Protein Kinase D3 Is Essential for Prostratin-Activated Transcription of Integrated HIV-1 Provirus Promoter via NF-κB Signaling Pathway
Figure 2
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.
