B-Myb is a transcription factor belonging to the myb family, whose activity has been associated with augmented DNA synthesis and cell cycle progression. We showed recently that B-Myb autoregulates its own expression through promoter transactivation. We report in this study that CDK9, the cyclin T associated kinase, which phosphorylates and activates RNA-Polymerase II, suppresses B-Myb autoregulation through direct interaction with the carboxyl-terminus of the B-Myb protein. Down-regulation of the transactivating ability of B-Myb is independent of the kinase activity of CDK9, because a kinase deficient mutant (dn-CDK9) also represses B-myb gene autoregulation. Overexpression of CDK9 did not result in suppression of p53-dependent transactivation or inhibition of the basal activity of the promoters tested so far, demonstrating that CDK9 is a B-Myb-specific repressor. Rather, transfection of the dominant negative dn-CDK9 construct inhibited the basal activity of the reporter genes, confirming an essential role for CDK9 in gene transcription. In addition, Cyclin T1 restores B-Myb transactivating activity when co-transfected along with CDK9, suggesting that the down-regulatory effect observed on B-Myb is specifically due to CDK9 alone. Thus, our data suggest that CDK9 is involved in the negative regulation of activated transcription mediated by certain transcription factors, such as B-Myb. This may indicate the existence of a feedback loop, mediated by the different activities of CDK9, which links basal with activated transcription.
B-Myb is a member of the Myb family that includes c-Myb and A-Myb (Nomura et al., 1988). v-myb, the prototype member of the family, is the transforming gene of the E26 and AMV viruses that cause myeloblastosis in birds, and has been described as the truncated derivative of the cellular gene c-myb (Gonda and Bishop, 1983; Klempnauer et al., 1983). Myb proteins are conserved from the fruitfly Drosophila melanogaster to vertebrates and presumably evolved from a primordial myb gene in a process of divergent evolution (Saville and Watson, 1988). Only one myb gene is present in Drosophila melanogaster, which shows structural and functional homology with all mammalian mybs (Katzen and Bishop, 1996).
Within the same species, Myb proteins display a very high degree of homology in the amino-terminal region of the protein, which corresponds to the sequence-specific DNA-binding domain (Klempnauer and Sippel, 1987). This suggests that all Mybs may interact with the regulatory region of an overlapping range of target genes (Lyon et al., 1994). The C-terminal domain of the different Myb proteins shows less structural and functional similarities, except for a short stretch of homology, which is termed CR (Conserved Region). Mybs are molecules negatively regulated by their carboxyl-termini, and protein truncation results in activation of the transcriptional activity of both c-Myb and B-Myb (Kalkbrenner et al., 1990; Ziebold et al., 1997; Lane et al., 1997). Several studies have demonstrated that B-Myb is a substrate for the cdk2/cyclinA kinase and that phosphorylated B-Myb is more active, in terms of its transactivating activity, than the unphosphorylated form (Ziebold et al., 1997; Lane et al., 1997; Sala et al., 1997). c-Myb expression is high in the hematopoietic lineages, especially in immature cell types (Gonda and Metcalf, 1984). Down regulation of c-myb levels correlates with induction of terminal differentiation of hemopoietic cells (Hoffman-Liebermann and Liebermann, 1991). Unlike the other members of the family, B-Myb expression is ubiquitous and strictly correlates with cell proliferation (Sala and Watson, 1999), being highest at the G1/S border of the cell cycle (Nomura et al., 1988; Reiss et al., 1991; Watson et al., 1993). Analysis of B-Myb mRNA levels in non-dividing tissues and in the whole mouse embryo confirms that B-Myb expression is linked to DNA synthesis (Sitsmann et al., 1996; Bouwmeester et al., 1992).
