S Sarcoma-Associated Herpesvirus Lana2 Protein Interacts with the Pocket Proteins and Inhibits Their Sumoylation

ORIGINAL ARTICLE Kaposi’s sarcoma-associated herpesvirus lana2 protein interacts with the pocket proteins and inhibits their sumoylation

L Marcos-Villar1,5, P Gallego1,5, C Mun˜ oz-Fontela2, CF de la Cruz-Herrera1, M Campagna1, D Gonza´lez1, F Lopitz-Otsoa3, MS Rodrı´guez3,4 and C Rivas1

The pocket proteins retinoblastoma protein (pRb), p107 and p130 are the key targets of oncoproteins expressed by DNA tumor viruses. Some of these viral proteins contain an LXCXE motif that mediates the interaction with the three pocket proteins and the inhibition of the pRb SUMOylation. Kaposi’s sarcoma herpesvirus (KSHV) contains at least two proteins that can regulate pRb function but, so far, a KSHV-encoded protein targeting p107 and p130 has not been identified. Here, we show that the KSHV latent protein LANA2 binds to pRb, p107 and p130. LANA2 contains an LXCXE motif that is required for bypassing pRb-mediated cell-cycle arrest and for inhibiting pRb SUMOylation. Finally, we demonstrate that, in addition to pRb, both p107 and p130 can be SUMOylated, and this modification is also inhibited by LANA2 in an LXCXE-dependent manner. These results demonstrate, for the first time, the SUMOylation of p107 or p130 and, so far, they represent the first example of a KSHV protein able to interact with the three pocket proteins and to inhibit their conjugation to SUMO.

Oncogene (2014) 33, 495–503; doi:10.1038/onc.2012.603; published online 14 January 2013 Keywords: pocket proteins; KSHV; LANA2; pocket proteins; sumoylation; LXCXE domain

INTRODUCTION vIRF3, interacts with pRb, p130 and p107 in vitro and in vivo. The retinoblastoma protein (pRb) family is constituted by pRb, LANA2 contains an LXCXE domain that is required for bypassing p107 and p130.1–4 The three proteins show maximal identity in the cell growth arrest induced by reintroduction of pRb into the the conserved pocket region, through which pRb, p107 and p130 pRb-null SAOS-2 cell line and to inhibit pRb SUMOylation. Finally, interact with viral oncoproteins and cellular factors, such as the we demonstrate, for the first time, that the pocket proteins p107 E2F family of transcription factors.5 Although the three members and p130 can be SUMOylated and that the LXCXE domain in share high homologies and overlapping features and activities, LANA2 is also important to regulate their SUMOylation. each protein has a unique function and has a unique, non- redundant role.6 pRb family members are post-translationally regulated proteins. Phosphorylation of the pocket proteins is regulated in a cell-cycle dependent manner and leads to the RESULTS functional inactivation of pRb, p107 or p130.7–11 pRb can be also LANA2 interacts with pRb, p107 and p130 in vitro and in vivo acetylated and SUMOylated but while the former modification To test whether LANA2 interacted with pRb, p107 and p130 might prevent its inactivation by phosphorylation, the function of in vitro, we carried out a glutathione S-transferase (GST)-pull down pRb SUMOylation is still unknown.12–14 Although acetylation of assay using in vitro translated [35S] methionine-labeled LANA2 p130 has been recently demonstrated,15 whether p107 and/or protein and GST-pRb, GST-p107, GST-p130 or GST beads. LANA2 p130 undergo SUMOylation remains to be elucidated. protein efficiently bound to GST-pRb, GST-p107 or GST-p130 resin Small DNA tumor viruses encode oncoproteins able to regulate but did not adhere to the GST resin (Figure 1a), indicating that the functions of the pocket proteins. A characteristic feature of LANA2 can interact with the three pocket proteins in vitro. In order many of these viral oncoproteins is an LXCXE sequence motif that to determine whether LANA2 could also interact with the pocket is necessary for high affinity binding to the pRb family members proteins in vivo, we carried out co-immunoprecipitation experi- and for inhibition of pRb SUMOylation.14,16,17 Kaposi’s sarcoma ments in KSHV-positive primary effusion lymphoma (PEL) cells herpesvirus (KSHV) contains two genes able to target the pRb: (BCP-1). As shown in Figure 1b, LANA2 was detected in the cell ORF72 that encodes a D-type cyclin that can phosphorylate extracts immunoprecipitated with anti-pRb, anti-p107 or anti-p130 pRb18–21 and ORF73 that encodes LANA1, which is able to bind antibodies. In addition, pRb, p107 and p130 were detected in the pRb and neutralize E2F regulation.22 However, these proteins do cell extracts immunoprecipitated with anti-LANA2 antibody not contain the interaction domain LXCXE characteristic of tumor (Figure 1b). These results demonstrate that LANA2 expressed in viruses and they cannot target p107 or p130. In this paper, we KSHV-infected B cells can interact with all the three endogenous demonstrate that the KSHV latent protein LANA2, also called pocket proteins in vivo.

1Centro Nacional de Biotecnologıá, CSIC, Campus Universidad Autońoma de Madrid, Madrid, Spain; 2Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany; 3Proteomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Buildin Derio, Spain and 4Ubiquitylation and Cancer Molecular Biology laboratory, Inbiomed, San Sebastian-Donostia, Gipuzkoa, Spain. Correspondence: Dr C Rivas, Molecular and Cellular Biology, Centro Nacional de Biotecnologıá, CSIC, Campus Universidad Autońoma de Madrid, Madrid 28049, Spain. E-mail: [email protected] 5These authors contributed equally to this work. Received 19 July 2012; revised 29 October 2012; accepted 7 November 2012; published online 14 January 2013 KSHV LANA2 protein modulates the pocket proteins L Marcos-Villar et al 496

Figure 1. LANA2 associates with pRb, p107 or p130. (a) In vitro-translated [35S]methionine-labeled LANA2 protein was incubated with GST, GST-pRb, GST-p107 or GST-p130 in a pull-down assay. Samples were resolved by SDS-PAGE gel and bands were visualized by autoradiography. (b) Protein extracts from KSHV infected BCP-1 cells were subjected to immunoprecipitation (IP) with anti-LANA2, anti-pRb, anti-p107, anti- p130 antibodies or with immunoglobulin G (IgG)s control, as indicated (IPs). Immunoprecipitated samples were run on a SDS-PAGE gel and western blotted with the indicated antibodies.

