cdk9 gene is widely expressed in human and murine tissues, *Correspondence: A Giordano, Sbarro institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, BioLife Science Bldg. Suite 333, 1900 N 12th Street, Philadelphia, PA 19122, USA; E-mail: [email protected] 5 The ®rst two authors contributed equally to this study. Received 19 June 2001; revised 13 March 2002; accepted 21 March 2002 with higher levels found in terminally dierentiated cells, and its promoter activity parallels the protein levels (De Luca et al., 1997; Bagella et al., 1998, 2000). The regulatory units of cdk9 are the T-family cyclins (T1, T2a and T2b) (Wei et al., 1998; Peng et al., 1998) and cyclin K (Edwards et al., 1998). The elevated levels of cdk9 and its regulatory subunits in terminally dierentiated cells, together with the fact that cdk9/cyclin T complexes are not cell cycle-regulated, distinguish cdk9 from the other cdks (MacLachlan et al., 1995; De Falco and Giordano, 1998). Moreover, unlike the other cdks, which regulate cell cycle progression and phosphorylate histone H1, cdk9 fails to phosphorylate H1 (Grana et al., 1994). Cyclin T levels are not cell cycle-regulated and, for this reason, cdk9 kinase activity does not change during the dierent phases of the cell cycle. Cdk9 is instead implicated in the regulation of transcriptional elongation via phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNApolII) and forms the positive transcription factor b (PTEF-b) with cyclin T (Dahmus, 1996; Marshall et al., 1996; Zhu et al., 1997; De Falco and Giordano, 2002). Kinase activity corresponding to cdk9 has been detected in the HIV Tatassociated kinase (TAK) complex, where cdk9 is required for Tat-dependent stimulation of transcriptional elongation (Yang et al., 1996; Zhu et al., 1997; Mancebo et al., 1997). CycT1 was the ®rst cyclin discovered to associate with cdk9 to form a complex interacting with HIV-1-Tat protein in the TAK complex (Wei et al., 1998). Human cycT1 stimulates Tat activation, while cycT2a and T2b fail to form a complex with Tat bound to the transactivation response RNA element (TAR) (Kwak et al., 1999; Wimmer et al., 1999). These data indicate functional dierences between cycT1 and cycT2, even if cycT2 function remains less understood. The catalytic activity of the cdk9 immunocomplexes results only from cdk9 and it is abolished using a dominant negative form (kinase inactive mutant, cdk9dn) (De Falco et al., 2000), which consists of a point mutation within the ATP-binding domain that changes the Asp to Asn at residue 167. Previous studies have already demonstrated that cdk9 is able to phosphorylate p56/pRb (a deletion mutant maintaining the A/B pocket and the C-terminal domain of pRb, 379 ± 928 aa) in vitro (Grana et al., 1994; De Luca et al., 1997). Phosphopeptide analysis of p56/pRb after phosphorylation by cdk9, compared to cdk9/cyclinT2 binds pRb C Simone et al that after cdk2 and cdc2 phosphorylation, indicates that, at least in vitro, the three cdk complexes share several target phosphoresidues, but cdk9 kinase activity involves only serine residues (De Luca et al., 1997). During preliminary studies of cdk9/cyclin T complexes, we performed immunoprecipitations (Ips) with anity puri®ed anti-cdk9 antibody from nuclear extracts of S35-labeled NIH3T3 cells. By comparing the speci®c pattern of immunoprecipitation with an Ip performed using pre-immune rabbit serum, we identi®ed several bands that could represent cdk9-interacting proteins. We started to consider bands with predicted molecular weights potentially corresponding to known proteins that may interact with cdk9. The predicted protein structure anity with the other cdks prompted us to consider of interest a band at 110 kD. Cdk4/ cyclin D, cdk2/cyclin E and cdk1/cyclin A complexes all bind and/or phosphorylate pRb during dierent phases of the cell cycle (Ezhevsky et al., 1997; Hatakeyama et al., 1994; Lundberg and Weinberg, 1998; Pan et al., 2001). The retinoblastoma gene codes for a 105 kD nuclear protein that is regulated by phosphorylation. Depending on the amount of phosphorylated residues, pRb shifts from an active hypophosphorylated