CDK9 (previously named PITALRE) is a cdc2-like kinase (Graña et al., 1994; De Falco and Giordano, 1998). It differs from other cdks because its expression is not cell cycle regulated and does not appear to be required during the cell cycle. In living cells, CDK9 is found associated with cyclins of the T class (T1, T2a and T2b) (Peng et al., 1998; Wei et al., 1998). CDK9 and cyclin T belong to the TAK complex, which is part of the basal transcription machinery (Yang et al., 1997). The TAK complex phosphorylates the Carboxyl Terminal Domain (CTD) of the RNA Polymerase II (RNA Pol II) converting the unphosphorylated, inactive pre-initiation complex into a phosphorylated and active form of the elongating complex (Dahmus, 1996). It was shown recently that CDK9 is actually the kinase of the complex responsible for RNA pol II activation (Wei et al., 1998). In addition, CDK9/Cyclin T binds the Tat proteins of the HIV virus, suggesting a possible role for this kinase in viral replication (Romano et al., 1999). This complex is also involved in the control of transcription elongation (Maiello et al., 1999).
Several studies have reported that B-Myb expression declines during cell differentiation, while ectopic expression of B-Myb results in the suppression of cell differentiation induced by different agents (Saville and Watson, 1998; Bies et al., 1996; Raschella’ et al., 1995). In contrast, CDK9, whose expression is ubiquitous (De Luca et al., 1997), shows an increased kinase activity in terminally differentiated tissues (Bagella et al., 1998). This is similar to the CDK5 kinase, whose expression also appears not to be cell cycle dependent, and is higher in differentiated tissues (Lew et al., 1994; Tsai et al., 1994). Because B-Myb is involved in mutually antagonistic interactions with factors associated with cell differentiation and gene transcription, such as the retinoblastoma family member p107 (Sala et al., 1996b; Raschella’ et al., 1997), we asked whether CDK9 could interfere with B-Myb activity. Here we report that: (i) CDK9 and B-Myb associate with each other in vitro and in vivo; and (ii) protein interaction is responsible for the suppression of B-Myb gene autoregulation.
These results reveal an unsuspected role for CDK9 as a suppressor of activated transcription and encourage further studies aimed at defining the role of CDK9 in mammalian cell differentiation.
CDK9 down regulates the transactivating activity of B-Myb
B-Myb has been reported to upregulate its own promoter (Sala et al., 1999). To evaluate the effects of CDK9 on the B-Myb promoter autoregulation, the luciferase reporter gene linked to a segment of the B-Myb promoter pGL2BMybPRO(wt), the pcDNA3-Bmyb and either pcDNA3-CDK9-HA or pcDNA3-dn-CDK9-HA were cotransfected in 293 cells.
Luciferase assays were performed and the results were evaluated to compare the effects of B-Myb on its own promoter with those resulting from the cotransfection of either wild-type CDK9 or dn-CDK9. Expression of wild-type CDK9 markedly decreased the capacity of B-Myb to transactivate its own promoter (Figure 1a). We next tested the capacity of CDK9 to repress B-Myb transactivation of a promoter containing Myb-binding sites cloned upstream the SV40 minimal promoter of the pGL2 vector (Sala et al., 1999). CDK9 was able to repress B-Myb-induced transactivation of the pGL-MIM-1 plasmid (Figure 1b). Notably, basal activity of the PGL-MIM-1 plasmid decreased in the presence of CDK9, suggesting that CDK9 might sequester the endogenous B-Myb to decrease the basal activation of the promoter.
CDK9 is essential for transcription
To demonstrate that the suppression of B-Myb gene autoregulation observed after cotransfection of wild-type CDK9 was directed on the B-Myb protein, and was not directed on the B-myb promoter, we cotransfected CDK9 with the B-Myb promoter alone as a negative control. In addition, a dominant negative form of CDK9 (dn-CDK9) was also used in these experiments. The dominant negative form of CDK9 consists of a single amino acid substitution in the catalytic domain, and lacks the kinase activity (De Falco and Giordano, 1998). The effects of both proteins (CDK9 and its dominant negative form) were also assessed.
