Although expression of certain viral proteins has been associated with sumoylation of cellular proteinsin vivo, a clear demonstration of a viral protein with SUMO E3 ligase activity has not previously been reported. KSHV, a gamma herpesvirus, is the causative agent for Kaposi’s sarcoma as well as primary effusion lymphoma and multicentric Rabbit polyclonal to MTH1 Castleman’s disease (16,17). the early stage of infection. In addition to functioning as a transcriptional factor, we show that K-bZIP carries a SIM (SUMO-interacting motif), which specifically binds to SUMO-2/3 but not SUMO-1. K-bZIP catalyzes its own SUMO modification as well as that of its interacting partners such as the cellular tumor suppressor proteins p53 and Rb, bothin vitroandin vivo. This reaction depends on an intact SIM. Sumoylation of p53 leads to its activation and K-bZIP is recruited to several p53 target chromatin sites AZD4547 in a SIM-dependent manner. In addition to the identification of a viral SUMO-2/3 E3 ligase, our results provide additional insights into the mechanisms whereby K-bZIP induces cell cycle arrest. Keywords:p53, Post-translational Modification, Signal Transduction, Sumoylation, Viral Protein, KSHV K-bZIP, SUMO E3 ligase, SUMO-2/3, SUMO-interacting Motifs == Introduction == Increasing evidence indicates that sumoylation,i.e.post-translational modification of proteins by the small ubiquitin-like modifier (SUMO)2plays a central role in cellular signal transduction. Like phosphorylation, sumoylation is rapid and reversible. In a manner similar to the binding of phosphorylated tyrosine by signal molecules carrying Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains, sumoylated proteins are specifically engaged by proteins with a SUMO-interacting motif (SIM). Modulation of sumoylation has a profound effect on protein-protein interactions and the propagation of cellular signals. Viruses have evolved different mechanisms to exploit the host sumoylation pathway to create a cellular environment that is favorable for viral replication by modulating the functions of viral and cellular proteins (reviewed in Ref.1). Many viral proteins are themselves sumoylated, and this post-translational modification affects specific functions of these targets. For DNA tumor viruses, the immediate-early and early gene products, which include transcriptional AZD4547 factors, are often sumoylated. Examples include immediate-early 1 (IE 1) and immediate-early 2 (IE 2) proteins of AZD4547 cytomegalovirus (CMV) (2,3), E1 and E2 of human papillomavirus (HPV) (4,5), BZLF1 of Epstein-Barr virus (EBV) (6), and K-bZIP of Kaposi’s sarcoma-associated herpesvirus (KSHV) (7). Some viral proteins indirectly modulate the sumoylation status of specific cellular proteins. For example, the HPV E7 protein and adenovirus E1A protein block sumoylation of the cellular tumor suppressor Rb (8). Additionally, the KSHV viral protein kinase (ORF36) inhibits the sumoylation of KAP-1 (9). Viruses can also affect global sumoylation of cellular proteins by directly modulating the cellular SUMO machinery. Avian adenovirus Gam1 protein inhibits the SUMO E1-activating enzyme by targeting the SAE1/SAE2 (AOS1/UBA2) heterodimer to cullin RING ligases (CRLs) and promoting SAE1 ubiquitylation and degradation (10). Here, we describe a unique mechanism by which a virus directly modulates sumoylation via the expression of a virally encoded SUMO E3 ligase. Analogous to ubiquitylation, conjugation of SUMO to target proteins is a multistep process involving an E1-activating enzyme heterodimer SAE1/SAE2, an E2-conjugating enzyme Ubc9 (ubiquitin-like protein SUMO-1-conjugating enzyme 9), and an E3 ligase, which is believed to provide specificity for the sumoylation pathway (11). In stark contrast to ubiquitin E3 ligases, which number in the hundreds to thousands, there are very few cellular SUMO E3 ligases. These include the PIAS (protein inhibitor of activated STATS) family (12,13), the RanBP2 (Ran-binding protein 2) family (14), and the Pc2 (polycomb 2) family (15). These ligases share the common properties of binding to 1 1) the SUMO moiety via their SIM domains, 2) Ubc9, the E2 conjugating enzyme, and 3) substrates to which SUMO is subsequently covalently attached. The ability of these ligases to catalyze the sumoylation reaction is demonstrated by reconstitutedin vitrosumoylation reactions using purified proteins, including the enzymes, E1, E2, and E3, substrate target proteins, and SUMO proteins. PIAS family members contain a RING domain commonly found in ubiquitin ligases, whereas RanBP2 and Pc2 do not share this feature. Although expression of certain viral proteins has been associated with sumoylation of cellular proteinsin vivo, a clear demonstration of a viral protein with SUMO E3 ligase activity has not previously been reported. KSHV, a gamma herpesvirus, is the causative agent for Kaposi’s sarcoma as well as primary effusion lymphoma and multicentric Castleman’s disease (16,17). This virus encodes two major transcriptional factors, K-Rta and K-bZIP (KSHV basic-leucine-zipper). K-Rta is a strong transactivator and a trigger of lytic replication (18,19), whereas K-bZIP is a strong transcriptional repressor that attenuates the activity of K-Rta (20). We previously reported that K-bZIP is highly sumoylated at a single lysine residue (Lys-158) and that its repressive activity depends on sumoylation (7,21). Upon phosphorylation of K-bZIP at Thr-111 by either the cell cycle protein CDK2 or the viral protein kinase (ORF36), the sumoylation of K-bZIP is inhibited; thus, phosphorylation converts AZD4547 K-bZIP from functioning as a transcriptional repressor to a transactivator (21). K-bZIP is a nuclear protein distributed in the nucleoplasm as.