state towards an inactive hyperphosphorylated state (Mittnacht, 1998; Stiegler and Giordano, 2001). The basic structure of pRb consists of the N-terminal, the pocket region (spanning the A and B domains, separated by a spacer) and the Cterminal region. The N-terminal region is the least understood in its functional role. The central region, which folds into a pocket, oers a docking site for several interacting proteins. Several residues within the pocket are responsible for the interaction with proteins containing a LXCXE docking site, such as E1A, E7, SV40 LTag, HDACs and cyclin D (Whyte et al., 1988; DeCaprio et al., 1998; Lee et al., 1998; MagnaghiJaulin et al., 1998; Ewen et al., 1993; Dowdy et al., 1993). The pocket region is also the binding site for other cellular factors lacking the LXCXE motif, such as E2F transcription factors (Stiegler and Giordano, 2001). Mutations in these pocket residues render pRb inactive or heavily impaired in controlling the cell cycle. The function to arrest the cell cycle is separate and independent from an intact LXCXE binding site, demonstrating that additional sequences besides the pocket are important for pRb function. The C-terminal region of pRb is involved in association with important cellular factors including E2F, Mdm2 and c-Abl (Qian et al., 1992; Xiao et al., 1995; Welch and Wang, 1993). The C-terminal region spanning residues 793 ± 928 is the smallest domain of pRb that is eciently phosphorylated by cdk4/cycD1, cdk2/cycE and cdk2/ cycA (Pan et al., 1998; Adams et al., 1999). Very recently, examination of the pRb C-terminus revealed that it contains sequence elements related to ZRXL, which is the E2F- and p21-conserved cyclin/cdkbinding motif (Adams et al., 1999). These ®ndings separate the C-terminal region into two subdomains, one, the putative cyclin/cdk-binding domain spanning residues 830 ± 928, and the other (780 ± 826 aa) contain- ing the S/TP phosphorylation sites for cdk/cyclin complexes (Adams et al., 1999). In addition to RIL (830 ± 2 aa), RVL (857 ± 9), KPLKKL (870 ± 5) and KHL (889 ± 91) cdk/cyclin-binding domains (Adams et al., 1999), a new domain surrounding residue L901 was found to be important for the interaction with the cdk4/cyclin D1 complex (Pan et al., 2001). pRb has been identi®ed to interact with several cellular factors that in¯uence gene expression. Many of these transcription factors, like E2F1, E2F2 and E2F3, transcribe RNApolII-dependent genes. This is another link between cdk9/cyclin T (P-TEFb) and pRb that suggests a possible functional/physical interaction during gene transcription. In this study, we demonstrate that cdk9/cycT2 binds to pRb, involving residues 129 ± 195 of cdk9, the region of cycT2 comprising amino acids 1 ± 642 and the Cterminal region of the retinoblastoma protein (835 ± 928) and that cdk9 complexes phosphorylate the pRb region spanning amino acids 793 ± 834. 4159 Results Cdk9 interacts in vitro with pRb through 129 ± 195 aa To understand if the 110 kD band (Figure 1) previously discussed is the protein product of the retinoblastoma protein, we performed an in vitro GST pull-down assay. To test whether a direct physical interaction between cdk9 and pRb occurs, we ®rst investigated if the proteins could interact in vitro. The in vitro translated (IVT) full-length cdk9 was wellprecipitated by GST-pRb (379 ± 928), while no interaction was found with GST alone Figure 2d, lanes 5 and 6). To map the pRb binding site on the cdk9 protein, three deletion mutants, cdk9 (129 ± 372), cdk9 Figure 1 Immunoprecipitations from nuclear extracts of S35labeled NIH3T3 cells. Immunoprecipitation (Ip) was performed with anity puri®ed anti-cdk9 antibody from nuclear extracts of S35-labeled NIH3T3 cells and compared to an Ip with preimmune rabbit serum (NRS). Bands corresponding to cdk9, cyclin T and a band at 110 kD were indicated Oncogene cdk9/cyclinT2 binds pRb C Simone et al 4160 Figure 2 Cdk9 interacts in vitro with pRb through the region corresponding to 129 ± 195 aa. (a) Summary of in vitro translated (IVT) S35-labeled cdk9 proteins and their ability to interact with GST-pRb (379 ± 928). (The PSTAIRE-like domain is referred to as (PITALRE)). (b) Summary of GST-fusion proteins and their ability to coprecipitate cdk9 (1 ± 372). (A: A domain; B: B domain; AB: pocket domain; C: C-terminal domain). (c) IVT-cyclin T2 isoforms (a and b). (The two splice isoforms are identical in the 1 ± 642aa region. 