Sumoylation has emerged as a major post-translational modification of cellular proteins, affecting a variety of cellular processes. Viruses have exploited the sumoylation pathway to advance their own replication by evolving several ways to perturb the host sumoylation apparatus. However, there has been no report of virally encoded enzymes directly involved in catalyzing the sumoylation reaction. Here, we report that the K-bZIP protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is a SUMO E3 ligase with specificity toward SUMO2/3. K-bZIP is a nuclear factor that functions to modulate viral gene expression and to prolong the G1 phase, allowing viral transcription and translation to proceed at 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, both in vitro and in 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 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.Increasing evidence indicates that sumoylation, i.e. posttranslational modification of proteins by the small ubiquitinlike modifier (SUMO) 2 plays 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 factors, are often sumoylated. Examples include immediate-early 1 (IE 1) and immediate-early 2 (IE 2) proteins of 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 sarcomaassociated 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 ...

Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to the development of Kaposi's sarcoma, a major AIDSassociated malignancy, and to hematologic malignancies, including primary effusion lymphoma and multicentric Castleman's disease. Like other herpesviruses, KSHV is capable of both latent and lytic replication. Understanding the molecular details associated with this transition from latency to lytic replication is key to controlling virus spread and can affect the development of intervention strategies. Here, we report that Kruppel-associated box domain-associated protein-1 (KAP-1)/ transcriptional intermediary factor 1B, a cellular transcriptional repressor that controls chromosomal remodeling, participates in the process of switching viral latency to lytic replication. Knockdown of KAP-1 by small interfering RNA leads to KSHV reactivation mediated by K-Rta, a key transcriptional regulator. In cells harboring latent KSHV, KAP-1 was associated with the majority of viral lytic-gene promoters. K-Rta overexpression induced the viral lytic cycle with concomitant reduction of KAP-1 binding to viral promoters. Association of KAP-1 with heterochromatin was modulated by both sumoylation and phoshorylation. During lytic replication of KSHV, KAP-1 was phosphorylated at Ser 824 . Several lines of evidence directly linked the viral protein kinase to this posttranslational modification. Additional studies showed that this phosphorylation of KAP-1 produced a decrease in its sumoylation, consequently decreasing the ability of KAP-1 to condense chromatin on viral promoters. In summary, the cellular transcriptional repressor KAP-1 plays a role in regulating KSHV latency, and viral protein kinase modulates the chromatin remodeling function of this repressor. [Cancer Res 2009;69(14):5681-9]

The switch between the latency and lytic cycles of Kaposi's sarcoma-associated herpesvirus (KSHV) is accompanied by specific alterations of histone codes. Recently, comprehensive analysis of histone modifications of KSHV showed the deposition of H3K27me3 across the KSHV genome with two specific regions occupied by the heterochromatin marker H3K9me3. Here, we show that knockdown of JMJD2A, an H3K9me3 demethylase, attenuates viral titers, whereas its overexpression increases KSHV reactivation. JMJD2A is localized in regions of latent viral chromosomes that are deficient in the H3K9me3 mark, indicating that JMJD2A may be responsible for the low level of this mark on viral chromatin. The presence of JMJD2A on the latent genome maintains H3K9 in unmethylated form and signals the readiness of specific sets of viral genes to be reactivated. The demethylase activity of JMJD2A is important for KSHV reactivation, because a demethylase-deficient mutant cannot restore the JMJD2A knockdown phenotype. Interestingly, we found that the KSHV encoded K-bZIP associated with JMJD2A, resulting in the inhibition of demethylase activity of JMJD2A both in vivo and in vitro. Inhibition of JMJD2A by K-bZIP is likely due to a physical interaction which blocks substrate accessibility. A consequence of such an inhibition is increasing global levels of H3K9me3 and gene silencing. Consistently, K-bZIP overexpression resulted in a repression of ϳ80% of the >2-fold differentially regulated genes compared to results for the uninduced control cells. The consequences of K-bZIP targeting JMJD2A during viral replication will be discussed. To our knowledge, this is the first description of a viral product shown to be a potent inhibitor of a host cellular histone demethylase.

