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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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PKD3 plays a crucial role in NF-κB activation. (a) Effect of knocking down PKD3 on prostratin-induced expression of NF-κB-luciferase gene. HeLa cells were cotransfected with 5×-κB-Luc reporter construct and indicated shRNA, followed by prostratin treatment as indicated. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (b) Effect of PKD3 on the expression of NF-κB-driven luciferase gene. HeLa cells were cotransfected with 5×-κB-Luc reporter construct and indicated PKD3 cDNA. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0. WT: wild-type; CA: constitutively active form of PKD3; KD: kinase-dead form of PKD3. (c) Effect of different forms of PKD3 on the expression of mutant HIV-LRT-Luc reporter with κB sites deleted. HeLa cells were cotransfected with indicated HA-PKD3 plus HIV-1 promoter-mutant reporter. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with PKD3-KD set to 1.0. (d) Effect of PKD3 silencing on prostratin-induced nuclear localization of NF-κB. HeLa cells were cotransfected with HA-RelA and shPKD3 and were treated with prostratin for 1 hr. The localization of NF-κB was examined by immunofluorescence detection with anti-HA antibody. The DAPI staining indicated the location of nucleus. (e) A model depicting the signaling pathway for prostratin-induced HIV-1 transcription. The extracellular prostratin first activates PKCε, which leads to the phosphorylation of the activation loop of PKD3. The phosphorylated PKD3 is now active and promotes the nuclear translocation of NF-κB, thereby leading to transcription activation of HIV-1 in a κB element dependent manner.
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fig5: PKD3 plays a crucial role in NF-κB activation. (a) Effect of knocking down PKD3 on prostratin-induced expression of NF-κB-luciferase gene. HeLa cells were cotransfected with 5×-κB-Luc reporter construct and indicated shRNA, followed by prostratin treatment as indicated. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (b) Effect of PKD3 on the expression of NF-κB-driven luciferase gene. HeLa cells were cotransfected with 5×-κB-Luc reporter construct and indicated PKD3 cDNA. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0. WT: wild-type; CA: constitutively active form of PKD3; KD: kinase-dead form of PKD3. (c) Effect of different forms of PKD3 on the expression of mutant HIV-LRT-Luc reporter with κB sites deleted. HeLa cells were cotransfected with indicated HA-PKD3 plus HIV-1 promoter-mutant reporter. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with PKD3-KD set to 1.0. (d) Effect of PKD3 silencing on prostratin-induced nuclear localization of NF-κB. HeLa cells were cotransfected with HA-RelA and shPKD3 and were treated with prostratin for 1 hr. The localization of NF-κB was examined by immunofluorescence detection with anti-HA antibody. The DAPI staining indicated the location of nucleus. (e) A model depicting the signaling pathway for prostratin-induced HIV-1 transcription. The extracellular prostratin first activates PKCε, which leads to the phosphorylation of the activation loop of PKD3. The phosphorylated PKD3 is now active and promotes the nuclear translocation of NF-κB, thereby leading to transcription activation of HIV-1 in a κB element dependent manner.

Mentions: To address the role of PKD3 on NF-κB-dependent transcription activation of HIV-1, we utilized a luciferase reporter construct that contains 5 tandem copies of κB enhancer (NF-κB-Luc). Remarkably, PKD3 silencing blocked the stimulatory effect of prostratin on the reporter expression (Figure 5(a)), indicating that PKD3 is required for this process. Moreover, the overexpression of either wild-type PKD3 or the constitutive active form of PKD3 (PKD3 CA) enhanced the expression of NF-κB-Luc, whereas the kinase-dead mutant (PKD3-KD) did not (Figure 5(b)), indicating that the kinase activity of PKD3 is required for activating NF-κB signaling pathway.


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)

PKD3 plays a crucial role in NF-κB activation. (a) Effect of knocking down PKD3 on prostratin-induced expression of NF-κB-luciferase gene. HeLa cells were cotransfected with 5×-κB-Luc reporter construct and indicated shRNA, followed by prostratin treatment as indicated. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (b) Effect of PKD3 on the expression of NF-κB-driven luciferase gene. HeLa cells were cotransfected with 5×-κB-Luc reporter construct and indicated PKD3 cDNA. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0. WT: wild-type; CA: constitutively active form of PKD3; KD: kinase-dead form of PKD3. (c) Effect of different forms of PKD3 on the expression of mutant HIV-LRT-Luc reporter with κB sites deleted. HeLa cells were cotransfected with indicated HA-PKD3 plus HIV-1 promoter-mutant reporter. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with PKD3-KD set to 1.0. (d) Effect of PKD3 silencing on prostratin-induced nuclear localization of NF-κB. HeLa cells were cotransfected with HA-RelA and shPKD3 and were treated with prostratin for 1 hr. The localization of NF-κB was examined by immunofluorescence detection with anti-HA antibody. The DAPI staining indicated the location of nucleus. (e) A model depicting the signaling pathway for prostratin-induced HIV-1 transcription. The extracellular prostratin first activates PKCε, which leads to the phosphorylation of the activation loop of PKD3. The phosphorylated PKD3 is now active and promotes the nuclear translocation of NF-κB, thereby leading to transcription activation of HIV-1 in a κB element dependent manner.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4127265&req=5

fig5: PKD3 plays a crucial role in NF-κB activation. (a) Effect of knocking down PKD3 on prostratin-induced expression of NF-κB-luciferase gene. HeLa cells were cotransfected with 5×-κB-Luc reporter construct and indicated shRNA, followed by prostratin treatment as indicated. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (b) Effect of PKD3 on the expression of NF-κB-driven luciferase gene. HeLa cells were cotransfected with 5×-κB-Luc reporter construct and indicated PKD3 cDNA. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0. WT: wild-type; CA: constitutively active form of PKD3; KD: kinase-dead form of PKD3. (c) Effect of different forms of PKD3 on the expression of mutant HIV-LRT-Luc reporter with κB sites deleted. HeLa cells were cotransfected with indicated HA-PKD3 plus HIV-1 promoter-mutant reporter. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with PKD3-KD set to 1.0. (d) Effect of PKD3 silencing on prostratin-induced nuclear localization of NF-κB. HeLa cells were cotransfected with HA-RelA and shPKD3 and were treated with prostratin for 1 hr. The localization of NF-κB was examined by immunofluorescence detection with anti-HA antibody. The DAPI staining indicated the location of nucleus. (e) A model depicting the signaling pathway for prostratin-induced HIV-1 transcription. The extracellular prostratin first activates PKCε, which leads to the phosphorylation of the activation loop of PKD3. The phosphorylated PKD3 is now active and promotes the nuclear translocation of NF-κB, thereby leading to transcription activation of HIV-1 in a κB element dependent manner.
Mentions: To address the role of PKD3 on NF-κB-dependent transcription activation of HIV-1, we utilized a luciferase reporter construct that contains 5 tandem copies of κB enhancer (NF-κB-Luc). Remarkably, PKD3 silencing blocked the stimulatory effect of prostratin on the reporter expression (Figure 5(a)), indicating that PKD3 is required for this process. Moreover, the overexpression of either wild-type PKD3 or the constitutive active form of PKD3 (PKD3 CA) enhanced the expression of NF-κB-Luc, whereas the kinase-dead mutant (PKD3-KD) did not (Figure 5(b)), indicating that the kinase activity of PKD3 is required for activating NF-κB signaling pathway.

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