1 ± 262: cyclin box region; 642 ± 663 for cycT2a and 642 ± 730 for cyclin T2b: C-terminal domain). (d) The interaction between GST-pRb (379 ± 928) and dierent IVT fragments of cdk9 was tested. GST alone was used as a negative control. The GSTfusion proteins were precipitated by glutathione-agarose anity chromatography and the interaction with the in vitro translated S35labeled proteins was visualized by autoradiography (218 ± 372) and cdk9 (1 ± 195) Figure 2a, lanes 8 and 12), were tested for their ability to interact with GSTpRb (379 ± 928) in a pull-down assay. GST alone was used as a negative control. An interaction, comparable to that with the full-length cdk9, was detected with two deleted forms, cdk9 (129 ± 372) and cdk9 (1 ± 195) Figure 2a), suggesting that the minimal binding region involves amino acids 129 ± 195. Minimal cdk9-binding region of pRb involves 835 ± 928 aa A similar experiment was performed to map the minimal cdk9-binding region of pRb and to address if pRb was also able to interact with cyclin T2, which is not extensively characterized in the literature. IVT S35labeled cdk9 and cyclin T2 (isoforms a and b) were tested for their ability to interact with GST-pRb (379 ± 928) Figure 3a) in a pull-down assay, using GST alone as a negative control. This experiment provided evidence that either cdk9 or cyclin T2a (and T2b) could directly interact with pRb and that they could form a trimeric complex in vitro. Afterwards, we investigated whether a pRb mutant (379 ± 928, Oncogene C706F), unable to interact through its pocket domain (Magnaghi-Jaulin et al., 1998), could still bind the cdk9/cyclin T2 complex. Under the same experimental conditions, the GST-pRbC706F mutant was still able to recruit both cdk9 and cyclin T2 (a and b), as the wild type does Figure 3b). These data indicate that the possible region of interaction could involve the Cterminus of pRb. To address this point, we tested three deletion mutants of pRb. IVT S35-labeled cdk9 and cyclin T2 (isoforms a and b) were tested with GST-pRb (793 ± 928), GST-pRb (768 ± 834) and GST-pRb (835 ± 928) Figure 3c ± e) in a pull-down assay, using GST alone as a negative control. Like GST-pRb (379 ± 928), GST-pRb (793 ± 928) and GST-pRb (835 ± 928) were able to precipitate cdk9/cycT2 proteins; the third deletion mutant, GST-pRb (768 ± 834), did not interact with PTEF-b components Figure 3c ± e). These data indicate that the regions mediating the binding between cdk9/cycT2 and pRb encompass the C-terminal domain of pRb (835 ± 928 aa), cyclinT2 (1 ± 642) and the central region (129 ± 195 aa) of cdk9 surrounding the ATP-binding kinase domain (see Figures 2 and 3). cdk9/cyclinT2 binds pRb C Simone et al 4161 Figure 3 Minimal cdk9/cycT2-binding region of pRb involves the region containing 835 ± 928 aa. (a) IVT S35-labeled proteins used in the GST pull-down experiments. (Lane 1: cdk9; lane 2: reticulocyte lysate; lane 2:cyclin T2a; lane 4: cyclin T2b). (b ± f) The GSTpRb fusion proteins indicated in Figure 2b were tested for their ability to coprecipitate IVT-cdk9, IVT-cycT2a and IVT-cycT2b. GST alone was used as a negative control. The GST-fusion proteins were precipitated by glutathione-agarose anity chromatography and the interaction with the in vitro translated S35-labeled proteins was visualized by autoradiography Cdk9, cyclin T2 and pRb form a multimeric nuclear complex in human cells Cdk9 complexes phosphorylate pRb on the C-terminal domain To verify that the interaction described above was present in vivo, we performed a GST pull-down of human nuclear cell extracts. GST-pRb was able to co-precipitate cdk9 from nuclear extracts of Jurkat and HeLa cells Figure 4a), while GST alone, used as a negative control, was not. As a positive control, the same ®lter was blotted with antiHDAC1 antibody, revealing the interaction as well (data not shown). Increasing