Protein Kinase D3 Is Essential for Prostratin-Activated Transcription of Integrated HIV-1 Provirus Promoter via NF-κB Signaling Pathway
Figure 3
Prostratin activates PKD3 via PKC 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 PKC or PKC 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.
