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Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway.

Wang H, Zhu X, Zhu Y, Liu J, Hu X, Wang Y, Peng S, Chen Y, Chen R, Ding F, Liu R - Biomed Res Int (2014)

Bottom Line: Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation.The molecular mechanism of this activation, however, is far from clear.In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China ; Department of Neurobiology, Xuzhou Medical College, Xuzhou, Jiangsu 221009, China.

ABSTRACT
Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus. First, silencing PKD3, but not the other members of PKD family, blocked prostratin-induced transcription of HIV-1. Second, overexpressing the constitutively active form of PKD3, but not the wild-type or kinase-dead form of PKD3, augmented the expression of HIV-1. Consistent with this observation, we found that prostratin could trigger PKD3 activation by inducing the phosphorylation of its activation loop. In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation. Finally, the activation effect of PKD3 on HIV-1 transcription was shown to depend on the presence of κB element and the prostratin-induced activation of NF-κB, as indicated by the fact that silencing PKD3 blocked prostratin-induced NF-κB activation and NF-κB-dependent HIV-1 transcription. Therefore, for the first time, PKD3 is implicated in the transcription activation of latent HIV-1 provirus, and our results revealed a molecular mechanism of prostratin-induced HIV-1 transcription via PKCε/PKD3/NF-κB signaling pathway.

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Prostratin activates PKD3 via PKCε of novel PKC subfamily. (a) Effect of PKC/PKD inhibitor on prostratin-stimulated HIV-1 expression in HeLa cells. HeLa HIV-LTR-Luc cells were pretreated with indicated inhibitor for 1 hr, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (b) Effect of PKC/PKD inhibitor on prostratin-stimulated expression of latent HIV-1 provirus in 2D10 Jurkat cells. 2D10 cells were cotreated with indicated inhibitor plus 0.5 μM prostratin for 16 hrs, followed by flow cytometry assay. The percentage of GFP-positive cells was plotted based on 3 independent experiments. (c) Effect of PKC inhibitors on prostratin-induced PKD3 activation. HeLa cells transfected with GFP-PKD3 were pretreated with indicated kinase inhibitor for 1 hr, followed by 2 μM prostratin treatment for 2 hrs. The phosphorylation levels of Ser731/735 of expressed GFP-PKD3 in cell lysates were analyzed by Western blotting, with the levels of bulk GFP-PKD3 shown at the bottom. (d) Effect of nPKC knockdown on prostratin-induced HIV-1 expression. HeLa cells were cotransfected HIV-LTR-luciferase construct with shRNA for PKCθ or PKCε for 48 hrs, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (e) Effect of nPKCs on PKD3 activation. HeLa cells were cotransfected with GFP-PKD3 plus indicated constitutive active form of PKC construct tagged with V5 (V5-PKC-CA). The phosphorylation levels of Ser731/735 of GFP-PKD3 were analyzed as (c). (f) Effect of knocking down PKD3 on nPKC-stimulated HIV-1 expression. HeLa cells were cotransfected HIV-LTR-Luc reporter plus indicated shPKD3 and PKC-CA constructs. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0.
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fig3: Prostratin activates PKD3 via PKCε of novel PKC subfamily. (a) Effect of PKC/PKD inhibitor on prostratin-stimulated HIV-1 expression in HeLa cells. HeLa HIV-LTR-Luc cells were pretreated with indicated inhibitor for 1 hr, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (b) Effect of PKC/PKD inhibitor on prostratin-stimulated expression of latent HIV-1 provirus in 2D10 Jurkat cells. 2D10 cells were cotreated with indicated inhibitor plus 0.5 μM prostratin for 16 hrs, followed by flow cytometry assay. The percentage of GFP-positive cells was plotted based on 3 independent experiments. (c) Effect of PKC inhibitors on prostratin-induced PKD3 activation. HeLa cells transfected with GFP-PKD3 were pretreated with indicated kinase inhibitor for 1 hr, followed by 2 μM prostratin treatment for 2 hrs. The phosphorylation levels of Ser731/735 of expressed GFP-PKD3 in cell lysates were analyzed by Western blotting, with the levels of bulk GFP-PKD3 shown at the bottom. (d) Effect of nPKC knockdown on prostratin-induced HIV-1 expression. HeLa cells were cotransfected HIV-LTR-luciferase construct with shRNA for PKCθ or PKCε for 48 hrs, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (e) Effect of nPKCs on PKD3 activation. HeLa cells were cotransfected with GFP-PKD3 plus indicated constitutive active form of PKC construct tagged with V5 (V5-PKC-CA). The phosphorylation levels of Ser731/735 of GFP-PKD3 were analyzed as (c). (f) Effect of knocking down PKD3 on nPKC-stimulated HIV-1 expression. HeLa cells were cotransfected HIV-LTR-Luc reporter plus indicated shPKD3 and PKC-CA constructs. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0.