the time of the binding reaction from 2 h to overnight incubation, GST-pRb co-precipitated more cdk9, as was demonstrated by Western blot with anity puri®ed anti-cdk9 antibody Figure 4a, compare lanes 2 and 4, and 7 and 9), while GST alone continued to not interact with cdk9 Figure 4a, lanes 1, 3, 8, and 10). To con®rm the interaction, we performed Ips from nuclear extracts of proliferating HeLa and Jurkat cells using anti-cdk9 anity puri®ed antibody. Western blot with anti-pRb antibody revealed the presence of the retinoblastoma gene product in association with cdk9 in the nucleus of human proliferating cells Figure 4b). Further analysis of the a-cdk9 antibody immunoprecipitates revealed the presence of cdk9 and cycT2 (data not shown). Previous reports indicate that p56/pRb is a good substrate in vitro for the cdk9 complex (Grana et al., 1994; De Luca et al., 1997). Using the reported GSTpRb fusion proteins as substrates Figure 2b), we performed a kinase assay with immunoprecipitated cdk9 complexes from HeLa nuclear extracts Figure 5) (pre-immune rabbit serum used as a negative control, data not shown). Cdk9 complexes phosphorylated pRb (793 ± 928) as well as p56/pRb (379 ± 928), con®rming that, at least in vitro, this complex behaves the same as other cdk/cyclin complexes. Very interestingly, neither of the two deleted forms of the pRb C-terminus, pRb (835 ± 928) and pRb (768 ± 834), was phosphorylated. The other cdk/cyclin complexes are unable to phosphorylate the region 794 ± 829 in the absence of recognition motifs contained between pRb residues 829 ± 928 (Adams et al., 1999). Our data suggest that this is true also for cdk9 complexes. In fact, cdk9/cyclin T2 binds pRb (835 ± 928), phosphorylates the C-terminal domain (793 ± 928), but fails to phosphorylate the two parts that compose it. The region of pRb corresponding to 793 ± 834 aa contains at least three proline-directed serines (sp), S795, S807 and S811, which have been reported to be phosphorylated in vivo (Hollingsworth Oncogene cdk9/cyclinT2 binds pRb C Simone et al 4162 Figure 4 Cdk9, cyclin T2 and pRb form a multimeric nuclear complex in human cells. (a) GST pull-down was performed with human nuclear cell extracts. GST-pRb (378 ± 928) was able to coprecipitate cdk9 from nuclear extracts of Jurkat and HeLa cells. The same ®lter was blotted with anti-HDAC1 antibody as a positive control (immunoblot not shown). GST alone was used as a negative control. (b) Jurkat and HeLa nuclear cell extracts were precleared and incubated with anity puri®ed anti-cdk9 and preimmune serum (NRS) (used as a negative control). After extensive washes, the samples were resuspended in Laemmli buer and loaded on acrylamide gel. The associated proteins were detected by immunoblot by using anti-pRb, anti-cycT2 and anti-cdk9 antibodies (cdk9 and cycT2 immunonoblots are not shown) the C-terminus of pRb to phosphorylate serine residues present between 793 and 834 aa. Discussion Figure 5 Cdk9 immunocomplexes phosphorylate pRb in vitro on the C-terminal domain. Ips were performed with anti-cdk9 in logarithmically growing HeLa cells. (Pre-immune rabbit serum used as a negative control, data not shown). The immunocomplexes were extensively washed, incubated in kinase assay reaction buer with 0.5 mg of the reported GST-fusion proteins and resolved on 13% gel et al., 1993) and could be targeted by cdk9 complexes. From our data, we cannot exclude that in the pRb amino acid sequence there are other serines targeted by cdk9 complexes and that this phosphorylation has a speci®c role in vivo; in our model, cdk9 complexes use the recognition sites of Oncogene Cdk9 protein levels and kinase activity are high in terminally dierentiated cells, as are cyclin T family protein levels (De Luca et al., 1997; Bagella et al., 1998; Wei et al., 1998; Peng et al., 1998). PTEF-b kinase activity is constant throughout the cell cycle, depending on cdk9 and cyclin T protein levels, which do not change during the dierent phases (Grana et al., 1994). In addition, cdk9 complexes fail to phosphorylate histone H1, but exert their kinase activity on the C-terminal domain of RNA polymerase