Functional Inactivation of Cdk9 Through Oligomerization Chain Reaction

Functional inactivation of Cdk9 through oligomerization chain reaction

Giuliana Napolitano1, Alberto Mazzocco1, Alessandro Fraldi1, Barbara Majello1 and Luigi Lania*,1

1Department of Genetics, Molecular and General Biology, University of Naples ‘Federico II’ Naples, Italy

The oligomerization chain reaction (OCR) strategy is a oligomerization is mediated by the CC domain of recently described technique for inactivation of target PML (Lin and Evans, 2000; Minucci et al., 2000, proteins that function as homoassociate complexes. This 2001; Salomoni and Pandolfi, 2000). Since the CC novel strategy is based on the fusion of self-associating domain is sufficient to induce oligomerization of the coiled-coil (CC) domain of the nuclear factor promyelo- RAR protein, it has been suggested that the presence cytic leukemia (PML) to target proteins. Here, we present of the CC domain of PML might be used to modify the the successful application of the OCR strategy for function of a target protein (Lin and Evans, 2000; inactivation of the heterodimeric Cdk9/cyclin T1 com- Minucci et al., 2000; Contegno et al., 2002). Indeed, plex. Cyclin T1/Cdk9 (P-TEFb) complex is a positive fusion of CC to p53 protein leads to formation of high- regulator of gene transcription, whose function is under- molecular complexes with a consequent inhibition of the lined by the ability to phosphorylate the carboxyl-terminal p53 function (Contegno et al., 2002). OCR strategy domain (CTD) of the RNA polymerase II conferring would, in principle, inactivate target proteins that are productive transcript elongation. Fusion of the CC domain able to self-associate. However, hetero-oligomeric to Cdk9 leads to the formation of high molecular factors, such as Cdk–cyclin complexes, may also be complexes to which the endogenous cyclin T1 is recruited. considered as a valid target for OCR. As a test case, we The CC-Cdk9 chimera effectively inhibits HIV-1 Tat investigated the possibility that the CC of PML might be activation, whose transcription activity is exquisitely used to modify the function of Cdk9/cyclinT (P-TEFb) dependent upon cyclin T1/Cdk9 function. Furthermore, complex. expression of CC-Cdk9 protein inhibits cell proliferation, Cdk9 in association with cyclin T (T1 or T2) as shown by colony-formation assay. Collectively, our constitutes the P-TEFb complex that stimulates findings add further support to the OCR strategy for transcription elongation of RNAPII transcripts functional inactivation of hetero-associated factors such by phosphorylating the carboxyl-terminal domain as the Cdk9/cyclin T1 complex, and highlight a putative (CTD)of RNAPII (Price, 2000 and references function of Cdk9 in cell growth control. therein). Hypophosphorylated RNAPII (IIa) is Oncogene (2003) 22, 4882–4888. doi:10.1038/sj.onc.1206785 recruited to promoters (Conaway et al., 2000 and references therein), shortly after the CTD becomes Keywords: Cdk9; OCR; P-TEFb; coiled-coil domain; extensively phosphorylated (IIo), primarily at Ser2 PML and Ser5 of the heptapeptide repeat, to overcome proximal promoter pausing and confer productive transcript elongation (Wada et al., 1998; Yamaguchi et al., 1999). P-TEFb appears to be required for transcription of most class II genes (Majello et al., Introduction 1999; Lis et al., 2000; Price, 2000; Shim et al., 2002; Taube et al., 2002)and the human Cdk9/cyclin T1 The oligomerization protein inactivation (OCR)is a complex is a direct cellular target of the HIV-1 activator, recently devised strategy to achieve functional ablation Tat (Mancebo et al., 1997; Zhu et al., 1997; Bieniasz of a protein in vivo (Contegno et al. , 2002). This