Protein Kinase D3 Is Essential for Prostratin-Activated Transcription of Integrated HIV-1 Provirus Promoter via NF-κB Signaling Pathway
Figure 5
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 PKC, 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.
