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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-induced HIV-1 transcription depends on NF-κB. (a) Schematics of HIV-1 promoter deletion mutations. dSp1 (without Sp1 binding sites), dEnh (without NF-κB enhancer element), dTAR (without TAR RNA sequences), or dSp1/dEnh (without Sp1 or NF-κB enhancer element). (b) Effect of promoter mutation on prostratin-induced HIV-1 transcription. HeLa cells were transfected with HIV-LTR-Luc reporter constructs containing the indicated promoter deletions, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of prostratin treatment on RelA and Sp1 recruitment to promoter. HIV-LTR-Luc cells were treated with prostratin for 1 hr and subjected to chromatin immunoprecipitation (ChIP) analysis with indicated antibody. The levels of DNA isolated by ChIP were analyzed by quantitative PCR (qPCR) with the primers targeting promoter region of HIV-LTR as indicated on the left and plotted based on 2 independent experiments, with the level of untreated cells set to 1.0. (d) Effect of inhibiting NF-κB signaling on prostratin-stimulated HIV-1 transcription. HIV-LTR-Luc cells were pretreated with inhibitor BAY, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.
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fig4: Prostratin-induced HIV-1 transcription depends on NF-κB. (a) Schematics of HIV-1 promoter deletion mutations. dSp1 (without Sp1 binding sites), dEnh (without NF-κB enhancer element), dTAR (without TAR RNA sequences), or dSp1/dEnh (without Sp1 or NF-κB enhancer element). (b) Effect of promoter mutation on prostratin-induced HIV-1 transcription. HeLa cells were transfected with HIV-LTR-Luc reporter constructs containing the indicated promoter deletions, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of prostratin treatment on RelA and Sp1 recruitment to promoter. HIV-LTR-Luc cells were treated with prostratin for 1 hr and subjected to chromatin immunoprecipitation (ChIP) analysis with indicated antibody. The levels of DNA isolated by ChIP were analyzed by quantitative PCR (qPCR) with the primers targeting promoter region of HIV-LTR as indicated on the left and plotted based on 2 independent experiments, with the level of untreated cells set to 1.0. (d) Effect of inhibiting NF-κB signaling on prostratin-stimulated HIV-1 transcription. HIV-LTR-Luc cells were pretreated with inhibitor BAY, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.

Mentions: A previous report showed that prostratin enhanced nuclear translocation of NF-κB to activate HIV-1 transcription [10]. Since both PKD1 and PKD2 were shown to activate NF-κB [32, 33] and recently PKD2 and PKD3 were reported to activate NF-κB pathway in prostate cancer cells [34], we suspected that prostratin may enhance HIV-1 expression through PKD3's activation effect on NF-κB pathway. To test this, we first examined whether the prostratin's effect relies on the cis-element κB of HIV-1 promoter. Using COP mutagenesis methods [22], the following HIV-1 promoter mutant constructs were made: dSp1 (without Sp1 binding sites), dEnh (without κB enhancer element), dTAR (without TAR sequences), or dSp1/dEnh (Figure 4(a)). These constructs were transiently transfected into HeLa cells followed by the treatment with 2 μM prostratin for 6 hrs, and the cells were harvested for luciferase assay. As shown in Figure 4(b), the deletion of either Sp1 or TAR element did not interfere with the activation effect of prostratin, whereas the deletion of κB enhancer element (dEnh) or the combined deletion of Sp1 and κB enhancer (dSp1/dEnh) reduced the activation effect of prostratin. Hence, the activation effect of prostratin depends on the presence of κB enhancer element of the HIV-1 promoter.


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-induced HIV-1 transcription depends on NF-κB. (a) Schematics of HIV-1 promoter deletion mutations. dSp1 (without Sp1 binding sites), dEnh (without NF-κB enhancer element), dTAR (without TAR RNA sequences), or dSp1/dEnh (without Sp1 or NF-κB enhancer element). (b) Effect of promoter mutation on prostratin-induced HIV-1 transcription. HeLa cells were transfected with HIV-LTR-Luc reporter constructs containing the indicated promoter deletions, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of prostratin treatment on RelA and Sp1 recruitment to promoter. HIV-LTR-Luc cells were treated with prostratin for 1 hr and subjected to chromatin immunoprecipitation (ChIP) analysis with indicated antibody. The levels of DNA isolated by ChIP were analyzed by quantitative PCR (qPCR) with the primers targeting promoter region of HIV-LTR as indicated on the left and plotted based on 2 independent experiments, with the level of untreated cells set to 1.0. (d) Effect of inhibiting NF-κB signaling on prostratin-stimulated HIV-1 transcription. HIV-LTR-Luc cells were pretreated with inhibitor BAY, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.
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Related In: Results  -  Collection

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fig4: Prostratin-induced HIV-1 transcription depends on NF-κB. (a) Schematics of HIV-1 promoter deletion mutations. dSp1 (without Sp1 binding sites), dEnh (without NF-κB enhancer element), dTAR (without TAR RNA sequences), or dSp1/dEnh (without Sp1 or NF-κB enhancer element). (b) Effect of promoter mutation on prostratin-induced HIV-1 transcription. HeLa cells were transfected with HIV-LTR-Luc reporter constructs containing the indicated promoter deletions, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of prostratin treatment on RelA and Sp1 recruitment to promoter. HIV-LTR-Luc cells were treated with prostratin for 1 hr and subjected to chromatin immunoprecipitation (ChIP) analysis with indicated antibody. The levels of DNA isolated by ChIP were analyzed by quantitative PCR (qPCR) with the primers targeting promoter region of HIV-LTR as indicated on the left and plotted based on 2 independent experiments, with the level of untreated cells set to 1.0. (d) Effect of inhibiting NF-κB signaling on prostratin-stimulated HIV-1 transcription. HIV-LTR-Luc cells were pretreated with inhibitor BAY, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.
Mentions: A previous report showed that prostratin enhanced nuclear translocation of NF-κB to activate HIV-1 transcription [10]. Since both PKD1 and PKD2 were shown to activate NF-κB [32, 33] and recently PKD2 and PKD3 were reported to activate NF-κB pathway in prostate cancer cells [34], we suspected that prostratin may enhance HIV-1 expression through PKD3's activation effect on NF-κB pathway. To test this, we first examined whether the prostratin's effect relies on the cis-element κB of HIV-1 promoter. Using COP mutagenesis methods [22], the following HIV-1 promoter mutant constructs were made: dSp1 (without Sp1 binding sites), dEnh (without κB enhancer element), dTAR (without TAR sequences), or dSp1/dEnh (Figure 4(a)). These constructs were transiently transfected into HeLa cells followed by the treatment with 2 μM prostratin for 6 hrs, and the cells were harvested for luciferase assay. As shown in Figure 4(b), the deletion of either Sp1 or TAR element did not interfere with the activation effect of prostratin, whereas the deletion of κB enhancer element (dEnh) or the combined deletion of Sp1 and κB enhancer (dSp1/dEnh) reduced the activation effect of prostratin. Hence, the activation effect of prostratin depends on the presence of κB enhancer element of the HIV-1 promoter.

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