et al., 1998; Garber et al., 1998; Wei et al., 1998; Zhou strategy relies on the ability of the coiled-coil (CC) et al., 1998). region of the nuclear factor promyelocytic leukemia Here we present evidences for effective inactivation (PML)to form self-associating oligomeric complexes. of Cdk9/cyclin T1 activity in cell culture through In acute promyelocytic leukemia (APL), the PML is OCR. We demonstrate that fusion of CC to Cdk9 fused to the retinoic acid receptor (RAR). The triggers the formation of large molecular weight PML–RAR forms self-associated complexes and heterocomplexes, leading to a functional inactivation of the Cdk9/cyclin T1 transcription regulatory properties. Based on these findings, we propose that *Correspondence: L Lania, Department of Genetics, Molecular and OCR strategy may be of general application for General Biology, University of Naples ‘Federico II’, Via Mezzocan- none 8, 80134 Naples, Italy; E-mail: [email protected] functional inactivation of heterocomplexes such as the Received 18 February 2003; revised 9 May 2003; accepted 12 May 2003 Cdk/cyclin complexes. OCR-mediated inactivation of Cdk9 G Napolitano et al 4883 Materials and methods volumes from nuclear and cytoplasmic fractions were analysed by Western blotting using anti-cyclin T1 (sc 10750, Santa Constructs Cruz). Binding was visualized by enhanced chemiluminescence (ECL-plus Kit, Amersham). The Flag-Cdk9 was constructed by insertion of the full-length Cdk9 cDNA (Majello et al., 1999)into the EcoRV site of p3xFlag-CMV10 vector (Clontech). To generate the Flag-CC- Colony-formation assay Cdk9, the PML CC cDNA region (encoding amino acids from aa200 to aa339 of the PML protein)was isolated by PCR using HeLa and H1299 cells were transfected by calcium phosphate the PML cDNA as template, and then inserted between the with the indicated expression vectors. At 48 h post-transfec- EcoRI–BgIII sites of the Flag-Cdk9. The EcoRI–BamHI tion, cells were counted and seeded at 20 000 cells per 10 cm fragment of this plasmid (encoding the CC-Cdk9 fusion)was culture dish, grown in medium containing G418 (500 mg/ml) then inserted into the pEGFP-C1 (Clontech)vector to generate for 2 weeks and then stained with Crystal Violet dye. Colonies GFP-CC-Cdk9 plasmid. CC-VDR, GAL4-CycT1, G5-83HIV- were counted from duplicate plates and values are reported for Luc and GFP-Cdk9 vectors have been previously described three independent experiments. (Majello et al., 1999; Napolitano et al., 2000, 2002; Contegno et al., 2002). All vectors were analysed by DNA sequencing. Results Cell culture and transfections HeLa, H1299 and CHO cells were grown in Dulbecco’s Oligomerization chain reaction modified Eagle medium supplemented with 10% FBS. For luciferase assays and GFP localization, cells were transfected The OCR is a novel strategy based on the capacity of the in 35 mm multiwells by the liposome method using LipofectA- CC domain of the nuclear factor PML, to induce MINE reagent (Invitrogen, Life Technologies), as previously oligomerization. It has been recently shown that the described (Napolitano et al., 2000, 2002). For luciferase assays, fusion of p53 with the CC domain of PML inhibits p53 100 ng of reporter DNA and different amounts as indicated in activity, thus indicating that the OCR strategy could the figure legends of each effector were transfected along with represent a valid tool for the functional inactivation of 20 ng of pRL (Renilla Luciferase)for internal transfection proteins that oligomerizes naturally in vivo (Contegno control (Dual Reporter Luciferase Assay, Promega). GFP et al., 2002). It has been suggested that the presence of analysis was carried out as recently described (Napolitano an oligomerization