Interaction between LANA2 and pRb do not depend exclusively cellular proteins or viral oncoproteins containing the LXCXE on the LXCXE domain domain.14 As LANA2 contains an LXCXE domain, we wanted to In silico analysis of the LANA2 amino-acid sequence revealed that evaluate whether LANA2 would affect SUMOylation of pRb. First, LANA2 contained a putative LXCXE motif between 109–113 amino- we carried out an in vitro SUMOylation assay with in vitro acid residues (LLCAE) characteristic for the interaction with the translated [35S] methionine-labeled pRb as a substrate and in the pocket proteins. Mutation of the cysteine residue has been shown presence of GST or increasing doses of GST-LANA2. As a negative to disrupt LXCXE domain function.23,24 In order to determine control, we performed a parallel in vitro SUMOylation assay using whether the interaction between LANA2 and the pocket proteins in vitro translated [35S] methionine–labeled promyelocytic leuke- requires this motif, we generated a LANA2 construct with a C111A mia (PML) protein, in the presence of GST or increasing doses of mutation in the LXCXE domain. Then, we carried out a GST-pull GST-LANA2. Addition of SUMO1 to the SUMOylation reaction down assay using [35S] methionine-labeled pRb, p107 or p130 induced the appearance of a higher molecular weight band protein and GST or increasing amounts of GST-LANA2 or GST- corresponding with pRb-SUMO1 or PML-SUMO1, as expected LANA2C111A beads. The results showed an increase in the amount (Figure 3a). Incubation of the SUMOylation reactions with LANA2 of pRb, p107 or p130 bound to GST-LANA2 that correlated directly resulted in a decrease of the intensity of the pRb-SUMO1 band with increased levels of GST-LANA2 protein (Figure 2a), indicating concomitant with increased levels of GST-LANA2 but did not that the interaction between LANA2 and the pocket proteins is cause a reduction of the intensity of the PML-SUMO1 bands specific. In addition, we also observed that, although the (Figure 3a), indicating that LANA2 can specifically reduce the LANA2C111A mutant bound pRb, p107 or p130 with slightly SUMOylation of pRb in vitro. To evaluate whether LANA2 could reduced efficiencies, there was also a specific dose–response also inhibit pRb SUMOylation in vivo, HEK-293 cells were binding (Figure 2a). These results indicate that the LXCXE motif in transfected with pcDNA or Ubc9 and His6-SUMO2, in the presence LANA2 is not essential for its interaction with pRb, p107 or p130 or absence of LANA2, and 48 h after transfection, histidine-tagged in vitro. Then, we decided to analyze whether the LANA2 mutant in proteins were purified. SUMOylation of endogenous pRb was then the LXCXE domain still interacted with the pocket proteins in vivo. analyzed by western blot using anti-pRb antibody. As shown in HEK-293 cells were co-transfected with either LANA2 wild type (WT) Figure 3b, expression of SUMO2 led to the formation of slower or the mutant in the LXCXE domain together with pRb, p107 or migrating pRb species with an apparent molecular weight of p130, and 48 h after transfection, the cell extracts were subjected to about 130 kDa that corresponded with pRb-SUMO2 protein, as immunoprecipitation using anti-LANA2, anti-pRb, anti-p107 or anti- previously reported.25 Expression of LANA2 induced a clear p130 antibodies. Analysis of the immunoprecipitated extracts with reduction in the levels of pRb-SUMO2 bands, indicating that either anti-LANA2 or anti-pRb antibodies revealed that both LANA2 LANA2 can inhibit the SUMOylation of pRb in vivo. We next and the LXCXE LANA2 mutant bound to pRb with similar efficiency analyzed whether a mutant of LANA2 in the LXCXE domain could (Figure 2b, upper panel). However, we did not detect interaction also inhibit pRb SUMOylation. HEK-293 cells were transfected with between the LXCXE mutant of LANA2 and p107 (Figure 2b, middle pcDNA or Ubc9 and His6-SUMO2 and in the presence or absence panel), and we observed a clear reduction in the interaction of p130 of LANA2 WT or the LXCXE LANA2 mutant. Forty-eight hours after with the mutant of LANA2 in comparison with the WT protein transfection, histidine-tagged proteins were purified. LANA2 can (Figure 2b, lower panel). These results indicated that the LXCXE conjugate SUMO,26 and western blot analysis of the His-purified motif in LANA2 is not required for its interaction with pRb but may proteins using anti-LANA2 antibody detected bands correspon- be important for the interaction with p107 and p130. ding to LANA2-SUMO2 protein in the lanes corresponding to cells co-transfected with SUMO2 and LANA2 WT or the LXCXE LANA2 mutant. We then analyzed SUMOylation of endogenous pRb by LANA2 inhibits the SUMOylation of pRb in an LXCXE-dependent western blotting using anti-pRb antibody. Transfection of SUMO2 manner induced the appearance of a band corresponding with pRb- pRb protein can be post-translationally modified by SUMO SUMO2 protein that was clearly reduced in the presence of WT conjugation and this modification is negatively regulated by LANA2 (Figure 3c), reinforcing our previous observation. However,

Oncogene (2014) 495 – 503 & 2014 Macmillan Publishers Limited KSHV LANA2 protein modulates the pocket proteins L Marcos-Villar et al 497

Figure 2. In vitro and in vivo LANA2: pRb interaction does not require LXCXE. (a) In vitro-translated [35S]methionine-labeled pRb, p107 or p130 proteins were incubated with GST or increasing doses of WT or mutant C111A GST-LANA2 fusion proteins in a pull-down assay. Samples were resolved by SDS-PAGE gel, and the bands were visualized by autoradiography. The bottom panel shows western blot (WB) analysis of GST and GST-LANA2 in the pull-down assay with anti-GST antibody. (b) Co-immunoprecipitation analysis between LANA2 WT or LANA2C111A and pRb, p107 or p130 in HEK-293 cells co-transfected with pRb, p107 or p130 and LANA2 WT or LANA2C111A. Input represents 10% of lysate used in the immunoprecipitation (IP) reactions.

we did not observe a clear reduction of pRb SUMOylation after LANA2 blocks the cell growth arrest mediated by pRb in an expression of the LANA2 mutant in the LXCXE domain. All LXCXE-dependent manner together these results demonstrated that LANA2 inhibits pRb One of the most relevant abilities of the pocket proteins is to SUMOylation in an LXCXE-dependent manner. control the cell cycle by negative modulation of the transition