The results show that there was no variation in the activity of the B-Myb promoter after co-transfection with CDK9, but a decrease in its activity was observed after transfection of the dominant negative form (Figure 1). After cotransfection of both CDK9 wild-type and its dominant negative form, promoter activity was intermediate (Figure 1a).
This suggests that kinase activity of CDK9 is required for promoter-regulated transcription activity. This hypothesis also was supported by the results of other experiments, using a different set of promoter/proteins, such as p53. p53-dependent transactivation was not suppressed by CDK9, but a decrease of the activity of the p53-responsive promoter was observed after transfection with dn-CDK9 (Figure 2).
These results reveal two different activities of CDK9. First, there is a general requirement for CDK9 in promoter-regulated transcription, and second the down regulation of B-Myb transactivating ability is likely consequent to specific effect on the B-Myb protein and not to the interaction with B-Myb promoter elements.
Dominant negative CDK9 strongly down-regulates the B-Myb transactivating activity
We found that the B-Myb transactivating activity was down-regulated strongly by CDK9-dominant negative, and even more by the addition of both wild-type CDK9 and dn-CDK9. The strong down-regulation resulting from the combination of the two CDK9 constructs may depend in part on the direct action of CDK9 on B-Myb, and in part on the inhibitory effect on promoter transcription by the dominant negative form of CDK9 (Figure 1a).
Cyclin T1 overrides the ability of CDK9 to down-regulate B-Myb transactivating activity
To test whether CDK9 alone or the complex CDK9/Cyclin T1 could be recruited to the B-Myb promoter by B-Myb, Cyclin T1 was co-transfected with the B-Myb promoter alone, or in combination with B-Myb or both B-Myb and CDK9. Cyclin T1 did not significantly affect either the basal promoter activity, or the B-Myb transactivating activity. Interestingly, when co-transfected with CDK9, Cyclin T1 restored the transactivating activity of B-Myb, suggesting that the down-regulation of B-Myb activity is specifically due to CDK9 alone (Figure 1a). In fact when Cyclin T1 was co-transfected along with CDK9 and B-Myb, it restored the B-Myb transactivating capacity, leading to the conclusion that the effect observed on the B-Myb activity was due to the squelching of CDK9 from the CDK9/Cyclin T1 complex.
The overexpression of CDK9 decreases the level of mRNA B-Myb
The levels of the endogenous B-Myb mRNA were also assessed in a human T-lymphocyte cell line stable overexpressing CDK9 and compared to those in wild-type, non transfected cells. By Northern blot analysis, a decrease in the levels of B-Myb transcripts was noted in cells overexpressing CDK9 (Figure 3), consistent with the ability of CDK9 to suppress B-Myb promoter activity (see Figure 1a). Similar results were obtained in Jurkat cells (data not shown).
CDK9 interacts in vitro with the C-terminal domain of B-Myb
To test whether down-regulation of B-Myb transactivation by CDK9 reflected a physical interaction between the two proteins, we assessed whether CDK9 and B-Myb interact directly in vitro. The B-Myb protein and the two deletion mutants, one lacking the C-terminus (ΔC) and the other lacking the N-terminus (ΔN) and both fused to the HA-Tag, were translated in vitro. GST-CDK9 was expressed bacterially and GST alone was used as a negative control. Full-length B-Myb protein interacted with GST-CDK9, but not with GST alone (Figure 4a). The binding also occurred with the B-Myb mutant lacking the N-terminus (ΔN), whereas the interaction was undetectable with B-Myb truncated at the C-terminus (ΔC). This experiment demonstrates that there is a direct interaction between B-Myb and CDK9, and that the binding occurs through the carboxyl-terminus of B-Myb. A schematic representation of the constructs used in the pull-down assay is shown in Figure 4b.