Mentions: To address the role of PKC in activating PKD3 during prostratin-induced transcription activation of latent HIV-1 provirus, both HIV-LTR-Luc and J-Lat 2D10 cells were pretreated with various PKC inhibitors followed by prostratin incubation (Figures 3(a) and 3(b)). Gö6983 which inhibits all three subfamilies of PKC but not PKD1 [29] and Gö6850 which inhibits classic PKC (cPKC) and novel PKC (nPKC) but not atypical PKC (aPKC) subfamily [30] blocked prostratin-induced HIV-1 expression, suggesting that aPKC subfamily is not required for this process. Consistent with the fact that both HeLa and Jurkat cells lack PKD1 expression (Figure 2(a)), the inhibitor Gö6976 which inhibits cPKC and PKD1, but not nPKC [30], did not block prostratin-induced HIV-1 expression but enhanced HIV-1 expression. These data indicated that nPKC subfamily is required for prostratin-stimulated HIV-1 expression.


Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway.

Wang H, Zhu X, Zhu Y, Liu J, Hu X, Wang Y, Peng S, Chen Y, Chen R, Ding F, Liu R - Biomed Res Int (2014)

Prostratin activates PKD3 via PKCε of novel PKC subfamily. (a) Effect of PKC/PKD inhibitor on prostratin-stimulated HIV-1 expression in HeLa cells. HeLa HIV-LTR-Luc cells were pretreated with indicated inhibitor for 1 hr, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (b) Effect of PKC/PKD inhibitor on prostratin-stimulated expression of latent HIV-1 provirus in 2D10 Jurkat cells. 2D10 cells were cotreated with indicated inhibitor plus 0.5 μM prostratin for 16 hrs, followed by flow cytometry assay. The percentage of GFP-positive cells was plotted based on 3 independent experiments. (c) Effect of PKC inhibitors on prostratin-induced PKD3 activation. HeLa cells transfected with GFP-PKD3 were pretreated with indicated kinase inhibitor for 1 hr, followed by 2 μM prostratin treatment for 2 hrs. The phosphorylation levels of Ser731/735 of expressed GFP-PKD3 in cell lysates were analyzed by Western blotting, with the levels of bulk GFP-PKD3 shown at the bottom. (d) Effect of nPKC knockdown on prostratin-induced HIV-1 expression. HeLa cells were cotransfected HIV-LTR-luciferase construct with shRNA for PKCθ or PKCε for 48 hrs, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (e) Effect of nPKCs on PKD3 activation. HeLa cells were cotransfected with GFP-PKD3 plus indicated constitutive active form of PKC construct tagged with V5 (V5-PKC-CA). The phosphorylation levels of Ser731/735 of GFP-PKD3 were analyzed as (c). (f) Effect of knocking down PKD3 on nPKC-stimulated HIV-1 expression. HeLa cells were cotransfected HIV-LTR-Luc reporter plus indicated shPKD3 and PKC-CA constructs. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0.
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fig3: Prostratin activates PKD3 via PKCε of novel PKC subfamily. (a) Effect of PKC/PKD inhibitor on prostratin-stimulated HIV-1 expression in HeLa cells. HeLa HIV-LTR-Luc cells were pretreated with indicated inhibitor for 1 hr, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (b) Effect of PKC/PKD inhibitor on prostratin-stimulated expression of latent HIV-1 provirus in 2D10 Jurkat cells. 