II (Dahmus, 1996; Marshall et al., 1996; Zhu et al., 1997) and MyoD, a bHLH transcription factor part of the Muscle Regulatory Factor (MRF) family (Simone et al., 2002; Simone and Giordano, 2001). Despite these relevant functional dierences with the other cdk/cyclin complexes, cdk9 shares 41 ± 43% identity (61 ± 65% similarity) with human cdc2, cdk2, cdk3, cdk5 and 38% with cdk4 (Grana et al., 1994). Similarly, cyclin T has an amino-terminal cyclin box motif sharing 39% identity with the human cyclins and a carboxyl- cdk9/cyclinT2 binds pRb C Simone et al terminal PEST sequence (709 ± 726 aa), which is frequently found in G1 cyclins to regulate their turnover by cellular ubiquitination and proteolytic pathways (Wei et al., 1998). In past years no functional reason has been found to justify cdk9 phosphorylation of pRb in vitro, probably due to the complexity of an in vivo study of pRb phosphorylation and regulation. In this study, we characterize the physical interaction between cdk9, cyclin T2 and pRb, demonstrating that the C-terminus of pRb (835 ± 928) directly interacts with the region surrounding the kinase domain of cdk9 (129 ± 195) and with both isoforms of cyclin T2 (a and b), suggesting that the region of interaction involves the common sequence comprising 1 ± 642 aa Figure 2a ± c). In addition, cdk9 complexes phosphorylate the C-terminus of pRb (793 ± 928) in vitro and our data suggest that the phosphorylated region spans amino acids 793 ± 834 Figure 6). This evidence is intriguing because it con®rms the division of the pRb C-terminus into two subdomains. In fact, several groups have reported that pRb Cterminus (793 ± 928) could be separated into two subdomains with speci®city for cdk/cyclin complexes. The ®rst region (793 ± 829) contains the cdk phosphorylation sites and the second (829 ± 928) is the recognition/binding site for cdk/cyclin complexes Figure 6). This subdivision has been proven to be important for cdk2/cycE, cdk2/cycA and cdk4/cycD1 (Pan et al., 1998, 2001; Adams et al., 1999). During the cell cycle, pRb phosphorylation mediated by cdk/cyclin complexes is required for the cell to progress through the dierent phases. pRb shifts from a hypophosphorylated active form to a hyperphosphorylated inactive form and allows E2F transcription factors to be free from pRb control and bind to speci®c gene promoters. Several pieces of evidence might help to elucidate the meaning of cdk9-mediated phosphorylation of pRb in vivo. First, pRb has been identi®ed to functionally interact with factors which in¯uence RNA polymerase II-dependent gene transcription (De Luca et al., 1998; Fanciulli et al., 2000). This important eect of pRb on RNApolII transcription could represent a functional link with PTEF-b. In fact, cdk9 complexes have been characterized as transcriptional elongation factors involved in transcription of speci®c genes (Lis et al., 2000; Kanazawa et al., 2000; Foskett et al., 2001). Most likely, cdk9 complexes exert their action through the hyperphosphorylation of the CTD of RNApolII, allowing passage from the initiation to the elongation phase of RNA transcription (Lis et al., 2000; Napolitano et al., 2000). The cdk9 phosphorylation of pRb could be another signal that allows conversion from an inactive complex into a fully active complex that is ready to induce RNA transcription. The second point of interest is the involvement of pRb during cellular dierentiation and in terminally dierentiated cells. Several pieces of evidence have implicated pRb as an essential cofactor during muscle dierentiation. Muscle cells derived from pRb7/7 mice fail to irreversibly exit the cell cycle (Schneider et al., 1994), express reduced levels of late dierentiation markers and display impaired fusion into multinucleated myotubes (Novitch et al., 1996). Several hypotheses have been proposed to justify pRb involvement in dierentiation, including a physical binding with MyoD (Gu et al., 1993) and the inhibition of the anti-myogenic activity of E2F (Shin et al., 1995; Corbeil et al., 1995; Puri et al., 1997). More recently, pRb has been shown to promote functional synergism between MyoD and MEF2 