B-Myb and CDK9 are found associated in tumor cell lines
To test whether B-Myb could interact with CDK9 in vivo, we immunoprecipitated endogenous B-Myb protein and subjected the immunoprecipitated material to Western blot analysis using an anti-CDK9 antibody. We used Jurkat cells for this assay because this cell line contains high levels of endogenous B-Myb. The extracts were precleared with a normal rabbit serum and, as a negative control, the normal rabbit serum was used again in IP. Consistent with the results obtained in vitro, a substantial amount of endogenous CDK9 protein was found associated with endogenous B-Myb in Jurkat cells, supporting the hypothesis that the two proteins form a heterodimer in the native condition (Figure 5).
The N-terminal region of B-Myb does not interact with CDK9 in vivo
In order to map the in vivo binding between CDK9 and B-Myb we cotransfected CDK9 and either B-Myb or its deletion mutant ΔC in 293 cells. The cells were collected 36 h after transfection, lysed and subjected to immunoprecipitation with the anti-B-Myb antibody or the anti-CDK9, followed by Western blot with anti CDK9 or the N-B-Myb antibody. The results of these experiments indicate that the interaction occurred between CDK9 and full length B-Myb (Figure 6a), while no interaction was detected with the N-terminal region of B-Myb (Figure 6b), consistent with the data in vitro (see Figure 4a).
The in vivo interaction is not dependent on the enzymatic activity of CDK9
To test whether the binding occurring between CDK9 and B-Myb was dependent on the kinase activity of CDK9, we performed a series of kinase assays to test whether B-Myb could be a substrate of CDK9.
Jurkat cell extracts, after pre-clearing with normal rabbit serum, were subjected to immunoprecipitation with anti-B-Myb and then were subjected to the kinase assay. Normal rabbit serum also was used in IP as a negative control. No phosphorylation of B-Myb was visible, while it was possible to see the autophosphorylation of CDK9 itself. These assays showed that B-Myb is not a substrate of CDK9 but, rather, CDK9 is able to phosphorylate itself (Figure 7).
In this study, we demonstrate the occurrence of a direct binding between CDK9 and B-Myb, through the C-terminal region of B-Myb.
B-Myb belongs to the Mybs family of proteins and generally is involved in G1-S transition (Sala et al., 1996a). CDK9 is the kinase of the TAK complex, responsible for the activation of the RNA Polymerase II (Yang et al., 1997; Romano et al., 1999). Several new pieces of information result from this study. First, by means of the kinase-deficient mutant of CDK9 (dn-CDK9), we show that endogenous CDK9 activity is required for promoter-regulated transcription. This is consistent with the postulated role for CDK9 in phosphorylating and in activating RNA Polymerase II in concert with cyclin T. From our experiments, it also is clear that CDK9 is not present in limiting amount in the cell, because ectopic expression of CDK9 does not increase the rate of transiently transfected promoters linked to the luciferase reporter. Secondly, we identify a novel role for CDK9, that is, to antagonize B-Myb activated transcription. Although at first glance this appears to contradict the CDK9 role in gene transcription, we hypothesize that this effect may be part of a distinct role for CDK9 in basal and activated transcription. In other words, we postulate that CDK9 might promote basal levels of gene expression, regardless of the type of promoter, but also antagonize activated transcription of specific transcription factors. The down-regulatory effect observed on B-Myb transactivating activity seems to be due to CDK9 alone, not to the complex CDK9/Cyclin T1. In fact, Cyclin T1 is able to restore the B-Myb transactivating activity when co-transfected along with CDK9. Generally, a native complex containing a cyclin should have a positive effect on transcription. Our results of CDK9-mediated down-regulation of B-Myb transactivation activity and the binding occurring in vivo between the two proteins suggest that B-Myb is sequestering CDK9 from the complex with Cyclin T1 and the negative effect that we observed is due to the squelching of the native complex, due to a competition with CDK9/Cyclin T1. This may be important during cell differentiation, when increased amounts of CDK9 may reflect the need to modulate the transcription of cell cycle-regulated and differentiation-associated genes. Physical association with B-Myb might be required to repress the activated transcription of genes that interfere with the differentiation program. Consistent with this hypothesis, CDK9 seems to be involved in differentiation, as shown by an increase of its kinase activity in differentiating myoblasts (MacLachlan et al., 1998). On the other hand, the B-Myb role in contrasting cell differentiation is well established (Bies et al., 1996; Raschella’ et al., 1995). In summary, we show that CDK9 and B-Myb bind to each other in vitro and in vivo, resulting in suppression of B-Myb gene autoregulation. Whether or not this association has a physiological meaning, especially with respect to cell differentiation, will be addressed by future experiments.