& 2014 Macmillan Publishers Limited Oncogene (2014) 495 – 503 KSHV LANA2 protein modulates the pocket proteins L Marcos-Villar et al 498

Figure 3. LANA2 inhibits the SUMOylation of pRb in a LXCXE-dependent manner. (a) In vitro-translated [35S]methionine-labeled pRb protein were subjected to in vitro SUMOylation in the presence of GST or increasing doses of GST-LANA2. The bottom panel shows western blot (WB) analysis of GST and GST-LANA2 in the in vitro SUMOylation assay with anti-GST antibody. (b) HEK-293 cells were transfected with pcDNA or Ubc9 and His6-SUMO2 in the presence or absence of LANA2. Lysates and histidine-SUMO conjugates recovered from Niquel-agarose beads were separated by SDS-PAGE and probed with anti-pRb antibody. (c) HEK-293 cells were transfected with pcDNA or Ubc9 and His6-SUMO2 in the presence or absence of LANA2 WT or the LXCXE mutant. Lysates and histidine-SUMO conjugates recovered from Niquel-agarose beads were separated by SDS-PAGE and probed with anti-pRb antibody. Note that unmodiﬁed pRb protein binds nonspeciﬁcally to the nickel beads.

between the G1 and S phases.27–30 Our results indicated that the SAOS-2 cells resulted in a decrease in the number of colonies LXCXE domain in LANA2 was not required for interacting with pRb compared with pcDNA. Similarly, co-transfection of LANA2 WT but but it was essential to inhibit pRb SUMOylation. We then sought to not the LANA2C111A mutant significantly increased the number determine the consequences of the interaction between LANA2 of antibiotic-resistant colonies (Figure 4b). These results indicate and pRb and the role of the LXCXE motif on the LANA2 effect. pRb that LANA2 neutralize the growth inhibitory effect of pRb in an expression in SAOS-2 cells has been shown to induce cell growth LXCXE-dependent manner. arrest. Thus, SAOS-2 cells were co-transfected with green fluorescent protein (GFP) and pcDNA or pRb in the presence or absence of LANA2 WT or LANA2C111A, and at 48 h after Both p107 and p130 can be SUMOylated, a modification that can transfection, the DNA content of the GFP-positive cells was be inhibited by LANA2 in an LXCXE-dependent manner determined by analytical flow cytometry. As expected, We then decided to evaluate whether the pocket proteins p107 or reintroduction of pRb in SAOS-2 cells induced a G1 cell-cycle p130 could be also modified by SUMO. It is known that most arrest that was reversed after transfection of LANA2 WT but not SUMOylated proteins interact directly with the E2 SUMO the LANA2C111A mutant (Figure 4a). In addition, SAOS-2 cells conjugating enzyme Ubc9.31 Then, we first evaluated the were transfected with pcDNA or co-transfected with pcDNA-pRb putative interaction between p107 or p130 and Ubc9. HEK-293 and pcDNA, pcDNA-LANA2 WT or pcDNA-LANA2C111A, selected cells were co-transfected with a plasmid encoding p107 or p130 with G418 for 2 weeks, and then the number of antibiotic-resistant and Ubc9-SV5, and 48 h after transfection, protein extracts colonies was counted. Introduction of pRb into the pRb-null were incubated with either anti-SV5 or a control antibody.

Oncogene (2014) 495 – 503 & 2014 Macmillan Publishers Limited KSHV LANA2 protein modulates the pocket proteins L Marcos-Villar et al 499 molecular weight bands that corresponded to p107-SUMO2 or p130-SUMO2 conjugates (Figure 5c), indicating both p107 and p130 pocket proteins can be also SUMOylated in vivo. Then, we decided to evaluate whether LANA2 could also repress the SUMOylation of p107 or p130. HEK-293 cells were transfected with p107 or p130 and pcDNA or Ubc9 and His6-SUMO2, in the presence or absence of LANA2. Forty-eight hours after transfection, histidine-tagged puriﬁed proteins were analyzed by western blot using anti-LANA2 and anti-p107 or anti-p130 antibodies. Western blot analysis using anti-LANA2 antibody detected bands corresponding to LANA2-SUMO2 protein in the lanes corresponding to cells co-transfected with SUMO2 and LANA2 WT or the LXCXE LANA2 mutant. Bands corresponding with p107-SUMO2 or p130-SUMO2 proteins were detected in those cells transfected with Ubc9 and SUMO2, as previously observed (Figure 5d). Expression of LANA2 WT resulted in a clear reduction in the intensity of the p107-SUMO2- or p130-SUMO2-modiﬁed bands that was not observed after expression of the mutant in the LXCXE domain, indicating that LANA2 partially inhibits the SUMOylation of p107 and p130 in an LXCXE-dependent manner (Figure 5d).