protein, a coactivator of speci®c gene transcription (Novitch et al., 1999). However, the molecular mechanisms through which pRb promotes myogenic transcription remain poorly understood. We found that cdk9/cycT2 complex activates MyoD-mediated transcription and that cdk9 enzymatic activity is necessary for the myogenic program. In fact, cells expressing the kinase-inactive mutant of cdk9 (which behaves as a dominant negative) fail to dierentiate (Simone et al., manuscript submitted). Immunoprecipitated cdk9 complexes from C2C12 mouse myoblasts induced to dierentiate have an increasing kinase activity on p56/pRb with a peak of phosphorylation at 96 h of the myogenic program (Bagella et al., 1998). During muscle dierentiation, pRb is present in the active hypophosphorylated form, especially due to down-regulation of cyclins A, E and D1 and the up-regulation of cdk inhibitors. On the other hand, MyoD is inactivated during the cell cycle by cdk/cyclin complexes through direct phosphorylation or physical binding (Puri and Sartorelli, 2000), while the kinase activity of cdk9 is a signal that triggers myogenic transcription (Simone and Giordano, 2002). It is possible that cdk9/cycT2 activity is involved in the basal phosphorylation of the retinoblastoma protein and that pRb and cdk9/cycT2 cooperate to support MyoD-mediated myogenic transcription. Future studies are awaited to explain the relevance of cdk9/ cycT2-pRb interaction and to clarify the importance of pRb phosphorylation in dierent pathways other than the cell cycle, ideally by the in vivo characterization of the phosphoresidues targeted by cdk/cyclin complexes. 4163 Materials and methods Figure 6 C-terminal domain of pRb protein. The putative proline-directed serines and the RXL domains are in bold and underlined Cell lines Jurkat and HeLa cells were grown, respectively, in RPMI 1640 and in DMEM supplemented with 10% FBS, LOncogene cdk9/cyclinT2 binds pRb C Simone et al 4164 glutamine and antibiotics. All cell lines were obtained from ATCC. The constructs pcDNA3-cdk9wt-HATag expressing fulllength wild type cdk9 were previously described (De Falco et al., 2000). The construct pcDNA3-cdk9wt-HATag was cleaved with HindIII or KpnI and the fragments were recircularized with T4 ligase generating the construct pcDNA3cdk9 (129 ± 372)-HATag and pcDNA3-cdk9 (218 ± 372)HATag, respectively. The fragments of 582 bp obtained after cleavage of pcDNA3-cdk9wt-HATag with KpnI were subcloned in the KpnI site of pcDNA3 vector (Invitrogen) generating pcDNA3-cdk9 (1 ± 195). The constructs to generate the GST-Rb fusion proteins GST-Rb (379 ± 928), GSTRb (793 ± 928), GST-Rb (768 ± 834) and GST-Rb (835 ± 928) were obtained by PCR ampli®cation using, respectively, the following couples of primers: GGATCCATGAACACTATCCAA, GAGCTCTCATTTCTCTTCCTTG; GATCCCCTAGTTCACCCTTAC, GAGCTCTCATTTCTCTTCCTTG; GATCCATTTTGCAGTATGCTTC, GAGCTCGATACTAAGATTCTTG; GGATCCATTGGTGAATCATTCG, GGATCCTCATTTCTCTTCCTTG. The PCR products were cloned into pGEX 4T-1 or pGEX 2T (Pharmacia) to obtain, respectively, pGEX-4T-1 Rb-D(1 ± 378), pGEX-4T-1 Rb-D(1 ± 792), pGEX-4T-1 Rb-D(1 ± 767, 835 ± 928) and pGEX-2T Rb-D(1 ± 834). The construct pGEX-4T-1 Rb-C706F-D(1 ± 378) to generate GST-Rb (379 ± 928) C706F was performed using a QuickChange Site-Directed Mutagenesis Kit (Promega). The following primers were used: GGACCAAATTATGATGTTTTCCATGTATGGCATATG and CATATGCCATACATGGAAAACATCATAATTTGGTCC. The constructs expressing HA-cyclin T2a and HA-cyclin T2b were previously described (Peng et al., 1998). determined by Bradford assay (Biorad, CA, USA), following the manufacturer's instructions and by using BSA as a standard. GST-pRb (379 ± 928), GST-pRb (379 ± 928) C706F, GSTpRb (793 ± 928), GST-pRb (768 ± 834) and GST-pRb (835 ± 928) were expressed in bacteria and puri®ed as described above (De Falco et al., 2000). Nuclear cell extracts were incubated with the reported GST fusion protein for 2 h and/ or overnight at 48C. Bond proteins were recovered by centrifugation at 14 000 r.p.m. for 30 s at 48C and washed three times with Lysis Buer (50 mM Tris, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton, 0.1 mM Na3VO4, and 