Materials and methods
Two hundred and ninety-three cells were grown in DMEM supplemented with 10% FBS, L-glutamine and antibiotics; Jurkat cells and SUPT1 cells were grown in RPMI supplemented with 10% FBS, L-glutamine and antibiotics. All cell lines were obtained from ATCC.
Stable SUPT1 cells (a human T-lymphocytes cell line), overexpressing both CDK9 and its dominant negative, were obtained by electroporating the cells at 200 V and 960 μFa. The stable transfectants were selected with G-418 (Sigma, MO, USA) at the concentration of 0.5 mg/ml.
The construct consisting of the B-Myb promoter linked to a luciferase reporter [pGL2BMybPRO(wt)] was obtained by subcloning the −277/−304 B-myb promoter segment in the SacI site of the pGL2-promoter vector (Sala et al., 1999b). Full-length wild-type CDK9 and its kinase deficient counterpart (dn-CDK9 containing a point mutation at nucleotide 563 which converts Asp into Asn) were amplified by PCR from the clone PK14 (Graña et al., 1994) using the oligonucleotide sequences: 5’-TAggatccATGGCAAAGCAGTAC-3’ and 5’-ATatcgatGAAGACGCGCTCAAACTC-3’ (lowercase letters indicate the BamHI and ClaI sites, respectively). The point mutation was PCR generated using oligonucleotide sequences: 5’-GGTCCTGAAGCTGGCAAAC-3’ and 5’-CGGGCCSGCCCAAAGTTTG (bold letters indicate the point mutation) (Ho et al., 1989). Amplified products (1.1 kb) were restricted with BamHI and ClaI and were subcloned into a pcDNA3 HATag (BamHI-ClaI). The sequences of the resulting plasmid (pcDNA3-CDK9wt-HATag and pcDNA3-dnD167NCDK9-HATag) were confirmed by dideoxynucleotide DNA sequencing using the Applied Biosystems model 373A DNA Sequencer (Sanger et al., 1977). The plasmids pcDNA3-BMyb-HATag, pcDNA3-ΔC-BMyb-HATag and pcDNA3-ΔN-BMyb-HATag were constructed by PCR. The plasmid pcDNA3-BMyb-HATag was constructed using the primer 5HA-Myb 5’-cgc-aag-ctt-atg-tct-cgg-cgg-acg-cgc-3’ and the primer 3HA-Myb 5’-cgc-gat-atc-cta-agc-ata-atc-agg-aac-atc-ata-agg-ata-gga-caa-gat-gag-ggt-ccg-aga-tgt-3’. The HA tag epitope is identified in bold letters. The plasmid pcDNA3-ΔC-BMyb-HATag (nt 128–709) was made by PCR using the primer NM5 5’-tat-cct-tat-gat-gtt-cct-gat-tat-gct-tag-gat-atc-3’ and the primer NM3 5’-ttt-gga-ctc-gct-caa-gaa-gcc-tcc-tgt-3’. The plasmid pcDNA3-ΔN-BMyb-HATag (nt 710–2230) was prepared by PCR using the primer CM5 5’-atg-gac-tgc-aag-ccc-cca-gtg-tac-ttg-3’ and the primer CM3 5’-aag-ctt-ggg-tct-ccc-tat-agt-gag-tcg-3’. All BMyb-HAtag constructs were confirmed by direct automated DNA sequencing.