DISCUSSION The retinoblastoma family of proteins represents key molecules in tumor suppression. Irrespective of their structural and biochemical similarities, each pocket protein has a unique function and has a 27 Figure 4. LANA2 can overcome the cell growth arrest induced by unique, non-redundant role. Many of the sequence similarities pRb expression in SAOS-2 cells in a LXCXE-dependent fashion. (a) reside in a homologous functional domain known as the pocket SAOS-2 cells were co-transfected with GFP and the indicated region that is responsible for the interaction with cellular proteins plasmids, and at 48 h after transfection, cell-cycle distribution of the and oncoproteins from DNA tumor viruses.17,32 A characteristic GFP-positive cells was analyzed by flow cytometry. (b) SAOS-2 cells feature of many viral and cellular proteins that target the pocket were transfected with the indicated plasmids, 48 h after transfection proteins is the presence of a short LXCXE sequence motif, which cells were incubated in the presence of G418 and at 14 days after 16,17 selection the number of colonies were counted. Data were normal- binds to a hydrophobic grove in the B domain of the pocket. KSHV encodes two latent proteins, LANA and vCyc, which target ized against the colony number of the SAOS-2 cells transfected with 19,21,22 empty vector, normalized as 1. The data shown are the average of the pRb. However, to date, no KSHV proteins able to target three independent experiments with error bars representing the s.d. the other pocket proteins p107 and p130 have been described. *Po0.05, Student’s t-test. Here we demonstrate that the latent protein LANA2 is able to interact with all the three pocket proteins pRb, p107 and p130 both in vitro and in KSHV-infected PEL cells. In addition, we show Inmunoprecipitated proteins were then analyzed by western that LANA2 inhibits the cell growth arrest mediated by pRb in an blotting using anti-p107 or anti-p130 antibodies. As shown in LXCXE-dependent manner; however, mutation of this motif in Figure 5a, both p107 and p130 were detected in the protein LANA2 did not abolish the interaction of LANA2 with pRb, extracts immunoprecipitated with anti-SV5 antibody but not in indicating that the interaction between LANA2 and pRb does not those immunoprecipitated with control antibody. These results depend on the LXCXE motif. The interaction between pRb and indicated that p107 and p130 interact in vivo with Ubc9. We next cellular proteins containing an LXCXE motif sometimes does not evaluated the SUMO modification of p107 or p130 in a require this domain as it has been reported for BCRA1.33 reconstituted in vitro system, where in vitro-translated [35S] Furthermore, the interaction of viral oncoproteins containing methionine-labeled p107 or p130 protein was incubated with LXCXE domains through additional regions has been previously SUMO1 or SUMO2 in the presence of the recombinant SUMOyla- reported. Thus, the interaction between pRb and adenovirus E1A tion machinery. As shown in Figure 5b, addition of SUMO1 or or polyomavirus SV40 large T antigen is mediated not only by an SUMO2 to the p107 in vitro SUMOylation reaction led to the LXCXE domain but also by additional regions.34–37 appearance of additional higher molecular weight bands, indicat- All of the three pocket proteins can be regulated at the post- ing that p107 can be SUMOylated in vitro. To further confirm that translational level. pRb is regulated by phosphorylation, acetyla- these bands corresponded with SUMOylated protein, p107- tion and SUMOylation.12 SUMOylation targets a specific residue in SUMO1 and p107-SUMO2 proteins obtained as described above the lysine cluster of the pocket B region of pRb, preferentially were incubated in the presence of GST-SENP1 in an in vitro modifies the hypophosphorylated form of pRb and requires the deSUMOylation reaction. The p107-SUMO1 and p107-SUMO2 integrity of the pocket domain.14 Although the consequences of bands disappeared after incubation with SENP1 (Figure 5b), pRb SUMOylation are not clear, it is known that the level of pRb confirming that p107 is SUMOylated in vitro. Similarly, incubation SUMOylation is controlled by the interaction of pRb with viral and of p130 with SUMO1 or SUMO2 resulted in a smear of additional cellular proteins harboring the LXCXE-motif.14 LANA2 reduced higher molecular weight bands that disappeared after incubation SUMOylation of pRb in an LXCXE-dependent manner, confirming with SENP1, indicating that p130 is SUMOylated in vitro again the functionality of this domain. Both p107 and p130 (Figure 5b). Then, we decided to evaluate whether p107 and proteins are also regulated by phosphorylation, but while p130 p130 could be also SUMOylated in vivo. HEK-293 cells were can be also acetylated,15 no acetylation of p107 has been transfected with p107 or p130 and pcDNA or Ubc9 and His6- reported, and so far no SUMOylation of p107 or p130 has been SUMO2, and 48 h after transfection, histidine-purified proteins demonstrated. The SUMOylation of pRb led us to evaluate the were analyzed using anti-p107 or anti-p130 antibodies. Consistent putative SUMOylation of p107 and p130. A significant fraction of with our in vitro data, transfection of SUMO2 resulted in higher the proteins that are modified by SUMO interact directly with the

& 2014 Macmillan Publishers Limited Oncogene (2014) 495 – 503 KSHV LANA2 protein modulates the pocket proteins L Marcos-Villar et al 500

Figure 5. Both p107 and p130 undergo SUMO modification, and LANA2 inhibits its SUMOylation in an LXCXE-dependent manner. (a) Both p107 and p130 interact with Ubc9 in vivo. p107 or p130 are detected in Ubc9-SV5 immunoprecipitates (IPs) from HEK-293 cells co-transfected with Ubc9-SV5 and p107 or p130, respectively, by western blotting (WB). By contrast, control IPs using an irrelevant mouse monoclonal antibody yielded no p107 or p130 bands. (b) Both p107 and p130 are SUMOylated in vitro. In vitro-translated [35S]-labeled p107 or p130 were subjected to in vitro SUMOylation assay in the presence of SUMO1 or SUMO2. SUMO-conjugated p107 and p130 proteins were then incubated with GST or GST-SENP1 as described in Materials and Methods. Proteins were resolved by SDS-PAGE and visualized by autoradiography. (c) Both p107 and p130 are SUMOylated in vivo. HEK-293 cells were transfected with p107 or p130 and pcDNA or Ubc9 and His6-SUMO2. Whole protein extracts or histidine-tagged purified proteins were analyzed by western blot using anti-p107 or p130 antibodies. (d) LANA2 inhibits the SUMOylation of p107 or p130 in a LXCXE-dependent manner. HEK-293 cells were transfected with p107 or p130 and pcDNA or Ubc9 and His6-SUMO2, in the presence or absence of LANA2 WT or LANA2C111A, as indicated. Lysates and histidine-SUMO conjugates recovered from Niquel-agarose beads were separated by SDS-PAGE and probed with anti-LANA2, anti-p107 or anti-p130 antibodies. Note that unmodified p107 and p130 proteins bind nonspecifically to the nickel beads.

E2 SUMO conjugation enzyme, Ubc9.31 Our results demonstrate proteins will help us to evaluate the consequences of the that both p107 and p130 interact with Ubc9, pointing to these two inhibition of SUMOylation of the pocket proteins by viral proteins as putative SUMO targets. Indeed, we demonstrated that oncoproteins. both p107 and p130 proteins can be SUMOylated in vitro and Targeting of all pRb family members by LANA2 could be seen as in vivo. Furthermore, LANA2 expression was also able to inhibit a viral need for blocking a redundant function of the pocket p107 and p130 SUMOylation, an effect that was clearly reduced proteins, for instance, the imposition of G1/S arrest. In addition to after expression of the LXCXE LANA2 mutant. All together these its role on the cell cycle, LANA2 might also inhibit apoptosis by results indicate that LANA2 inhibits the SUMOylation of the three targeting Rb.38–43 Several reports evidence the multiple anti- pocket proteins in an LXCXE-dependent manner. The functions of apoptotic and pro-proliferative activities of LANA2. Thus, for pRb SUMOylation are not clear, but it has been suggested that example, LANA2 inhibits the transcriptional activation and conjugation of SUMO2 to pRb may have a role in premature apoptosis mediated by p53,44 inhibits the apoptosis mediated senescence25 and that inhibition of pRb SUMOylation by oncoviral by the double-stranded RNA-dependent protein kinase R,45 proteins may represent a strategy used by virus to suppress cell interferes with the PML-mediated transcriptional repression of senescence. Further work on the activities of p107 or p130- SUMO survivin,46 enhances hypoxia-inducible factor-1-alpha-mediated

