EBNA3C Augments Pim-1 Mediated Phosphorylation and Degradation of p21 to Promote B-Cell Proliferation

Abstract

Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, can latently infect the human population. EBV is associated with several types of malignancies originating from lymphoid and epithelial cell types. EBV latent antigen 3C (EBNA3C) is essential for EBV-induced immortalization of B-cells. The Moloney murine leukemia provirus integration site (PIM-1), which encodes an oncogenic serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis. Notably, enhanced expression of Pim-1 kinase is associated with numerous hematological and non-hematological malignancies. A higher expression level of Pim-1 kinase is associated with EBV infection, suggesting a crucial role for Pim-1 in EBV-induced tumorigenesis. We now demonstrate a molecular mechanism which reveals a direct role for EBNA3C in enhancing Pim-1 expression in EBV-infected primary B-cells. We also showed that EBNA3C is physically associated with Pim-1 through its amino-terminal domain, and also forms a molecular complex in B-cells. EBNA3C can stabilize Pim-1 through abrogation of the proteasome/Ubiquitin pathway. Our results demonstrate that EBNA3C enhances Pim-1 mediated phosphorylation of p21 at the Thr145 residue. EBNA3C also facilitated the nuclear localization of Pim-1, and promoted EBV transformed cell proliferation by altering Pim-1 mediated regulation of the activity of the cell-cycle inhibitor p21/WAF1. Our study demonstrated that EBNA3C significantly induces Pim-1 mediated proteosomal degradation of p21. A significant reduction in cell proliferation of EBV-transformed LCLs was observed upon stable knockdown of Pim-1. This study describes a critical role for the oncoprotein Pim-1 in EBV-mediated oncogenesis, as well as provides novel insights into oncogenic kinase-targeted therapeutic intervention of EBV-associated cancers.

The regulatory role of protein phosphorylation in human gammaherpesvirus associated cancers

“…Upon EBV latent infection, not only Pim-1 is required for LMP1-induced cell survival, but both Pim-1 and Pim-2 are also upregulated, and in turn enhance the activity of EBNA2 in driving EBVinduced cell immortalization (Rainio et al, 2005;Kim et al, 2010). Further studies reveal that other latent antigen EBNA3C interacts with and stabilizes Pim-1, which leads to Pim-1-mediated phosphorylation of the Cyclin inhibitor p21 at the threonine 145 residue for promoting B-cell proliferation (Banerjee et al, 2014). Surprisingly, inhibition of ATM/ChK2 instead of ATR/Chk1 will markedly increase EBV-transformation efficiency of primary B cells (Nikitin et al, 2010;Mordasini et al, 2017).…”

Epstein–Barr Virus: Diseases Linked to Infection and Transformation

“…However, only EBNA3A and EBNA3C are essential for viral transformation of B-lymphocytes, and all appear to significantly contribute to maintaining the viability of transformed cells, suggesting an important role in oncogenesis (Tomkinson et al, 1993). EBNA3C has been reported to interact with many cellular factors (Robertson et al, 1995; Choudhuri et al, 2007; Saha et al, 2011, 2015; Banerjee et al, 2013, 2014; Jha et al, 2013a, 2014, 2015a,b,c). One of these cellular antigens is RBP-JK, which binds to all the EBNA3 proteins (Robertson et al, 1995, 1996).…”

“…The available database included cellular players that are significantly associated ( p -value is ∼10 -13 ) with transformation and proliferation of tumor cell lines ( p -value is ∼10 -12 ) ( Figure 2 ). Our lab and others have demonstrated that EBNA1, EBNA2, EBNA3C, and LMP1 are potent EBV antigens associated with a number of transcription factors including E2F1, Tp73, MDM2, Tp53, IRF4, Myc, HDAC1, and GMNN (Robertson et al, 1995, 1996; Kaiser et al, 1999; Ma et al, 2000; Plaxco et al, 2000; Zhou et al, 2000, 2005; Knight et al, 2005; Liu et al, 2005; Saridakis et al, 2005; Choudhuri et al, 2007; Ding et al, 2007; Chau et al, 2008; Allday, 2009; Forte and Luftig, 2009; Borestrom et al, 2012; Frappier, 2012a; Accardi et al, 2013; Banerjee et al, 2013; Tursiella et al, 2014; Jha et al, 2015b,c; Saha et al, 2015), kinases including Aurora Kinase B, Pim1, GSK3B (Saha et al, 2011; Jha et al, 2013a; Banerjee et al, 2014; Edwards et al, 2015), and forms complexes with cellular factors whcih includes the tumor suppressor and oncoproteins, Rb, Skp2, and CyclinD (Arvanitakis et al, 1995; Ruf and Sample, 1999; Prathapam et al, 2002; Knight et al, 2005; Choudhuri et al, 2007; Kang et al, 2011; Saha and Robertson, 2011; Saha et al, 2011, 2015; Sun et al, 2015; Figure 2 ). Cell transformation is regulated through a broad range of interacting partners including Rb, E2F1, Aurora Kinase B, Tp73, Mdm2, Pim1, Tp53, IRF4, Skp2, and Myc (Halazonetis and Kandil, 1991; Lahoz et al, 1994; Hoang et al, 1995; Pirollo et al, 1997; De Laurenzi et al, 1998; Givol et al, 1998; Mochizuki et al, 1999; Lin et al, 2000; Gstaiger et al, 2001; Kanda et al, 2005; Zhang et al, 2005; Xu et al, 2011).…”

Epstein-Barr virus latency: current and future perspectives

“…The recent finding that EBNA3C can bind to and stabilize IRF4 through degradation of its IRF8 partner is consistent with a concept that IRF4 recruits EBNA3C to chromatin (6). Furthermore, the induction of the cellular oncoprotein Pim-1 by EBNA3C results in increased p21 phosphorylation and its degradation mediated by the proteosomal degradation pathway (7). These studies are consistent with a role for EBNA3C in regulating a number of major cellular transcription and cell cycle regulatory factors for driving proliferation and survival of EBV infected cells.…”