induced by the CC domain of PML et al., 2002). Briefly, after transfection cells were washed in 1 Â cold PBS, fixed in PFA 4% in PBS and after extensive washes would lead to the formation of aberrant, nonfunctional (1 Â PBS), nuclei were stained with Hoechst 33258 (SIGMA). high molecular mass complexes (Contegno et al., 2002). Slides were analysed by fluorescence microscopy and images Since the OCR strategy is effective on target proteins were digitally processed using Adobe Photoshop Software. that oligomerizes naturally, we reasoned that possible targets for OCR could also be hetero-oligomers such as Glycerol gradient and coimmunoprecipitation Cyc/Cdk complexes. In principle, the oligomerization (CC)domain of PML fused to Cdk should impose an Whole-cell extracts were prepared from HeLa cells transfected altered oligomerization state not only of the CC-fused with Flag-Cdk9 or Flag-CC-Cdk9 as indicated. Extracts were obtained using buffer A (Nguyen et al., 2001). As indicated, chimera, but also of its natural regulatory cyclin RNase A (1 mg/ml)was added and extracts were incubated at subunit, leading to functional inhibition of the complex 371C for 10 min. Protein extracts were then ultracentrifuged at through the formation of inactive OCR aggregates 40 000 r.p.m. for 16 h at 41C in SW40.1 rotor on a preformed (Figure 1). As a test case, we sought to inhibit the 5–45% discontinuous glycerol gradient containing buffer A. activity of the cyclinT/Cdk9 (P-TEFb)complex. P- Samples were collected in 10 fractions (1 ml each starting from TEFb is a multiprotein complex involved in transcrip- the top), and the residual pellets were resuspended in buffer A. tion regulation. Cdk9 in association with cyclin T (T1 or In all, 20 ml of each fraction were separated by SDS–PAGE T2), constitutes the P-TEFb complex that stimulates and analysed by Western blotting using appropriate anti- transcription elongation of RNAPII transcripts by bodies. The following antibodies were used: anti-Flag M2 phosphorylating the CTD of RNAPII (Price, 2000 and monoclonal antibody peroxidase conjugate (Sigma), anti- cyclin T1 (sc 10750, Santa Cruz)and anti-Cdk9 (sc 8338, references therein). Santa Cruz). Coimmunoprecipitation analyses were performed A strict requirement for the chimeric Cdk9 protein using whole-cell extracts incubated with the anti-Flag-M2 would be the capacity to associate with the regulatory monoclonal antibody attached to agarose beads (Sigma). After cyclin T partner. We fused the CC region of PML at the extensive washes, the recovered beads were resuspended in amino-terminal of the full-length Cdk9 to generate the Laemmli buffer resolved on SDS–PAGE and analysed by Flag-CC-Cdk9, and then compared its functional Western blotting with anti-CycT1 antibody. For coimmuno- activity to the isogenic Flag-Cdk9, vector. HeLa cells precipitation of 7SK snRNA, total RNA was isolated from were transfected with the Flag-Cdk9 and the Flag-CC- Flag-immuno-complexes and subjected to DNase I treatment, Cdk9 vectors, respectively, and interactions with the followed by reverse transcription using antisense oligonucleo- endogenous regulative partner cyclin T1 were monitored tides for 7SK snRNA. The PCR primers were: sense GGATGTGAGGC- by coimmunoprecipitation. As shown in the upper panel GATCTGGCTG; antisense, TAAAGAAAGGCAGACTGC- of Figure 2a, we found that CC-Cdk9 interacts as CAC. PCR products were analysed on agarose gels. Cell efficiently as Cdk9 with the endogenous cyclin T1. fractionation into cytoplasmic and nuclear fractions was It has been recently shown that almost half of the P- performed as described (Napolitano et al., 2002). Equal cell TEFb in vivo is associated with the small nuclear 7SK

