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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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The active form of PKD3 is required for prostratin-induced HIV-1 transcription activation. (a) Expression profile of 3 PKDs in HeLa and Jurkat cells. RNA isolated from HeLa or Jurkat cells was analyzed by qRT-PCR for the expression levels of PKD1, PKD2, or PKD3, respectively. Data from 3 independent experiments were averaged and plotted. (b) Effect of shRNA knockdown on prostratin-activated HIV-1 expression in HeLa cells. The cells were cotransfected with HIV-LTR-Luc reporter construct and indicated shRNA for 48 hrs, followed by a 6 hr treatment of 2 μM prostratin as indicated. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of shRNA knockdown on prostratin-activated expression of latent HIV-1 provirus in J-Lat 2D10 cells. The cells were infected with indicated shRNA for 48 hrs, followed by a 16 hr treatment of 0.5 μM prostratin as indicated. The GFP-positive cells were detected by flow cytometry and plotted based on 3 independent experiments as in Figure 1(b). (d) Prostratin activates PKD3 by inducing the phosphorylation at Ser731/735 of its activation loop. HeLa cells transfected with GFP-PKD3 cDNA were treated with 2 μM of prostratin for indicated time. The phosphorylation levels of Ser731/735 of GFP-PKD3 in cell lysates were measured by Western blotting, with the levels of bulk GFP-PKD3 shown below. (e) Effect of PKD3 activity on HIV-1 expression. HeLa cells were cotransfected with HIV-LTR-Luc reporter plus indicated HA-tagged PKD3 constructs for 48 hrs. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0. The expression levels of transfected HA-PKD3 were detected by Western blot with anti-HA antibody and shown at the bottom. V: empty vector; WT: wild-type; CA: constitutively active form of PKD3 containing S731E/S735E mutations; KD: kinase-dead form of PKD3 containing S731A/S735A mutations.
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fig2: The active form of PKD3 is required for prostratin-induced HIV-1 transcription activation. (a) Expression profile of 3 PKDs in HeLa and Jurkat cells. RNA isolated from HeLa or Jurkat cells was analyzed by qRT-PCR for the expression levels of PKD1, PKD2, or PKD3, respectively. Data from 3 independent experiments were averaged and plotted. (b) Effect of shRNA knockdown on prostratin-activated HIV-1 expression in HeLa cells. The cells were cotransfected with HIV-LTR-Luc reporter construct and indicated shRNA for 48 hrs, followed by a 6 hr treatment of 2 μM prostratin as indicated. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of shRNA knockdown on prostratin-activated expression of latent HIV-1 provirus in J-Lat 2D10 cells. The cells were infected with indicated shRNA for 48 hrs, followed by a 16 hr treatment of 0.5 μM prostratin as indicated. The GFP-positive cells were detected by flow cytometry and plotted based on 3 independent experiments as in Figure 1(b). (d) Prostratin activates PKD3 by inducing the phosphorylation at Ser731/735 of its activation loop. HeLa cells transfected with GFP-PKD3 cDNA were treated with 2 μM of prostratin for indicated time. The phosphorylation levels of Ser731/735 of GFP-PKD3 in cell lysates were measured by Western blotting, with the levels of bulk GFP-PKD3 shown below. (e) Effect of PKD3 activity on HIV-1 expression. HeLa cells were cotransfected with HIV-LTR-Luc reporter plus indicated HA-tagged PKD3 constructs for 48 hrs. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0. The expression levels of transfected HA-PKD3 were detected by Western blot with anti-HA antibody and shown at the bottom. V: empty vector; WT: wild-type; CA: constitutively active form of PKD3 containing S731E/S735E mutations; KD: kinase-dead form of PKD3 containing S731A/S735A mutations.

Mentions: PKDs have been implicated as the downstream effectors of novel PKC (nPKC) subfamily [11–13], which has been shown to be involved in prostratin-induced transcription activation of HIV-1. There are three isoforms of PKD with different expression profiles in various human tissues and cell lines. To explore the role of PKDs in prostratin-induced activation of latent HIV-1, we first examined the expression profile of PKDs in HeLa and Jurkat cells. qRT-PCR analysis indicated that PKD2 and PKD3, but not PKD1, are expressed in these two cell lines (Figure 2(a)). Hence, PKD1 was ruled out from the list of candidates.