The promoter containing Myb-binding sites cloned upstream the SV40 minimal promoter of the pGL2 vector has been described (Sala et al., 1996b).
The construct pCMV/p53 and the promoter of WAF-1 linked to a CAT reporter were previously described (Sala et al., 1996a).
The construct expressing Cyclin T1 has been previously described (Napolitano et al., 1999).
Transient transfections and luciferase assay
Transient transfections and luciferase assays were performed using kits according to manufacturer’s instructions (Invitrogen, CA, USA and Promega, WI, USA respectively). Transfections were normalized by cotransfecting the pSVβGAL plasmid (Promega, WI, USA) and by performing a standard β-galactosidase assay (Lam and Watson, 1993). Two μg of each plasmid were transfected, triplicate of transfection-luciferase assays were performed five times. The total amount of CMV promoter was maintained constant in each transfection. There was no significant difference in the transfection efficiencies among the various constructs as determined by the β-gal activity. No evidence of CDK9 altering the β-gal activity was observed.
Western blot and immunoprecipitation (IP)
Two hundred and ninety-three extracts were used for Western blot and IPs. These extracts were obtained by lysing the cells in PLC-LB [50 mM HEPES pH 7.5, 150 mM sodium chloride, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1.5 mM magnesium chloride, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 10 μg/ml aprotinin, leupeptin and phenylmethylsulphonyl fluoride (PMSF)]. The protein concentration was estimated by Bradford assay (Bio-Rad, CA, USA), following the manufacturer’s instructions and by using Bovine Serum Albumin as a standard.
The IPs were performed after preclearing with normal rabbit serum, and after using anti-CDK9 (Bagella et al., 1998) or anti B-Myb (Raschella’ et al., 1995). Antibody incubations were performed overnight, at 4°C, followed by the addition of protein A-sepharose for 60 min, rocking, at 4°C. Beads were washed five times with an excess of PBS, and loading buffer (50 mM Tris/HCl pH 6.8, 2% SDS, 10% glycerol and 5% β-mercaptoethanol) was added to the samples. The samples were resolved by 7 or 10% SDS–PAGE and were transferred to a PVDF (Immobilon, Millipore, MA, USA) membrane, using CAPS 10 mM and methanol 20% v/v, pH 11.0 as a buffer. The transfer was performed at 4°C and at 70 V for 2–3 h. 0.5% Ponceau Red was used to ensure equal transferring. The blots then were blocked with TBST containing 5% non-fat dry milk. Anti-CDK9 (1 : 100) or anti-B-Myb (1 : 1000), were used in TBST containing 5% non-fat dry milk, according to the Western blot conditions suggested by Santa Cruz for polyclonal antibodies (Santa Cruz, CA, USA). Anti-rabbit peroxidase conjugated (1 : 20 000) (Amersham, IL, USA) and the ECL detection system (NEN, Du Pont MA, USA) were used to detect the signal.
In vitro translation
The TNT coupled reticulocyte kit was used for in vitro translation (Promega, WI, USA), according to the manufacturer’s instructions. All the samples were labeled using 35S-Methionine (Amersham, IL, USA).
In vitro binding
The labeled samples were incubated with GST-CDK9, using GST as a negative control, for 2 h at 4°C, rocking, for in vitro binding. The samples then were washed extensively in NENT buffer (20 mM Tris/HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40), containing fresh inhibitors and 1 mM DTT, and were resolved on 10% SDS–PAGE. The gels then were fixed and amplified following the conditions suggested by Amersham. Finally the gels were dried and exposed at −80°C to an intensifier screen, using Kodak film (Kodak, NY, USA).