Oncogene (2014) 495 – 503 & 2014 Macmillan Publishers Limited KSHV LANA2 protein modulates the pocket proteins L Marcos-Villar et al 501 cell proliferation,47 increases c-Myc signaling,48,49 inhibits nuclear Vidal (University of Santiago de Compostela, Spain). pcDNA-His6-SUMO1, factor-kB activation50 and interferon transcription and pro- pcDNA-His6-SUMO2 and pcDNA-Ubc9-SV5 plasmids have been previously duction.51–53 The ability of LANA2 to target the pocket proteins described.70–72 together with its additional activities might then account for the reduction of proliferation and apoptosis detected in PEL cells in GST pull-down 54 response to downmodulation of LANA2 levels. Moreover, several GST-pull down experiments were performed using [35S]methionine-labeled lines of research suggest that the simultaneous inactivation of all in vitro transcribed/translated LANA2, pRb, p107 or p130 proteins and GST, the three pRb family members may be necessary to manifest the GST-pRb, GST-p107, GST-p130, GST-LANA2 or GST-LANA2C111A, as fully transformed phenotype of mammalian cells,37,55–57 described previously.46 In vitro transcription/translation of proteins were suggesting that targeting of the pocket proteins might have performed using 1 mg of plasmid DNA and rabbit reticulocyte-coupled important implications for the oncogenic activity of LANA2. transcription/translation system according to the instructions provided by In addition to overlapping functions, p107 and p130 could have the manufacturer (Promega, Madrid, Spain). specific activities with important consequences for KSHV infection. In this regard, p107, but not pRb, itself can directly bind c-Myc and In vitro SUMO conjugation assay 58,59 prevent its transactivation. LANA2 has been shown to stabilize In vitro SUMO conjugation assay was performed on [35S]methionine- and induce the transactivation of c-Myc, in part, by its association labeled in vitro transcribed/translated pRb, p107 or p130 proteins as with the transcriptional co-factor for c-myc, Skp2 or by inhibiting described previously73 using recombinant E1 (Biomol, Enzo Life Sciences, the association between c-Myc-suppressor MM-1 alpha and Lausen, Switzerland), Ubc9 and SUMO1 or SUMO2. c-Myc.48,49 It is then tempting to speculate that the interaction of LANA2 with p107 may also contribute to the transcriptional In vitro deSUMOylation assay activation of c-Myc. p107-SUMO1, p107-SUMO2, p130-SUMO1 or p130-SUMO2 proteins KSHV establishes a long-term latent infection in B lymphocytes, obtained in an in vitro SUMOylation reaction as described above was and the regulation of reactivation of the lytic cycle from latency is incubated with 2 mg of GST-SENP1 (Biomol) in 30 ml reaction buffer considered to be controlled, at least in part, through epigenetic containing 50 mM Tris (pH 7.5), 2 mM MgCl and 5 mM b-mercaptoethanol. 60,61 2 factors. In this sense, the upregulation of the levels of DNA Reactions were incubated at 37 1C for 1 h and terminated with sodium methyltransferase-3a and -3b induced as a consequence of the dodecyl sulfate (SDS) sample buffer containing mercaptoethanol. Reac- downmodulation of p130 by the KSHV miRNA K12-4-5p has been tions products were then fractionated by SDS-PAGE (polyacrylamide gel suggested to contribute to maintain the latent state of the KSHV electrophoresis) and detected by autoradiography. genome.62,63 Based on these data, we speculate that LANA2 could also help establish viral latency by targeting the pocket proteins. Purification of His-tagged conjugates In summary, the inhibition of the pocket proteins might help Purification of His-tagged conjugates using Ni2 þ -NTA-agarose beads was LANA2 to create a cellular environment supportive of DNA performed as described.46 replication and could contribute to the establishment of viral latency. This work enlarges the list of viral proteins capable of interacting with the pocket proteins through an LXCXE domain, Western blot and immunoprecipitation analysis and antibodies and the list of KSHV proteins with the capacity to interact with For western blot analysis, cells were washed in phosphate-buffered saline, pRb. Furthermore, this is the first example of a KSHV protein that scraped in SDS-PAGE loading buffer and boiled for 5 min. Proteins of total can modulate the SUMOylation of the pocket proteins. Although extracts were separated by 8% SDS-PAGE and transferred to nitrocellulose membrane. For immunoprecipitation analysis, cells were washed in the classical tumor-suppressor proteins are often mutated in phosphate-buffered saline and then lysed in RIPA buffer containing fresh human cancers, pRb is not mutated in KSHV-associated malig- proteinase inhibitor mixture. After a brief sonication, the lysates were 64 nancies. Furthermore, p130 is infrequently mutated, and no cleared by high-speed centrifugation. A 50% protein A-sepharose slurry, p107 mutations in human cancers have been identified.65–69 Thus, which had been incubated with the appropriate antibody at 4 1C modulation of their activities by post-translational mechanisms overnight, was added to the cleared lysates and incubated at 4 1C for provides a new mechanism by which LANA2 could contribute to 2 h. Afterwards, the sepharose beads were collected and washed five times KSHV pathogenesis. in RIPA buffer. The precipitated protein complexes were recovered by boiling them in SDS sample buffer and analyzed by western blotting. Western blotting was carried out using standard methods. All antibodies were used at a dilution of 1:1000 of the stock in blocking buffer (5% skim MATERIALS AND METHODS milk prepared in 1 Â TBS-Tween). The following antibodies were used: anti- Cell lines p107 (sc-318, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p130 The human osteosarcoma cell line SAOS-2 and the human embryonic (sc-317, Santa Cruz Biotechnology), anti-pRb (554136, BD Pharmigen, BD kidney cell line HEK-293 were grown in Dulbecco’s modified Eagle medium Biosciences, Madrid, Spain), anti-LANA2 (NB200-167, Novus Biologicals, (Gibco BRL, Madrid, Spain) supplemented with 10% fetal bovine serum, Cambridge, UK) and anti-alpha-tubulin antibody (MCA78G, Serotec, penicillin (100 units/ml), streptomycin (100 mg/ml) and 2 mM L-glutamine. Du¨sseldorf, Germany). BCP-1 PEL cells were grown in RPMI supplemented with 20% fetal calf serum, glutamine and penicillin-streptomycin. Transfection of HEK-293 cells was done using Xtreme (Roche, Madrid, Spain) following the Colony-formation assays manufacturer’s instructions. In all, 1 Â 106 SAOS-2 cells were transfected SAOS-2 cells were transfected with the indicated expression vectors. All the by electroporation, using a Bio-Rad Gene Pulser at 250 mV constant current transfections were performed using the same amount of plasmids carrying (Bio-Rad Laboratories SA, Madrid, Spain). G-418 resistance. G-418 selection was initiated 2 days after transfection, dishes were stained with crystal violet 14 days later and colonies exhibiting G-418 resistance were counted. Plasmids Plasmids pcDNA-LANA2 and GST-LANA2 have been previously described.44 Plasmids pcDNA-LANA2C111A and GST-LANA2C111A were generated Cell-cycle analysis using the QuickChange PCR-based mutagenesis kit (Stratagene, La Jolla, SAOS-2 cells in 10 mm dishes were transfected with GFP and the indicated CA, USA) and the pcDNA-LANA2 and GST-LANA2 plasmids as template, plasmids, and 48 h after transfection, cells were harvested, fixed and respectively. Oligonucleotides used in the site-directed mutagenesis stained by using propidium iodine. The distribution of GFP-positive cells in are LANA2C111AF: 50-GGAATTCATGGCGGGACGCAGGCTTACCTGG-30 and the different phases of the cell cycle was determined by analytical flow LANA2C111AR: 50-GGGATCCGGTCATCACATGTAACTGAACG-30. Plasmids cytometry using a CYAN (DAKO Cytomation, Glostrup, Denmark), and data pcDNA-pRb, pcDNA-p107 and pcDNA-p130 were a generous gift of Anxo were analyzed by using Summit software (DAKO Cytomation).

