We have previously demonstrated that

We have previously demonstrated that p30II interacts with TIP60 and enhances c-MYC-dependent transcriptional activation and oncogenic potential (Awasthi et al., 2005). However, the molecular mechanism(s) by which p30II cooperates with c-MYC remains to be fully elucidated. Herein, we have shown that p30II is recruited to c-MYC/TIP60/p300 transcriptional complexes on E-box enhancer elements within the endogenous cyclin D2 promoter in HTLV-1-transformed HuT-102 T-lymphocytes using dual-ChIPs (Fig. 1H). p30II induces acetylation of the c-MYC oncoprotein in transfected 293 cells (Fig. 4C); and c-MYC is also strongly acetylated in the HTLV-1-infected T-cell-lines HuT-102 and MJG11 (Fig. 4D). In the Awasthi et al. (2005) study, we demonstrated that amino elastase residues 99–154 of p30II interact with the TIP60 acetyltransferase, which functions as a transcriptional cofactor and has been shown to acetylate the c-MYC protein (Patel et al., 2004; Frank et al., 2003). The specific sites of TIP60-mediated acetylation within c-MYC are yet to be identified (Patel et al., 2004). Furthermore, we found that the acetylation-defective Lys→Arg substitution mutants of c-MYC (R5 and K323R/K417R) are impaired for oncogenic cellular transformation/foci formation with p30II in cotransfected c-myc HO15.19 fibroblasts (Fig. 2A−C).
We did not observe discernable differences in cellular apoptosis induced by wildtype c-MYC or the acetylation-defective c-MYC mutants K323/R/K417R or R5 in the presence of p30II in cotransfected c-myc HO15.19 fibroblasts (Fig. 5A and B). As Datta et al. (2007) have reported that p30II expression inhibits apoptosis induced by genotoxic stress in Camptothecin-treated primary T-lymphocytes transduced with an HTLV-1 ACH proviral clone, we next tested whether c-MYC-acetylation plays a role in the ability of p30II to protect against cell-death induced by DNA-damage-inducing agents. Interestingly, p30II significantly inhibited c-MYC-dependent apoptosis caused by prolonged exposure to BrdU, which induces single-strand DNA breaks (Fig. 5C–F; Ackland et al., 1988). Neither of the acetylation-defective c-MYC mutants resulted in increased apoptosis in BrdU-treated cells (Fig. 5C–F). This could be attributed to an inability of these mutants to restore normal cell-cycle functions and genomic replication in transfected c-myc HO15.19 fibroblasts, as compared to wildtype c-MYC. We also observed that p30II results in an increased number of multinucleate cells in the presence of the genotoxic chemical, etoposide (Fig. 6A and B). These findings collectively agree with our previous results (Awasthi et al., 2005) and those in Datta et al. (2007), as well as the report by Baydoun et al. (2011) that p30II promotes error-prone nonhomologous-end-joining DNA-repair.
Doueiri et al. (2012) have reported a list of proteins detected in the interactomes of S-tagged HTLV-1 p30II and related HTLV-2 p28II proteins, based upon S-tag affinity purification and mass spectrometric/proteomic analyses. Noticeably, however, these studies failed to detect interactions between p30II and TIP60, c-MYC, or other known p30II-binding partners including p300/CBP, CREB, PU.1, and large ribosomal subunit protein L18a (Awasthi et al., 2005; Michael et al., 2006; Zhang et al., 2000, 2001; Ghorbel et al., 2006; Datta et al., 2006; Doueiri et al., 2012). Using the I-TASSER computational protein-folding and structure prediction algorithm (University of Michigan, 〈http://zhanglab.ccmb.med.umich.edu/I-TASSER/〉), we determined that the TIP60-binding domain (aa residues 99–154; Awasthi et al., 2005) of the S-tagged p30II protein is predicted to be misfolded and likely inaccessible, compared to the wildtype p30II protein elastase structure (Supplementary Fig. S1). Thus, albeit intriguing, the significance of the interacting factors identified through this proteomic screen remains to be determined until the biological functionality (e.g., inhibition of Tax-dependent transactivation, cooperation with oncoproteins, or nuclear sequestration of tax/rex mRNA) of the S-tagged p30II protein has been demonstrated (Doueiri et al., 2012).