We have previously demonstrated that human H2-relaxin can mediate androgen-independent growth of LNCaP through a mechanism that involves the activation of the androgen receptor (AR) signaling pathway. The goal of the current study is to elucidate the mechanism(s) by which H2-relaxin causes activation of the AR pathway. Our data indicate that there is cross-talk between AR and components of the Wnt signaling pathway. Addition of H2-relaxin to LNCaP cells resulted in increased phosphorylation of protein kinase B (Akt) and inhibitory phosphorylation of glycogen synthase kinase-3b (GSK-3b) with subsequent cytoplasmic accumulation of b-catenin. Immunoprecipitation and immunocytochemical studies demonstrated that the stabilized b-catenin formed a complex with AR, which was then translocated into the nucleus. Chromatin immunoprecipitation analysis determined that the AR/b-catenin complex binds to the proximal region of the prostatespecific antigen promoter. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, using LY294002, prevented both H2-relaxin-mediated phosphorylation of Akt and GSK-3b and translocation of b-catenin/AR into the nucleus. Knockdown of b-catenin levels using a b-cateninspecific small interfering RNA inhibited H2-relaxin-induced AR activity. The combined data demonstrate that PI3K/ Akt and components of the Wnt pathway can facilitate H2-relaxin-mediated activation of the AR pathway.

Androgen-independent (AI)2 prostate cancer (CaP) typically develops from the selective outgrowth of tumor cells to castrate levels of testosterone in response to androgen-deprivation therapy (1). AI CaP cells have evolved different strategies for overriding the androgen-dependent (AD) growth and survival characteristics of early stage, organ-confined CaP or early stage metastatic CaP (2). Aberrant AR activation is the primary mechanism for the growth and survival of AI CaP in response to castrate levels of androgen (2). With the exception of prostatic small cell neuroendocrine carcinoma (3), the most parsimonious model of AI CaP arises from the inappropriate activation of AR-dependent cell growth and survival pathways. Unfortunately, this model is incomplete because examples of AR-independent CaP do exist. Most notably, the well established human CaP cell lines DU145 and PC3, which were derived from brain and bone metastases, respectively (4, 5), are supposedly devoid of AR mRNA and protein (6, 7) and thus represent bona fide models of AR-independent CaP. Interestingly, this classification was recently called into question as detectable levels of AR mRNA and protein were observed in both DU145 and PC3 cells (8). More importantly, several studies have demonstrated low AR activity in PC3 cells, suggesting these cells also utilize AR-dependent mechanisms for growth and survival similar to other established AD CaP cell lines (e.g. LNCaP, 22Rv1, and C4-2) (8 -11).AR displays variable immunoreactivity in individual tumor cells of AI CaP, with cells showing a strong, weak, or undetectable AR signal (12). These results illustrate the heterogeneity of AR expression in AI CaP and suggest that both AR-dependent and AR-independent mechanisms of growth and survival are present in AI CaP. Practically speaking, determining whether AI CaP utilizes AR-dependent or AR-independent mechanisms for growth and survival is a controversial and arduous endeavor that has profound clinical ramifications on how to treat advanced stage, AI CaP. Here we have investigated whether DU145 and PC3 cells are representative models of AR-independent CaP by testing if they respond to androgens or require AR expression for cell growth in vitro. We show that DU145 and PC3 cells are androgen-responsive and require AR for optimal growth in vitro, thus demonstrating each cell line is a bona fide model of androgen-responsive, AR-dependent CaP. We show AR gene transcripts in DU145 and PC3 cells harbor nucleotide transitions suggesting the AR pre-mRNA is the target of multiple RNA editing enzymes. We propose that RNA editing enzymes are modulators of AR activity through the introduction of loss-of-function or gain-of-function mutations into AR gene transcripts in advanced stage AI CaP.