Cell extracts (200 μg) prepared in EBC buffer (50 mM Tris/HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM PMSF, 10 μg/ml leupeptin, 200 mM DTT, 250 mM MgCl2) were used for immunoprecipitation with a polyclonal anti-B-Myb antibody, after pre-clearing with normal rabbit serum, O/N at 4°C. The immunocomplexes were pulled down with protein A-sepharose and washed with an excess of EBC buffer. The complexes also were washed once with kinase assay buffer (minus ATP) (20 mM HEPES pH 7.4, 10 mM Mg Acetate) and then were used for the assay. The kinase assay was performed in a volume of 20 μl, using 5 μCi/sample of γ-ATP (Amersham, IL, USA). No substrate was added to the samples and the same procedure was done in parallel on an extract pulled down with normal rabbit serum. The samples were incubated for 30 min at 30°C and then were washed with an excess of PBS. The reactions were stopped by adding 100 μl of Stop Solution (10 mM Na Phosphate pH 8.0, 10 mM Na Pyrophosphate, 10 mM EDTA, 1 mg/ml BSA). After the addition of 2× Laemmli buffer the samples were loaded and resolved on a 7% SDS–PAGE. The dried gel was then exposed at −80°C using Kodak film (Kodak, NY, USA).
Total RNA was prepared using the RNEasy kit (Qiagen, CA, USA). The probe was labeled with α32P-CTP (Amersham, IL, USA) using the kit Rediprimer II (Amersham, IL, USA), following the conditions described by the manufacturer.
Nonyl Phenoxy Polyetoxy Ethanol
Phosphate Buffer Saline
Tris Buffer Saline+Triton X-100
Bovine Serum Albumin
Normal Rabbit Serum
Bagella L, MacLachlan TK, Buono RJ, Pisano MM, Giordano A and De Luca A. . 1998 J. Cell. Physiol. 177: 206–213.
Bies J, Hoffman B, Amanullah A, Giese T and Wolff L. . 1996 Oncogene 12: 355–363.
Bouwmeester T, Guehmann S, el-Baradi T, Kalkbrenner F, van Wijk I, Moelling K and Pieler T. . 1992 Mech. Dev. 37: 57–68.
Dahmus ME. . 1996 J. Biol. Chem. 217: 19009–19012.
De Falco G and Giordano A. . 1998 J. Cell. Physiol. 177: 501–506.
De Luca A, Esposito V, Baldi A, Claudio PP, Fu Y, Caputi M, Pisano M, Baldi F and Giordano A. . 1997 J. Cell. Physiol. 172: 265–273.
Gonda TJ and Bishop JM. . 1983 J. Virol. 46: 212–220.
Gonda TJ and Metcalf D. . 1984 Nature 310: 249–251.
Graña X, De Luca A, Sang N, Fu Y, Claudio PP, Rosenblatt J, Morgan DO and Giordano A. . 1994 Proc. Natl. Acad. Sci. USA 91: 3834–3838.
Ho SN, Hunt HD, Orton RM, Pullen JK and Pease LR. . 1989 Gene 77: 51–59.
Hoffman-Liebermann B and Liebermann DA. . 1991 Oncogene 6: 903–909.
Kalkbrenner F, Guehmann S and Moelling K. . 1990 Oncogene 5: 657–661.
Katzen L and Bishop JM. . 1996 Proc. Natl. Acad. Sci. USA 93: 13955–13960.
Klempnauer KH and Sippel AE. . 1987 EMBO J. 6: 2719–2725.
Klempnauer KH, Ramsay G, Bishop JM, Moscovivi MG, McGrath JP and Levinson AD. . 1983 Proc. Natl. Acad. Sci. USA 33: 345–355.
Lam EW and Watson RJ. . 1993 EMBO J. 12: 2705.
Lane S, Farlie P and Watson R. . 1997 Oncogene 14: 2445–2453.