Oncogene OCR-mediated inactivation of Cdk9 G Napolitano et al 4884

Figure 1 A schematic representation of OCR targeting on Cdk/cyclin complexes. Fusion of the CC extraoligomerization domain to the kinase leads to the formation of aberrant, large molecular weight complexes

RNA (Nguyen et al., 2001; Yang et al., 2001). More- In the presence of overexpressed Flag-CC-Cdk9, over, association of the 7SK snRNA has been shown to the endogenous cyclin T1 displayed an aberrant inhibit the activity of P-TEFb (Nguyen et al., 2001; sedimentation profile. Cyclin T1 was found to Yang et al., 2001). To monitor the physical association partly cofractionate with the chimeric CC-Cdk9 protein. between the endogenous 7SK snRNA with the exogen- Given the capacity of CC-Cdk9 to associate with ously expressed CC-Cdk9, the presence of 7SK snRNA endogenous cyclin T1, we interpreted the widespread from immunoprecipitated materials was analysed by distribution of the two proteins as the result of RT–PCR. We found that the CC-Cdk9 chimera, like the populations of differently sized hetero-oligomeric parental Cdk9 protein, interacts with 7SK snRNA complexes. (Figure 2a, lower panel). These results demonstrated that the CC-Cdk9 chi- Transcription inactivation of P-TEFb by OCR targeting mera retains the ability to associate with cyclin T1 and 7SK snRNA, despite the presence of the PML-CC P-TEFb represents the cellular target of the viral extra-oligomerization domain. transcription transactivator Tat (Mancebo et al., 1997; To test if the fusion of Cdk9 to the CC domain of Zhu et al., 1997; Bieniasz et al., 1998; Garber et al., PML might induce the formation of high molecular 1998; Wei et al., 1998; Zhou et al., 1998). Indeed, Tat mass complexes, we analysed the sedimentation proper- strongly activates transcription of the HIV-1 LTR ties of Cdk9/cyclin T1 using glycerol gradient fractiona- promoter through the recruitment of P-TEFb on the tion. Cellular lysates from transiently transfected HeLa TAR RNA structure present at the 50 of all the nascent cells were ultracentrifugated on a glycerol gradient and viral transcripts. Since Tat-mediated activation of HIV- the recovered fractions were analysed by Western 1 LTR promoter is strictly dependent upon cyclin T1/ blotting. Accordingly with recent findings (Nguyen Cdk9 activity, we sought to determine the functional et al., 2001), the exogenous Flag-Cdk9 and the consequences of the overexpression of CC-Cdk9 endogenous cyclin T1 cosedimented in two sets of protein on HIV-1 LTR-driven gene transcription. fractions (Figure 2b). Flag-Cdk9 and cyclin T1 in As presented in Figure 3a, we found that Tat-mediated fractions 3 and 4 correspond to the active core P-TEFb transactivation of the HIV-1 LTR was effectively and fractions 6 and 7 to the inactive P-TEFb/7SK inhibited by the overexpression of Flag-CC-Cdk9 snRNA complex (Nguyen et al., 2001). Consistent in a dose-dependent manner. In contrast, overexpression with the presence of the PML CC oligomerization of Cdk9 protein did not significantly alter Tat domain, the Flag-CC-Cdk9 fusion was found in activity. As a control of the specificity of the CC-Cdk9 fractions (8 to the pellet)corresponding to an on Tat function, the chimera CC-VDR (Contegno et al., apparent molecular weight ranging from 500 kDa to 2002)encoding for an unrelated transcription 41 MDa (Figure 2c), while the endogenous Cdk9 factor (vitamin d receptor), fused to the PLM CC was mainly found in fractions (4 and 5)corresponding domain, had no effect on Tat activity (Figure 3a). To to the low molecular weight complexes (Figure 2c). further substantiate the inhibitory function of CC-Cdk9,

Oncogene OCR-mediated inactivation of Cdk9 G Napolitano et al 4885 A large number of evidences indicate that Tat transactivation is mainly due to the recruitment of the cellular complex P-TEFb to the LTR. Accordingly, it has been previously shown that direct recruitment of cyclin T1 to a promoter template by fusion to the GAL4 DNA-binding domain, activates transcription from an HIV-1 LTR reporter bearing GAL4 sites in the absence of Tat (Majello et al., 1999, Lis et al., 2000: Taube et al., 2002). We sought to determine the consequences of ectopically expressed CC-Cdk9 on P-TEFb-induced transcription in the absence of Tat. HeLa cells were transfected with the G5-83HIV-Luc reporter along with GAL4-cyclin T1 vector, in the presence of increasing amounts of Cdk9 and CC-Cdk9, respectively. As shown in Figure 3 panel c, we found that GAL4-CycT1 effectively activates transcription from the HIV-1 LTR reporter, and coexpression of either Cdk9 did not signiﬁcantly affect GAL4-CycT1- mediated activation. In contrast, coexpression of CC- Cdk9 strongly inhibited the GAL4-cyclin T1 activation of the HIV-1 promoter.

