SUMO-1 modification required for transformation by adenovirus type 5 early region 1B 55-kDa oncoprotein

Abstract

SUMO-1 is a small ubiquitin-related modifier protein that is covalently linked to many cellular and viral protein targets. Modification by SUMO-1 is proposed to play a role in protein targeting and͞or stability. We show here that adenovirus type 5 early region 1B 55-kDa (E1B-55kDa) oncoprotein can be covalently modified by SUMO-1 in vivo through a major attachment site comprising a single lysine residue at amino acid position 104. The sequence surrounding this lysine matches the proposed ⌿KxE consensus motif required for SUMO-1 conjugation. A single mutation (K104R) that abolishes SUMOylation of E1B-55kDa dramatically reduces the ability of the adenovirus type 5 protein to transform primary baby rat kidney cells in cooperation with E1A and to inhibit p53-mediated transactivation. Overexpression of SUMO-1 in adenovirus type 5 E1A͞E1B-55kDa-transformed baby rat kidney cells causes the relocalization of E1B-55kDa from the cytoplasm to the nucleus, where it accumulates with SUMO-1 in dot-or track-like structures. Significantly, when SUMO-1 is ectopically expressed in transformed rat cells no effect on the cytoplasmic localization of the E1B-K104R mutant protein is observed. Our results demonstrate that SUMO-1 modification is required for transformation by adenovirus type 5 E1B-55kDa and provide further evidence for the idea that this posttranslational modification plays a role in protein targeting to specific subcellular sites. T he 55-kDa phosphoprotein encoded in early region 1B (E1B-55kDa) from adenovirus type 5 (Ad5) is required for efficient viral DNA replication, selective viral late mRNA transport to the cytoplasm, and shut-off of host cell protein synthesis in productively infected cells (reviewed in ref. 1). In addition, the Ad protein provides functions for complete oncogenic transformation of mammalian cells in cooperation with Ad E1A (2). During the past few years it has been well established that the transforming potential of E1B-55kDa correlates with its ability to act as a direct transcriptional repressor targeted to p53-responsive promoters by binding to the tumor suppressor protein (3, 4). Considerable evidence suggests that these activities antagonize p53-induced apoptosis (5) and͞or cell cycle arrest (6). The regions required for transformation map to several segments in the Ad protein, including the p53-binding domains located around amino acid position 180 (Fig. 1A) and in the central part (4,7,8), plus two segments at the carboxy terminus that mediate inhibition of p53-dependent and p53-independent transactivation (5, 9, 10). Although the mechanism by which E1B-55kDa blocks transcription is still unclear, recent data suggest that its silencing activity requires a cellular corepressor that copurifies with RNA polymerase II (11) as well as interaction with cellular factors known to be involved in transcriptional repression such as histone deacetylase 1 (HDAC1) and mSin3A (12). Furthermore, consistent with its role in blocking p53-mediated transactivation, the Ad protein inhibits p53 acetylation b...

SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML

“…Direct immunoprecipitation of E1B-55K shows a sufficient and comparable amount of the adenoviral protein in all transfections ( Figure 2a, panel 1). Another band of B75 kDa was also evident most presumably corresponding to covalently SUMO1-modified E1B-55K (Figure 2a, panel 1) (Endter et al, 2001). Interestingly, the analysis of coprecipitated flag-PML reveals a highly specific interaction of E1B-55K with the PML isoforms IV and V ( Figure 2a, panel 2, lane 6/7), whereas some minor interactions with the isoforms I and III are visible in long exposure (data not shown).…”

“…Mutational inactivation of the NES leads to the accumulation of the viral protein in the nucleus accompanied by sequestration of different cellular proteins including PML (Endter et al, 2001(Endter et al, , 2005Ha¨rtl et al, 2008). To elucidate this phenomenon, stably transformed rat cell lines AB120/AB115 expressing E1A plus E1B-55K-wt/E1A plus E1B-55K-NES were analysed for steady-state localization of E1B-55K and PML (Figures 1A and B).…”

“…The pro-tumorigenic functions of the 55-kDa phosphoproteins encoded by the early region 1B (E1B-55K) of human adenoviruses are primarily linked to the modulation of the tumour suppressor p53 and consequently inhibition of programmed cell death and growth arrest. Subsequent steps of direct interaction (Sarnow et al, 1982a;Kao et al, 1990), transcriptional repression (Yew et al, 1994;Berk, 1998, 1999) and nuclear-cytoplasmic relocalization (Endter et al, 2001(Endter et al, , 2005 induce the complete silencing of p53-dependent tumour suppressive functions. However, previously described results suggest additional p53-independent mechanisms of E1B-55K induced cellular transformation involving cellular factors as Mre11 (Ha¨rtl et al, 2008) or the transcription factor Daxx (Sieber and Dobner, 2007).…”

“…Since these initial findings, it has become evident that PML is a general tumour suppressor frequently deregulated in various tumour types (Gurrieri et al, 2004) most presumably involving secondary effects of PML bodies as sites of protein degradation (Lallemand-Breitenbach et al, 2001), transcriptional regulation (Li et al, 2000;Zhong et al, 2000), cellular senescence (Ferbeyre et al, 2000;Pearson et al, 2000;Bischof et al, 2002;Langley et al, 2002), tumour suppression (Salomoni and Pandolfi, 2002;Salomoni et al, 2008), DNA repair (Bischof et al, 2001;Carbone et al, 2002), apoptosis (Hofmann and Will, 2003;Takahashi et al, 2004) and epigenetic regulation (Torok et al, 2009). Interestingly, functional inactivation of the E1B-55K leucine-rich nuclear export sequence (NES) induces enhanced posttranslational modification of E1B-55K by the small ubiquitin-related modifier 1 (SUMO1) at lysine 104 (SUMO-conjugation motif, SCM) as well as augments transformation of primary rat cells involving the accumulation of p53, E1B-55K and PML in subnuclear aggregates (Endter et al, 2001(Endter et al, , 2005. Consistently, mutational inactivation of the SCM completely abrogates the ability of E1B-55K to transform primary rodent cells in combination with E1A (Endter et al, 2001).…”

“…Interestingly, functional inactivation of the E1B-55K leucine-rich nuclear export sequence (NES) induces enhanced posttranslational modification of E1B-55K by the small ubiquitin-related modifier 1 (SUMO1) at lysine 104 (SUMO-conjugation motif, SCM) as well as augments transformation of primary rat cells involving the accumulation of p53, E1B-55K and PML in subnuclear aggregates (Endter et al, 2001(Endter et al, , 2005. Consistently, mutational inactivation of the SCM completely abrogates the ability of E1B-55K to transform primary rodent cells in combination with E1A (Endter et al, 2001). Although the role of E1B-55K SUMOylation is still unclear, several line of evidence indicates that this posttranslational modification regulates the targeting of E1B-55K to subnuclear structures and viral transcription/replication centers (Endter et al, 2005;Kindsmu¨ller et al, 2007).…”