Protein Phosphatase 2A Activates the HIV-2 Promoter through Enhancer Elements That Include the pets Site

Abstract

Human immunodeficiency virus type 2 (HIV-2) gene expression is regulated by upstream promoter elements, including the peri-Ets (pets) site, which mediate enhancer stimulation following treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We previously showed that the oncoprotein DEK binds to the pets site in a site-specific manner. In this report, we show that binding to the HIV-2 pets site is modulated by treatment of U937 monocytic cells with TPA, an activator of protein kinase C. TPA treatment resulted in a reduction in the levels of DEK and the formation of a faster migrating pets complex in gel shift assays. We show further that the actions of TPA on pets binding can be duplicated by phosphatase treatment of nuclear proteins and is blocked with okadaic acid, a protein phospatase-2A (PP2A) inhibitor. Finally, we demonstrate that ectopic expression of the catalytic domain of PP2A can activate the HIV-2 enhancer/promoter alone or in synergy with TPA, an effect mediated in part through the pets site. These results suggest that, through an interaction with the protein kinase C pathway, PP2A is strongly involved in regulating HIV-2 enhancer-mediated transcription. This is a consequence of its effects on DEK expression and binding to the pets site, as well as its effects on other promoter elements. These findings have implications not only for HIV-2 transcription but also for multiple cellular processes involving DEK or PP2A.

p300/CBP-associated Factor Drives DEK into Interchromatin Granule Clusters

“…These data indicate that the more acetylated form of DEK has a 3-to 4-fold decrease in affinity for DNA, as compared with less acetylated forms of DEK. These data are consistent with the general observation that TSA activates transcription, and the finding that DEK likely plays a role in transcriptional repression (15,16).…”

“…Acetylation of DEK Decreases Affinity for DNA-We have previously demonstrated that DEK binds to the TG-rich pets site from the HIV-2 promoter and that dephosphorylation of endogenous DEK results in release of DEK bound to this site (14,15). These data indicate that, similar to many other proteins, post-translational modifications may play a significant role in the function of DEK within cells.…”

“…Dissociation Constant Measurements Using Electrophoretic Mobility Shift Assay-Dissociation constant titrations were conducted by incubating immunoprecipitated FLAG-DEK (10-fold on either side of the dissociation constant) with a 32 P-end-labeled DNA probe (5 nM) (sense, 5Ј-TAT ACT TGG TCA GGG CGA ATT CTA ACT AAC AGA-3Ј) containing the pets site (bold letters (15)) at 22°C for 1 h in binding buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5% sucrose w/v; 10 l of total volume). The dependence of binding affinity on buffer ionic strength was assessed by performing a series of binding titrations as a function of NaCl concentration (85 mM to 350 mM).…”

“…Interestingly, DEK has been implicated in all three of these nuclear events. We have previously shown that DEK binds to the TGrich peri-ets (pets) site from the human immunodeficiency virus type 2 (HIV-2) 1 promoter and that this binding is responsive to cellular signals (14,15). In the case of HIV-2, DEK appears to function in transcriptional repression (15).…”

“…We have previously shown that DEK binds to the TGrich peri-ets (pets) site from the human immunodeficiency virus type 2 (HIV-2) 1 promoter and that this binding is responsive to cellular signals (14,15). In the case of HIV-2, DEK appears to function in transcriptional repression (15). Subsequently, DEK has also been isolated in a complex containing both hDaxx and HDAC2, essential proteins involved in transcriptional repression (16), lending further support to the role of DEK in this process.…”