Subcellular localization of CC-Cdk9 To further analyse the dominant-negative effect exerted by the PML CC oligomerization domain on Cdk9, we checked for proper subcellular localization of the CC- Cdk9. HeLa cells were transiently transfected with expression vectors for GFP-Cdk9 and GFP-CC-Cdk9 Figure 2 (a)Upper panel: whole-cell extracts (I)and Flag- and analysed by fluorescence imaging, to check if the immunoprecipitated materials (IP)from transiently transfected fusion of Cdk9 with the CC domain could alter the HeLa cells with Flag-Cdk9 (lanes 1, 2), Flag-CC-Cdk9 (lanes 3, 4) normal Cdk9 intracellular distribution. Accordingly and mock transfected (lanes 5, 6)were resolved on SDS–PAGE and analysed by Western blotting using anti-cyclin T1 antibody. Lower with previous studies (Herrmann and Mancini, 2001; panel, total RNA was isolated from the whole-cell extracts (I)and Marcello et al., 2001; Napolitano et al., 2002), we found from Flag-immunoprecipitated (IP)complexes and was subjected that GFP-Cdk9 displayed a substantial nuclear localiza- to RT–PCR using primers specific for 7SK snRNA. PCR products tion (Figure 4a, b). In sharp contrast, the GFP-CC- were analysed on 1% agarose gel. Panel b: HeLa cells were transfected with Flag-Cdk9 vector DNA and whole-cell extracts Cdk9 was almost entirely localized in the cytoplasm were fractionated by ultracentrifugation on a glycerol gradient. The (Figure 4c, d). Since GFP-CC-Cdk9 behaves identically distribution of Flag-Cdk9 and cyclin T1 in gradient fractions (1– to CC-Cdk9 in all transcriptional assays tested (data not 10)and pellet (P)was monitored by Western blotting using the shown), it is likely that the dominant-negative effect indicated antibodies. The fractions containing the small (SC)and of CC-Cdk9 is at least in part, achieved through large (LC)Cdk9/cyclin T1 complexes are indicated. Panel c: whole- cell extracts from Flag-CC-Cdk9 transiently transfected cells were the formation of subcellular delocalized oligomeric fractionated on a glycerol gradient. Fractions (1–10)and pellet (P) complexes. To address this point, the subcellular were analysed by Western blotting using specific antibodies as localization of the endogenous cyclin T1 was assessed indicated by Western blotting of cellular fractionated extracts derived from cells transfected with either GFP-Cdk9 or GFP-CC-Cdk9. Western blotting with anti-cycT1 in- we extended our analysis using the rodent CHO cells. dicated that endogenous cyclinT1 was, at least in part, Tat protein is a potent activator of HIV-1 LTR delocalized in the cytoplasm of cells transfected with the transcription in primate cells, but only poorly functional CC-Cdk9. In contrast, an exclusive nuclear localization in rodent cells (Alonso et al., 1992). However. was observed in the presence of GFP-Cdk9 (Figure 4 Tat-mediated activation can be rescued by enforced lower panel). expression of human cyclin T1 (Bieniasz et al., 1998; Finally, as previously discussed (Contegno et al., Garber et al., 1998; Zhou et al., 1998). As presented in 2002), in principle, it is possible that the CC-based Figure 3b, we found that Tat enhancement mediated by chimeras may interact with wt PML and interfere with hCycT1 was effectively abrogated by coexpression of its function. However, we found no evidence for CC-Cdk9. These findings demonstrate that the fusion of colocalization of GFP-CC-Cdk9 in the PML nuclear CC with Cdk9 results in a functional inactivation of P- bodies (Figure 4, panel d), and the CC-Cdk9 chimera TEFb function in the context of Tat-dependent tran- was not able to disrupt PML nuclear bodies (data not scription. shown).

Oncogene OCR-mediated inactivation of Cdk9 G Napolitano et al 4886

Figure 3 Effect of CC-Cdk9 on P-TEFb transcriptional activity. HeLa cells were transfected with the G5-83HIV-Luc reporter (100 ng)along with Tat (200 ng, lanes 2–8)and increasing amounts (100–300 ng)of Flag-Cdk9 (lanes 3, 4).Flag-CC-Cdk9 (lanes 5, 6) and CC-VDR (lanes 7, 8), respectively. (b) CHO cells were transfected with G5-83HIV-Luc reporter (100 ng) along with Tat (200 ng, lanes 2–7). CMV-cyclin T1 (200 ng, lanes 3–7) and increasing amounts (200–500 ng) of Flag-Cdk9 (lanes 4, 5) and Flag-CC-Cdk9 (lanes 6, 7)vectors. (c)HeLa cells were transfected with the G5-83HIV-Luc reporter (100 ng)along with GAL4-CycT1 (200 ng, lanes 2–6)and increasing amounts (200–500 ng)of Flag-Cdk9 (lanes 3, 4)and Flag-CC-Cdk9 (lanes 5, 6),respectively. Western blotting analysis of transfected cells showed comparable levels of expression of Flag-CC-Cdk9 and Flag-Cdk9 chimeras (data not shown). Each histogram bar represents the mean of at least three independent transfections made in duplicate