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)

The active form of PKD3 is required for prostratin-induced HIV-1 transcription activation. (a) Expression profile of 3 PKDs in HeLa and Jurkat cells. RNA isolated from HeLa or Jurkat cells was analyzed by qRT-PCR for the expression levels of PKD1, PKD2, or PKD3, respectively. Data from 3 independent experiments were averaged and plotted. (b) Effect of shRNA knockdown on prostratin-activated HIV-1 expression in HeLa cells. The cells were cotransfected with HIV-LTR-Luc reporter construct and indicated shRNA for 48 hrs, followed by a 6 hr treatment of 2 μM prostratin as indicated. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of shRNA knockdown on prostratin-activated expression of latent HIV-1 provirus in J-Lat 2D10 cells. The cells were infected with indicated shRNA for 48 hrs, followed by a 16 hr treatment of 0.5 μM prostratin as indicated. The GFP-positive cells were detected by flow cytometry and plotted based on 3 independent experiments as in Figure 1(b). (d) Prostratin activates PKD3 by inducing the phosphorylation at Ser731/735 of its activation loop. HeLa cells transfected with GFP-PKD3 cDNA were treated with 2 μM of prostratin for indicated time. The phosphorylation levels of Ser731/735 of GFP-PKD3 in cell lysates were measured by Western blotting, with the levels of bulk GFP-PKD3 shown below. (e) Effect of PKD3 activity on HIV-1 expression. HeLa cells were cotransfected with HIV-LTR-Luc reporter plus indicated HA-tagged PKD3 constructs for 48 hrs. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0. The expression levels of transfected HA-PKD3 were detected by Western blot with anti-HA antibody and shown at the bottom. V: empty vector; WT: wild-type; CA: constitutively active form of PKD3 containing S731E/S735E mutations; KD: kinase-dead form of PKD3 containing S731A/S735A mutations.
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fig2: The active form of PKD3 is required for prostratin-induced HIV-1 transcription activation. (a) Expression profile of 3 PKDs in HeLa and Jurkat cells. RNA isolated from HeLa or Jurkat cells was analyzed by qRT-PCR for the expression levels of PKD1, PKD2, or PKD3, respectively. Data from 3 independent experiments were averaged and plotted. (b) Effect of shRNA knockdown on prostratin-activated HIV-1 expression in HeLa cells. The cells were cotransfected with HIV-LTR-Luc reporter construct and indicated shRNA for 48 hrs, followed by a 6 hr treatment of 2 μM prostratin as indicated. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of shRNA knockdown on prostratin-activated expression of latent HIV-1 provirus in J-Lat 2D10 cells. The cells were infected with indicated shRNA for 48 hrs, followed by a 16 hr treatment of 0.5 μM prostratin as indicated. The GFP-positive cells were detected by flow cytometry and plotted based on 3 independent experiments as in Figure 1(b). (d) Prostratin activates PKD3 by inducing the phosphorylation at Ser731/735 of its activation loop. HeLa cells transfected with GFP-PKD3 cDNA were treated with 2 μM of prostratin for indicated time. The phosphorylation levels of Ser731/735 of GFP-PKD3 in cell lysates were measured by Western blotting, with the levels of bulk GFP-PKD3 shown below. (e) Effect of PKD3 activity on HIV-1 expression. HeLa cells were cotransfected with HIV-LTR-Luc reporter plus indicated HA-tagged PKD3 constructs for 48 hrs. The luciferase activities were plotted based on 3 independent experiments, with the level of cells transfected with empty vector set to 1.0. The expression levels of transfected HA-PKD3 were detected by Western blot with anti-HA antibody and shown at the bottom. V: empty vector; WT: wild-type; CA: constitutively active form of PKD3 containing S731E/S735E mutations; KD: kinase-dead form of PKD3 containing S731A/S735A mutations.
Mentions: PKDs have been implicated as the downstream effectors of novel PKC (nPKC) subfamily [11–13], which has been shown to be involved in prostratin-induced transcription activation of HIV-1. There are three isoforms of PKD with different expression profiles in various human tissues and cell lines. To explore the role of PKDs in prostratin-induced activation of latent HIV-1, we first examined the expression profile of PKDs in HeLa and Jurkat cells. qRT-PCR analysis indicated that PKD2 and PKD3, but not PKD1, are expressed in these two cell lines (Figure 2(a)). Hence, PKD1 was ruled out from the list of candidates.

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