2D10 cells were cotreated with indicated inhibitor plus 0.5 μM prostratin for 16 hrs, followed by flow cytometry assay. The percentage of GFP-positive cells was plotted based on 3 independent experiments. (c) Effect of PKC inhibitors on prostratin-induced PKD3 activation. HeLa cells transfected with GFP-PKD3 were pretreated with indicated kinase inhibitor for 1 hr, followed by 2 μM prostratin treatment for 2 hrs. The phosphorylation levels of Ser731/735 of expressed GFP-PKD3 in cell lysates were analyzed by Western blotting, with the levels of bulk GFP-PKD3 shown at the bottom. (d) Effect of nPKC knockdown on prostratin-induced HIV-1 expression. HeLa cells were cotransfected HIV-LTR-luciferase construct with shRNA for PKCθ or PKCε for 48 hrs, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (e) Effect of nPKCs on PKD3 activation. HeLa cells were cotransfected with GFP-PKD3 plus indicated constitutive active form of PKC construct tagged with V5 (V5-PKC-CA). The phosphorylation levels of Ser731/735 of GFP-PKD3 were analyzed as (c). (f) Effect of knocking down PKD3 on nPKC-stimulated HIV-1 expression. HeLa cells were cotransfected HIV-LTR-Luc reporter plus indicated shPKD3 and PKC-CA constructs. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0.
Mentions: To address the role of PKC in activating PKD3 during prostratin-induced transcription activation of latent HIV-1 provirus, both HIV-LTR-Luc and J-Lat 2D10 cells were pretreated with various PKC inhibitors followed by prostratin incubation (Figures 3(a) and 3(b)). Gö6983 which inhibits all three subfamilies of PKC but not PKD1 [29] and Gö6850 which inhibits classic PKC (cPKC) and novel PKC (nPKC) but not atypical PKC (aPKC) subfamily [30] blocked prostratin-induced HIV-1 expression, suggesting that aPKC subfamily is not required for this process. Consistent with the fact that both HeLa and Jurkat cells lack PKD1 expression (Figure 2(a)), the inhibitor Gö6976 which inhibits cPKC and PKD1, but not nPKC [30], did not block prostratin-induced HIV-1 expression but enhanced HIV-1 expression. These data indicated that nPKC subfamily is required for prostratin-stimulated HIV-1 expression.

Bottom Line: Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation.The molecular mechanism of this activation, however, is far from clear.In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China ; Department of Neurobiology, Xuzhou Medical College, Xuzhou, Jiangsu 221009, China.

ABSTRACT
Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus. First, silencing PKD3, but not the other members of PKD family, blocked prostratin-induced transcription of HIV-1. Second, overexpressing the constitutively active form of PKD3, but not the wild-type or kinase-dead form of PKD3, augmented the expression of HIV-1. Consistent with this observation, we found that prostratin could trigger PKD3 activation by inducing the phosphorylation of its activation loop. In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation. Finally, the activation effect of PKD3 on HIV-1 transcription was shown to depend on the presence of κB element and the prostratin-induced activation of NF-κB, as indicated by the fact that silencing PKD3 blocked prostratin-induced NF-κB activation and NF-κB-dependent HIV-1 transcription. Therefore, for the first time, PKD3 is implicated in the transcription activation of latent HIV-1 provirus, and our results revealed a molecular mechanism of prostratin-induced HIV-1 transcription via PKCε/PKD3/NF-κB signaling pathway.

Show MeSH
Related in: MedlinePlus