Figure 5 (a)H1299 and HeLa cells were transfected with empty control vector, Flag-Cdk9 and Flag-CC-Cdk9. respectively. At 36 h hours post-transfection, cells were split and placed in medium Figure 4 Representative ﬂuorescent microscopy images from containing G418 to select for transfected cells. At 2 weeks after HeLa cells transfected with GFP-Cdk9 (a, b)and GFP-CC-Cdk9 selection, colonies were counted and mean values from three (c, d)as indicated. Lower panel: at 36 h after transfection, cell independent experiments are reported in graph as percent of empty extracts were fractionated, and an equal cell volume from each control vector. (b)Whole-cell extracts were isolated from HeLa fraction (C, cytoplasmic fraction; N, nuclear fraction)was analysed cells transiently transfected with Flag-CC-Cdk9 vector and from a by Western blotting with anti-cyclin T1 pool of Flag-CC-Cdk9 drug resistant colonies after 2 weeks of selection with G418. The expression of CC-Cdk9 fusion proteins was detected with the anti-Flag antibody. The asterisk indicates the aberrant Flag-Cdk9 protein OCR-mediated inactivation of Cdk9 affects cell proliferation Several studies suggested a putative role of P-TEFb in development of the C.elegans embryo (Shim et al., apoptotic and cellular differentiation programs. An 2002). antiapoptotic function has been suggested upon activa- To evaluate the functional consequences of CC-Cdk9 tion of the NF-kB pathway in TNF-a-responsive cells expression on cell proliferation, we performed a colony- (Barboric et al., 2001)and during activation of formation assay (CFA)using HeLa cells and p53 À/À peripheral blood lymphocytes (Foskett et al., 2001). H1299 human cell lines. After 2 weeks of drug selection, More recently. P-TEFb activity has been involved in it was evident that the number of colonies in CC-Cdk9 differentiation of muscle cells (Simone et al., 2002), in transfections was reduced relative to the number of cardiac hypertrophy (Sano et al., 2002)and during colonies in control empty vector transfections. In