Lew J, Huang QQ, Qi Z, Winfkein RJ, Aebersold R, Hunt T and Wang JH. . 1994 Nature 371: 423–426.
Lyon J, Robinson C and Watson R. . 1994 Crit. Rev. Oncog. 5: 373–378.
MacLachlan TK, Sang N, Puri PL, De Luca A, Levrero M and Giordano A. . 1998 J. Cell. Biochem. 71: 467–478.
Majello B, Napolitano G, Giordano A and Lania L. . 1999 Oncogene 18: 4598–4605.
Napolitano G, Licciardo P, Gallo P, Majello B, Giordano A and Lania L. . 1999 AIDS 13: 1453–1459.
Nomura N, Takahashi M, Matsui M, Ishii S, Date T, Sasamoto S and Ishizaki R. . 1988 Nucleic Acid. Res. 16: 11075–11089.
Peng J, Zhu Y, Milton JT and Price DH. . 1998 Genes Dev. 12: 755–762.
Raschella’ G, Negroni A, Sala A, Pucci S, Romeo A and Calabretta B. . 1995 J. Biol. Chem. 270: 8540–8545.
Raschella’ G, Tanno B, Bonetto F, Amendola R, Battista T, De Luca A, Giordano A and Paggi MG. . 1997 J. Cell. Biochem. 67: 297–303.
Reiss K, Travali S, Calabretta B and Baserga R. . 1991 J. Cell. Physiol. 14: 338–343.
Romano G, Kasten M, De Falco G, Micheli P, Khalili K and Giordano A. . 1999 J. Cell. Biochem. 75: 357–368.
Sala A and Watson R. . 1999 J. Cell. Physiol. 179: 245–250.
Sala A, Saitta P, De Luca A, Casella I, Lewis RE, Watson R and Peschle C. . 1999 Oncogene 18: 1333–1339.
Sala A, Casella I, Grasso L, Bellon T, Reed JC, Miyashita T and Peschle C. . 1996a Cancer Res. 56: 1991–1996.
Sala A, De Luca A, Giordano A and Peschle C. . 1996b J. Biol. Chem. 271: 28738–28740.
Sala A, Kundu M, Casella I, Engelhard A, Calabretta B, Grasso L, Paggi MG, Giordano A, Watson RJ, Khalili K and Peschle C. . 1997 Proc. Natl. Acad. Sci. USA 94: 532–536.
Sanger F, Nicklen S and Coulson AR. . 1977 Proc. Natl. Acad. Sci. USA 74: 5463–5467.
Saville MK and Watson RJ. . 1988 Adv. Cancer Res. 72: 109–140.
Sitzmann J, Noben-Trauth K, Kamano H and Klempnauer KH. . 1996 Oncogene 12: 1889–1894.
Tsai LH, Delalle I, Cavines VJ, Chae T and Harlow E. . 1994 Nature 371: 419–423.
Watson RJ, Robinson C and Lam EW. . 1993 Nucleic. Acid. Res. 21: 267–272.
Wei P, Gerber ME, Fang SM, Fischer WH and Jones K. . 1998 Cell 92: 451–462.
Yang X, Gold MO, Tang DN, Lewis DE, Aguilar-Cordova E, Rice AP and Herrman C. . 1997 Proc. Natl. Acad. Sci. USA 94: 12331–12336.
Ziebold U, Bartsch O, Marais R, Ferrari S and Klempnauer KH. . 1997 Curr. Biol. 7: 253–260.
This work has been supported in part by grants from the National Institute of Health (A Giordano and B Calabretta). G De Falco was partially supported from an AIDS fellowship from the ‘Istituto Superiore di Sanita’. PP Claudio is the recipient of a fellowship from the ‘Associazione Leonardo di Capua’, Napoli, Italy. A De Luca is the recipient of a grant FIRC. L Bagella is supported by a Dottorato di Ricerca in Biologia Diagnostica Quantitiva from the Universita di Siena.
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