Expression of a Protein Phosphatase 1 Inhibitor, cdNIPP1, Increases CDK9 Threonine 186 Phosphorylation and Inhibits HIV-1 Transcription

Abstract

Cyclin-dependent kinase-9 (CDK9) 3 /cyclin T1 is a protein kinase that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II during transcription elongation (1). CDK9/cyclin T1 is part of positive transcription elongation complex (P-TEFb) and present in the cells within large and small complexes. The large complex contains 7SK RNA (2, 3) and the hexamethylene bisacetamide-induced protein (HEXIM1) (4, 5). The activity of CDK9 in the large complex is inhibited by the interaction with 7SK RNA and HEXIM1 (2-5). In stress-induced cells, CDK9/cyclin T1 dissociates from the 7SK RNA and HEXIM1 protein and then binds to the bromodomain protein 4 (Brd4) (6, 7), a member of bromodomain-containing extraterminal domain protein family that interact with acetylated histones. CDK9/cyclin T1 bound to Brd4 forms the transcriptionally active P-TEFb (7). Brd4 recruits P-TEFb to the promoters of cellular genes that are expressed at the end of mitosis (8). HIV-1 Tat protein can also recruit P-TEFb by binding directly to CDK9/cyclin T1 and targeting it to HIV-1 TAR RNA (7). Hence, Tat induces HIV-1 transcription by recruiting CDK9/cyclin T1 (9 -11) and histone acetyltransferases to the HIV-1 promoter (7,(12)(13)(14). The HIV-1 Tat prevents the formation of the large complex and promotes the dissociation of CDK9/cyclin T1 from 7SK RNA/HEXIM1 complex (15). (20) and in cultured cells (21). PP1 holoenzymes consist of a constant catalytic subunit (PP1␣, PP1␤/␦, or PP1␥) and a variable regulatory subunit that determines the localization, activity, and substrate specificity of the phosphatase (22, 23). One of the major nuclear regulators of PP1 is NIPP1 (nuclear inhibitor of PP1), which inhibits the dephosphorylation of a wide range of PP1 substrates (22, 23). Tat-mediated HIV-1 transcription is blocked by the expression of NIPP1, and the inhibition is reversed by the co-expression of PP1␥ (21). Tat contains a PP1-binding The abbreviations used are: CDK9, cyclin-dependent kinase-9; Brd4, bromodomain protein 4; cdNIPP1, central domain NIPP1; EGFP, enhanced green fluorescent protein; HEXIM1, hexamethylene bisacetamide-induced protein; NIPP1, nuclear inhibitor of PP1; P-TEFb, positive transcription elongation complex; PP1, protein phosphatase 1; PP1C, catalytic subunit of PP1; PP2B, protein phosphatase 2B; PPM1A, protein phosphatase M1A; TAR RNA, transactivation response RNA.

Role of Protein Phosphatase 1 in Dephosphorylation of Ebola Virus VP30 Protein and Its Targeting for the Inhibition of Viral Transcription

“…Second, we recently demonstrated that high doses of 1E7-03 administered to mice by injections do not result in a toxic effect (25). Third, a stable expression of PP1-sequestering cdNIPP1, although inhibitory for HIV-1, did not compromise cell growth (10). Fourth, viable cell lines stably expressing cdNIPP1 (10) and transgenic mice in which genetic PP1 inhibition through induced expression of inhibitor-1 (I-1) resulted in ϳ70% reduction of the PP1 activity (46) were developed.…”

“…We showed that short, 2-hlong, treatment with 100 nM okadaic acid induced VP30 phosphorylation, which has not been noted before. Moreover, we used a specific PP1 inhibitor, cdNIPP1, which we previously demonstrated to be non-toxic when stably expressed in 293T cells (10), to analyze the role of PP1 in VP30 phosphorylation. Expression of cdNIPP1 increased phosphorylation of VP30, suggesting that it is controlled by PP1.…”

“…Third, a stable expression of PP1-sequestering cdNIPP1, although inhibitory for HIV-1, did not compromise cell growth (10). Fourth, viable cell lines stably expressing cdNIPP1 (10) and transgenic mice in which genetic PP1 inhibition through induced expression of inhibitor-1 (I-1) resulted in ϳ70% reduction of the PP1 activity (46) were developed. Therefore, it is unlikely that the compound interferes with interactions of PP1 with its cellular regulatory subunits and its cellular substrates.…”

“…However, okadaic acid is known to preferentially inhibit PP2A over PP1 (21). Thus, to determine whether VP30 is dephosphorylated by PP1 in cultured cells, PP1-binding peptide, cdNIPP (10), was co-expressed with FLAG-tagged VP30 in 293T cells. Expression of cdNIPP significantly increased VP30 phosphorylation as compared with cdNIPP inactive mutant that does not bind PP1 (10) (Fig.…”

Viral–Host Interactions That Control HIV-1 Transcriptional Elongation

“…For example, the interaction of Tat with protein phosphatase PP1 56 , which contributes to stress-induced 7SK snRNP disruption through dephosphorylating Thr186 at the CDK9 T-loop 56 , could conceivably lend a helping hand to Tat in the extraction process. In agreement with this notion, PP1 has been shown to play a positive role in Tat transactivation and HIV replication 57 .…”