10 mg/ml aprotinin, leupeptin and phenylmethylsulphonyl ¯uoride (PMSF)). Immunoprecipitations were performed after preclearing the extracts with pre-immune rabbit serum. Anity puri®ed anti-cdk9 (1 : 50; De Falco et al., 2000) antibody incubations were performed overnight, at 48C, followed by the addition of protein A-sepharose for 60 min, rocking, at 48C. Beads were washed three times with Lysis Buer and twice with Lysis Buer containing 400 mM NaCl, and loading buer (50 mM Tris/HCl pH 6.8, 2% SDS, 10% glycerol) with 5% b-mercaptoethanol was added. The samples were resolved in 8 or 10% SDS-PAA gels and were transferred to a Hybond-ECL nitrocellulose ®lter (Amersham Life Science Inc.) at 48C and at 70 V for 2 h. 0.5% Ponceau Red was used to ensure equal transferring. The blots were blocked with TBST containing 5% non-fat dry milk. Anticdk9 (1 : 200), anti-cycT2 (1 : 2000; Peng et al., 1998) and antiHDAC1 (1 : 200, Santa Cruz Biotechnology, CA, USA) polyclonal antibodies and anti-pRb (1 : 500, Pharmingen) monoclonal antibody were used in TBST containing 5% nonfat dry milk, according to the Western blot conditions suggested by Santa Cruz (Santa Cruz Biotechnology, CA, USA). Anti-rabbit and anti-mouse peroxidase conjugated (1 : 20000) (Amersham, IL, USA) and ECL detection system (NEN, Du Pont, MS, USA) were used for detection. In vitro binding Kinase assay The TNT coupled reticulocyte kit was used for in vitro translation (Promega, WI, USA), according the manufacturer's instructions. All the samples were labeled using 35SMethionine (Amersham, IL, USA). The labeled samples were incubated with GST-fusion proteins, GST-pRb (379 ± 928) or GST-pRb (379 ± 928) C706F or GST-pRb (793 ± 928) or GST-pRb (768 ± 834) or GST-pRb (835 ± 928), using GST as a negative control, for 2 h at 48C, rocking, for in vitro binding. These fusion proteins were precipitated by glutathione-agarose anity chromatography and the immunoprecipitates were washed extensively in CPA buer (20 mM Tris-HCl pH 7.6, 250 mM NaCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.2% NP-40) containing fresh inhibitors and 1 mM DTT. Afterwards, the immunoprecipitates were resolved on 8 or 15% SDS ± PAGE and subjected to autoradiography. Cell extracts (200 mg) prepared in Lysis Buer were used for immunoprecipitations with a polyclonal anti-cdk9 antibody (negative control: pre-immune rabbit serum). The immunocomplexes were pulled down with protein A-sepharose and washed three times with Lysis Buer and twice with Lysis Buer containing 400 mM NaCl. The complexes were also equilibrated in kinase assay buer (minus ATP) (20 mM HEPES pH 7.4, 10 mM Mg Acetate). The kinase assay was performed in a volume of 20 ml, using 5 mCi/sample of g-ATP (Amersham, IL, USA). 0.1 mg of GST alone, GST-pRb (379 ± 928), GST-pRb (793 ± 928), GST-pRb (768 ± 834) and GST-pRb (835 ± 928) were added to the samples and incubated for 30 min at 308C. The reactions were stopped by adding 56 Laemmli Buer and the samples were resolved on a 13% SDS ± PAGE and subjected to autoradiography. Plasmids GST pull-down, immunoprecipitations and Western blot Jurkat and HeLa nuclear cell extracts were used for Western blot and Ips. Nuclear extracts were obtained by lysing the cells ®rst in RSB Buer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, phenylmethylsulphonyl ¯uoride (PMSF) and DTT); then, after centrifugation and three washes with RSB buer, the nuclei were lysed with Buer C ((20 mM HEPES, 0.2 mM EDTA, 420 mM NaCl, 1.5 mM MgCl2, 25% Glycerol, fresh aprotinin, leupeptin, DTT and phenylmethylsulphonyl ¯uoride (PMSF)). The protein concentration was Oncogene Abbreviations cdk, cyclin-dependent kinase; GST, glutathione S-transferase; pRb, retinoblastoma protein Acknowledgments We thank Marie Basso for her editorial assistance in the preparation of the manuscript and Dr Gaia Gallo for her technical support. We thank Dr David Price for generously cdk9/cyclinT2 binds pRb C Simone et al providing reagents. This work was supported in part by grants from the Sbarro Institute for Cancer Research and Molecular Medicine (to Dr Giordano). 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