& 2014 Macmillan Publishers Limited Oncogene (2014) 495 – 503 KSHV LANA2 protein modulates the pocket proteins L Marcos-Villar et al 502 Statistical analysis 21 Li M, Lee H, Yoon DW, Albrecht JC, Fleckenstein B, Neipel F et al. Kaposi’s sarcoma- For statistical analysis between control and different groups, the Student’s associated herpesvirus encodes a functional cyclin. J Virol 1997; 71: 1984–1991. t-test was applied. The significance level chosen for the statistical analysis 22 Radkov SA, Kellam P, Boshoff C. The latent nuclear antigen of Kaposi sarcoma- was Po0.05. associated herpesvirus targets the retinoblastoma-E2F pathway and with the oncogene Hras transforms primary rat cells. Nat Med 2000; 6: 1121–1127. 23 Moran E, Zerler B, Harrison TM, Mathews MB. Identification of separate domains in CONFLICT OF INTEREST the adenovirus E1A gene for immortalization activity and the activation of virus early genes. Mol Cell Biol 1986; 6: 3470–3480. The authors declare no conflict of interest. 24 Zhang B, Chen W, Roman A. The E7 proteins of low- and high-risk human papillomaviruses share the ability to target the pRB family member p130 for degradation. Proc Natl Acad Sci USA 2006; 103: 437–442. ACKNOWLEDGEMENTS 25 Li T, Santockyte R, Shen RF, Tekle E, Wang G, Yang DC et al. Expression of SUMO-2/ This project has been possible thanks to the generous support from Patrick 3 induced senescence through p53- and pRB-mediated pathways. J Biol Chem Moore and Yuan Chang (Pittsburg University, USA) in the initial experiments and to 2006; 281: 36221–36227. the reagents they provided. This work was supported by BFU-2008-03784 and BFU- 26 Marcos-Villar L, Campagna M, Lopitz-Otsoa F, Gallego P, Gonzalez-Santamaria J, 2011-27064. MC and LM-V are supported by Juan de la Cierva Program. PG is Gonzalez D et al. Covalent modification by SUMO is required for efficient dis- supported by JAE predoctoral fellowship from CSIC. CFC-H is supported by La Caixa ruption of PML oncogenic domains by Kaposi’s sarcoma-associated herpesvirus fellowship. latent protein LANA2. J Gen Virol 92(Part 1): 188–194. 27 Claudio PP, Howard CM, Baldi A, De Luca A, Fu Y, Condorelli G et al. p130/pRb2 has growth suppressive properties similar to yet distinctive from those of reti- REFERENCES noblastoma family members pRb and p107. Cancer Res 1994; 54: 5556–5560. 1 Ewen ME, Xing YG, Lawrence JB, Livingston DM. Molecular cloning, chromosomal 28 Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH. The retinoblastoma gene mapping, and expression of the cDNA for p107, a retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell 1991; product-related protein. Cell 1991; 66: 1155–1164. 67: 293–302. 2 Hannon GJ, Demetrick D, Beach D. Isolation of the Rb-related p130 through its 29 Starostik P, Chow KN, Dean DC. Transcriptional repression and growth suppres- interaction with CDK2 and cyclins. Genes Dev 1993; 7: 2378–2391. sion by the p107 pocket protein. Mol Cell Biol 1996; 16: 3606–3614. 3 Li Y, Graham C, Lacy S, Duncan AM, Whyte P. The adenovirus E1A-associated 130- 30 Zhu L, van den Heuvel S, Helin K, Fattaey A, Ewen M, Livingston D et al. Inhibition kD protein is encoded by a member of the retinoblastoma gene family and of cell proliferation by p107, a relative of the retinoblastoma protein. Genes Dev physically interacts with cyclins A and E. Genes Dev 1993; 7: 2366–2377. 1993; 7: 1111–1125. 4 Mayol X, Grana X, Baldi A, Sang N, Hu Q, Giordano A. Cloning of a new member of 31 Melchior F. SUMO--nonclassical ubiquitin. Annu Rev Cell Dev Biol 2000; 16: the retinoblastoma gene family (pRb2) which binds to the E1A transforming 591–626. 32 Weinberg RA. The molecular basis of oncogenes and tumor suppressor genes. domain. Oncogene 1993; 8: 2561–2566. Ann NY Acad Sci 1995; 758: 331–338. 5 Dyson N, Harlow E. Adenovirus E1A targets key regulators of cell proliferation. 33 Fan S, Yuan R, Ma YX, Xiong J, Meng Q, Erdos M et al. Disruption of BRCA1 LXCXE Cancer Surv 1992; 12: 161–195. motif alters BRCA1 functional activity and regulation of RB family but not RB 6 Claudio PP, De Luca A, Howard CM, Baldi A, Firpo EJ, Koff A et al. protein binding. Oncogene 2001; 20: 4827–4841. Functional analysis of pRb2/p130 interaction with cyclins. Cancer Res 1996; 56: 34 Barbeau D, Charbonneau R, Whalen SG, Bayley ST, Branton PE. Functional inter- 2003–2008. actions within adenovirus E1A protein complexes. Oncogene 1994; 9: 359–373. 7 Beijersbergen RL, Carlee L, Kerkhoven RM, Bernards R. Regulation of the 35 Dyson N, Guida P, McCall C, Harlow E. Adenovirus E1A makes two distinct con- retinoblastoma protein-related p107 by G1 cyclin complexes. Genes Dev 1995; 9: tacts with the retinoblastoma protein. J Virol 1992; 66: 4606–4611. 