Oncogene OCR-mediated inactivation of Cdk9 G Napolitano et al 4887 contrast, Cdk9 slightly increased the number of colonies Cdk9/cyclin T1 complex. The exogenously expressed (compared to the empty vector). Similar reduction of CC-Cdk9 chimera retains the ability to associate with cell colonies after transfection with CC-Cdk9 was the endogenous regulatory partner cyclin T1. Fusion of observed in both HeLa and H1299 cells (Figure 5a). Cdk9 with the CC domain induces the formation of Furthermore, although a full-length Flag-CC-Cdk9 aberrant large molecular weight complexes, to which the protein was clearly detectable by Western analysis at regulatory partner cyclin T1 is recruited. As a result of 36 h after transfections, an aberrant Flag-CC-Cdk9 titration of the regulatory partner cyclin T1, a significant chimera was detected on the pool of Flag-CC-Cdk9- dominant-negative effect takes place in vivo. We found transfected colonies after drug selection, suggesting that that the CC-Cdk9 effectively inhibits the Tat activity, expression of a full-length CC-Cdk9 fusion protein is which is specifically dependent upon the endogenous incompatible with a normal cell growth (Figure 5b). cyclin T1/Cdk9 activity (Zhu et al., 1997; Mancebo et al., Finally, we found that the cell growth properties of the 1997; Bieniasz et al., 1998; Garber et al., 1998; Wei et al., colonies formed in the CC-Cdk9 transfections were 1998; Zhou et al., 1998). Furthermore, promoter profoundly altered; that is, the cells grew very activation mediated by direct recruitment of cyclin T1 poorly and repeated attempts to isolate clones through GAL4 DNA-binding sites was also strongly stably expressing CC-Cdk9 were not successful, repressed by CC-Cdk9. Thus, the CC-Cdk9 chimera indicating that the dominant-negative effect of CC- functions as a strong and specific inhibitor of P-TEFb Cdk9 on P-TEFb activity inhibits cell proliferation. activity in vivo. Unlike the predominant nuclear Collectively, these findings demonstrated that constitu- localization of Cdk9 (Herrmann and Mancini. 2001; tive expression of CC-Cdk9 caused either growth arrest Marcello et al., 2001; Napolitano et al., 2002), the CC- or cell death. Cdk9 fusion was mainly found in the cytoplasm compartment, indicating that the dominant-negative effect of CC-Cdk9 might be at least in part, achieved through altered subcellular localization. Since the CC- Discussion p53 chimera was also delocalized into the cytoplasm (Contegno et al., 2002), it is conceivable that the high Cyclin-dependent kinases have emerged as key molecular weight complexes formed by the CC-chimeras regulators of cell cycle and they have been intensely might be inherently unable to enter into the nucleus, and studied as potential targets for cancer therapies. How- delocalization might represent a general feature of target ever, identification of a selective inhibitor for a proteins fused to the PML CC extra-oligomerization particular Cdk is a critical issue for the development domain. of a potential biological tool for an innovative The effective dominant-negative function of CC-Cdk9 therapeutic approach (Senderowicz and Sausville. on P-TEFb activity has a significant impact on cell 2000; Sausville, 2002). In our study, we have presented proliferation. Unlike the wt Cdk9, the CC-Cdk9 inhibits the successful application of the OCR strategy for an cell proliferation as tested by CFAs, suggesting that effective inhibition of a specific Cdk/cyclin complex constitutive expression of CC-Cdk9 causes either growth (Cdk9/cyclin T1). arrest or cell death. Clearly, further work will be The OCR is a recently described strategy based on the required to address this point, as well as uncover a fusion of the CC domain of the nuclear factor PML, to a potential direct mechanism of P-TEFb on cell prolifera- target protein capable of self-association. The CC tion. It is possible, if not likely, that the growth domain confers to the target an extra-oligomerization inhibition or cell death may result from a general domain, inducing the formation of aberrant large inhibition of RNAPII transcription mediated by domi- molecular weight complexes. OCR has been shown to nant-negative effect of CC-Cdk9 on P-TEFb activity. be an effective strategy for the in vivo inactivation of p53 Flavopiridol, a potential antiproliferative agent cur- (Contegno et al., 2002). Here we tested the function of rently being tested in clinical trials (Senderowicz and OCR in the contest of hetero-oligomers such as Cdk/ Sausville, 2000; Sausville, 2002), specifically inhibits P- cyclin complexes. The fusion of the CC domain to a Cdk TEFb activity and blocks most RNAPII transcription in (or to the cyclin)subunit should impose an altered vivo (Chao and Price, 2001). It is likely that the potential structure to the complex due to the presence of the CC of P-TEFb to affect cell proliferation is rather an extraoligomerization domain (Figure 1). indirect mechanism correlated to the global requirement As a test case, we carried out the functional for P-TEFb activity in RNAPII gene transcription inactivation of Cdk9/cyclin T1 (P-TEFb)complex. (Shim et al., 2002). Cdk9/cyclin T complex phosphorylates the CTD of While additional studies may help determine whether RNAPII, allowing productive transcription elongation Cdk9 is an effective antiproliferative target, other Cdks (Price, 2000). Moreover, the Cdk9/cyclin T1 is the directly involved in cell-cycle progression, represent cellular cofactor of the HIV-1 transactivator Tat valid antiproliferative targets for OCR-mediated inhibi- (Mancebo et al., 1997; Zhu et al., 1997; Bieniasz et al., tion. Recently, suppression of Cdk4 has been proposed 1998; Garber et al., 1998; Wei et al., 1998; Zhou et al., as a potential strategy to recruit cells to senescence (Zou 1998). Our results show that addition of the PML CC et al., 2002). The functional consequences of OCR- domain to the Cdk9 results in an oligomerization chain mediated inactivation of Cdk4, are currently under reaction that alters the functional properties of the investigation.

Oncogene OCR-mediated inactivation of Cdk9 G Napolitano et al 4888 Acknowledgements grants from the Italian Association for Cancer Research We thank Saverio Minucci and Pier Giuseppe Pelicci for (AIRC), the I.S.S. (Programma Nazionale di Ricerca AIDS, reagents and helpful discussions. This work was supported by Grant 40B.53)to LL.

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Oncogene