1340–1353. 36 Sullivan CS, Cantalupo P, Pipas JM. The molecular chaperone activity of simian 8 Chellappan SP, Hiebert S, Mudryj M, Horowitz JM, Nevins JR. The E2F transcription virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an factor is a cellular target for the RB protein. Cell 1991; 65: 1053–1061. ATP-dependent mechanism. Mol Cell Biol 2000; 20: 6233–6243. 9 Lundberg AS, Weinberg RA. Functional inactivation of the retinoblastoma protein 37 Zalvide J, Stubdal H, DeCaprio JA. The J domain of simian virus 40 large T antigen requires sequential modification by at least two distinct cyclin-cdk complexes. is required to functionally inactivate RB family proteins. Mol Cell Biol 1998; 18: Mol Cell Biol 1998; 18: 753–761. 1408–1415. 10 Smith EJ, Leone G, Nevins JR. Distinct mechanisms control the accumulation of 38 Araki K, Ahmad SM, Li G, Bray Jr. DA, Saito K, Wang D et al. Retinoblastoma RB94 the Rb-related p107 and p130 proteins during cell growth. Cell Growth Differ 1998; enhances radiation treatment of head and neck squamous cell carcinoma. Clin 9: 297–303. Cancer Res 2008; 14: 3514–3519. 11 Xiao ZX, Ginsberg D, Ewen M, Livingston DM. Regulation of the retinoblastoma 39 Bowen C, Spiegel S, Gelmann EP. Radiation-induced apoptosis mediated by protein-related protein p107 by G1 cyclin-associated kinases. Proc Natl Acad Sci retinoblastoma protein. Cancer Res 1998; 58: 3275–3281. USA 1996; 93: 4633–4637. 40 Hsieh JK, Chan FS, O’Connor DJ, Mittnacht S, Zhong S, Lu X. RB regulates the 12 Burkhart DL, Sage J. Cellular mechanisms of tumour suppression by the retino- stability and the apoptotic function of p53 via MDM2. Mol Cell 1999; 3: 181–193. blastoma gene. Nat Rev Cancer 2008; 8: 671–682. 41 Knudsen KE, Weber E, Arden KC, Cavenee WK, Feramisco JR, Knudsen ES. The 13 Chan HM, Krstic-Demonacos M, Smith L, Demonacos C, La Thangue NB. Acet- retinoblastoma tumor suppressor inhibits cellular proliferation through two dis- ylation control of the retinoblastoma tumour-suppressor protein. Nat Cell Biol tinct mechanisms: inhibition of cell cycle progression and induction of cell death. 2001; 3: 667–674. Oncogene 1999; 18: 5239–5245. 14 Ledl A, Schmidt D, Muller S. Viral oncoproteins E1A and E7 and cellular LxCxE 42 Lemaire C, Godefroy N, Costina-Parvu I, Rincheval V, Renaud F, Trotot P et al. proteins repress SUMO modification of the retinoblastoma tumor suppressor. Caspase-9 can antagonize p53-induced apoptosis by generating a p76(Rb) trun- Oncogene 2005; 24: 3810–3818. cated form of Rb. Oncogene 2005; 24: 3297–3308. 15 Schwarze F, Meraner J, Lechner M, Loidl A, Stasyk T, Laich A et al. Cell 43 Zhao X, Day ML. RB activation and repression of C-MYC transcription precede cycle-dependent acetylation of Rb2/p130 in NIH3T3 cells. Oncogene 2010; 29: apoptosis of human prostate epithelial cells. Urology 2001; 57: 860–865. 5755–5760. 44 Rivas C, Thlick AE, Parravicini C, Moore PS, Chang Y. Kaposi’s sarcoma-associated 16 Kim HY, Ahn BY, Cho Y. Structural basis for the inactivation of retinoblastoma herpesvirus LANA2 is a B-cell-specific latent viral protein that inhibits p53. J Virol tumor suppressor by SV40 large T antigen. Embo J 2001; 20: 295–304. 2001; 75: 429–438. 17 Lee JO, Russo AA, Pavletich NP. Structure of the retinoblastoma tumour- 45 Esteban M, Garcia MA, Domingo-Gil E, Arroyo J, Nombela C, Rivas C. The latency suppressor pocket domain bound to a peptide from HPV E7. Nature 1998; 391: protein LANA2 from Kaposi’s sarcoma-associated herpesvirus inhibits apoptosis 859–865. induced by dsRNA-activated protein kinase but not RNase L activation. J Gen Virol 18 Chang Y, Moore PS, Talbot SJ, Boshoff CH, Zarkowska T, Godden K et al. Cyclin 2003; 84(Pt 6): 1463–1470. encoded by KS herpesvirus. Nature 1996; 382: 410. 46 Marcos-Villar L, Lopitz-Otsoa F, Gallego P, Munoz-Fontela C, Gonzalez-Santamaria 19 Godden-Kent D, Talbot SJ, Boshoff C, Chang Y, Moore P, Weiss RA et al. The cyclin J, Campagna M et al. Kaposi’s sarcoma-associated herpesvirus protein LANA2 encoded by Kaposi’s sarcoma-associated herpesvirus stimulates cdk6 to phos- disrupts PML oncogenic domains and inhibits PML-mediated transcriptional phorylate the retinoblastoma protein and histone H1. J Virol 1997; 71: 4193–4198. repression of the survivin gene. J Virol 2009; 83: 8849–8858. 20 Jung JU, Stager M, Desrosiers RC. Virus-encoded cyclin. Mol Cell Biol 1994; 14: 47 Shin YC, Joo CH, Gack MU, Lee HR, Jung JU. Kaposi’s sarcoma-associated her- 7235–7244. pesvirus viral IFN regulatory factor 3 stabilizes hypoxia-inducible factor-1 alpha to

Oncogene (2014) 495 – 503 & 2014 Macmillan Publishers Limited KSHV LANA2 protein modulates the pocket proteins L Marcos-Villar et al 503 induce vascular endothelial growth factor expression. Cancer Res 2008; 68: 61 Pantry SN, Medveczky PG. Epigenetic regulation of Kaposi’s sarcoma-associated 1751–1759. herpesvirus replication. Semin Cancer Biol 2009; 19: 153–157. 48 Baresova P, Pitha PM, Lubyova B. Kaposi sarcoma-associated herpesvirus vIRF-3 62 Benetti R, Gonzalo S, Jaco I, Munoz P, Gonzalez S, Schoeftner S et al. A mammalian protein binds to F-box of Skp2 protein and acts as a regulator of c-Myc protein microRNA cluster controls DNA methylation and telomere recombination via function and stability. J Biol Chem 2012; 287: 16199–16208. Rbl2-dependent regulation of DNA methyltransferases. Nat Struct Mol Biol 2008; 49 Lubyova B, Kellum MJ, Frisancho JA, Pitha PM. Stimulation of c-Myc transcriptional 15: 268–279. activity by vIRF-3 of Kaposi sarcoma-associated herpesvirus. J Biol Chem 2007; 63 Lu F, Stedman W, Yousef M, Renne R, Lieberman PM. Epigenetic regulation of 282: 31944–31953. Kaposi’s sarcoma-associated herpesvirus latency by virus-encoded microRNAs 50 Seo T, Park J, Lim C, Choe J. Inhibition of nuclear factor kappaB activity by viral that target Rta and the cellular Rbl2-DNMT pathway. J Virol 2010; 84: 2697–2706. interferon regulatory factor 3 of Kaposi’s sarcoma-associated herpesvirus. 64 Platt G, Carbone A, Mittnacht S. p16INK4a loss and sensitivity in KSHV associated Oncogene 2004; 23: 6146–6155. primary effusion lymphoma. Oncogene 2002; 21: 1823–1831. 51 Joo CH, Shin YC, Gack M, Wu L, Levy D, Jung JU. Inhibition of interferon regulatory 65 Claudio PP, Howard CM, Fu Y, Cinti C, Califano L, Micheli P et al. Mutations in the factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi’s sarcoma- retinoblastoma-related gene RB2/p130 in primary nasopharyngeal carcinoma. associated herpesvirus viral IRF homolog vIRF3. J Virol 2007; 81: 8282–8292. Cancer Res 2000; 60: 8–12. 52 Lubyova B, Kellum MJ, Frisancho AJ, Pitha PM. Kaposi’s sarcoma-associated her- 66 Claudio PP, Howard CM, Pacilio C, Cinti C, Romano G, Minimo C et al. Mutations in pesvirus-encoded vIRF-3 stimulates the transcriptional activity of cellular IRF-3 the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of and IRF-7. J Biol Chem 2004; 279: 7643–7654. tumor growth in vivo by retrovirus-mediated gene transfer. Cancer Res 2000; 60: 53 Wies E, Hahn AS, Schmidt K, Viebahn C, Rohland N, Lux A et al. The Kaposi’s 372–382. sarcoma-associated herpesvirus-encoded vIRF-3 inhibits cellular IRF-5. J Biol Chem 67 Howard CM, Claudio PP, De Luca A, Stiegler P, Jori FP, Safdar NM et al. Inducible 2009; 284: 8525–8538. pRb2/p130 expression and growth-suppressive mechanisms: evidence of a pRb2/ 54 Wies E, Mori Y, Hahn A, Kremmer E, Sturzl M, Fleckenstein B et al. The viral p130, p27Kip1, and cyclin E negative feedback regulatory loop. Cancer Res 2000; interferon-regulatory factor-3 is required for the survival of KSHV-infected primary 60: 2737–2744. effusion lymphoma cells. Blood 2008; 111: 320–327. 68 Scambia G, Lovergine S, Masciullo V. RB family members as predictive and 55 Christensen JB, Imperiale MJ. Inactivation of the retinoblastoma susceptibility prognostic factors in human cancer. Oncogene 2006; 25: 5302–5308. protein is not sufﬁcient for the transforming function of the conserved region 2- 69 Yeung RS, Bell DW, Testa JR, Mayol X, Baldi A, Grana X et al. The retinoblastoma- like domain of simian virus 40 large T antigen. J Virol 1995; 69: 3945–3948. related gene, RB2, maps to human chromosome 16q12 and rat chromosome 19. 56 DeGregori J, Johnson DG. Distinct and overlapping roles for E2F family members Oncogene 1993; 8: 3465–3468. in transcription, proliferation and apoptosis. Curr Mol Med 2006; 6: 739–748. 70 Desterro JM, Rodriguez MS, Hay RT. SUMO-1 modiﬁcation of IkappaBalpha inhi- 57 Wirt SE, Sage J. p107 in the public eye: an Rb understudy and more. Cell Div bits NF-kappaB activation. Mol Cell 1998; 2: 233–239. 2010; 5:9. 71 Treier M, Staszewski LM, Bohmann D. Ubiquitin-dependent c-Jun degradation 58 Beijersbergen RL, Hijmans EM, Zhu L, Bernards R. Interaction of c-Myc with the in vivo is mediated by the delta domain. Cell 1994; 78: 787–798. pRb-related protein p107 results in inhibition of c-Myc-mediated transactivation. 72 Vertegaal AC, Andersen JS, Ogg SC, Hay RT, Mann M, Lamond AI. Distinct and Embo J 1994; 13: 4080–4086. overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative 59 Gu W, Bhatia K, Magrath IT, Dang CV, Dalla-Favera R. Binding and suppression of proteomics. Mol Cell Proteomics 2006; 5: 2298–2310. the Myc transcriptional activation domain by p107. Science 1994; 264: 251–254. 73 Campagna M, Herranz D, Garcia MA, Marcos-Villar L, Gonzalez-Santamaria J, 60 Miller G, El-Guindy A, Countryman J, Ye J, Gradoville L. Lytic cycle switches of Gallego P et al. SIRT1 stabilizes PML promoting its sumoylation. Cell Death Differ oncogenic human gammaherpesviruses. Adv Cancer Res 2007; 97: 81–109. 2011; 18:72–79.

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