2A (left panel), MycPKC was pulled down with both active RdxE (RdxT564E) and, to a minor extent, inactive RdxA (RdxT564A)

2A (left panel), MycPKC was pulled down with both active RdxE (RdxT564E) and, to a minor extent, inactive RdxA (RdxT564A). of PDK1, PKC, and radixin. A9 cells grown on spot slides were infected (or not) with Patchouli alcohol CsCl-purified MVM (30 pfu/cell) and examined 36 h p.i. by Patchouli alcohol confocal laser scanning microscopy to confirm colocalization of PDK1 (green), PKC (red), and Rdx (blue). Colocalization appears white in the merge and was quantified with Image J software. Scale bars: 30 and 15 m, as indicated. To test this hypothesis, we first examined whether Rdx or other ERM-family proteins might interact physically with PKC and modulate its activity. A9 cells and derivatives expressing Myc-tagged PKC (MycPKC), Hexarelin Acetate either alone or in the presence of a Flag-tagged ERM variant, were infected with MVM and harvested 24 h post-infection. Complexes containing Flag-tagged ERM were recovered by immunoprecipitation with anti-Flag and tested for the presence of MycPKC by western blotting with anti-Myc. As shown in Fig. 2A (left panel), MycPKC was pulled down with both active RdxE (RdxT564E) and, to a minor Patchouli alcohol extent, inactive RdxA (RdxT564A). No MycPKC was detected in the absence of recombinant Flag-ERM or in the presence of Flag-Ez or Flag-Moe. The specificity of the interaction was confirmed with the reverse co-immunoprecipitation assay with Myc (Fig. 2A right panel). While immunoprecipitation with MycPKC was able to capture significant amounts of endogenous Rdx, only minor quantities were detected in absence of Myc-tagged proteins or MycCKII. PKC thus appears to bind specifically to Rdx in MVM-infected A9 cells. We next tested how this binding might affect the properties of PKC. First, MVM-infected A9 cells and derivatives expressing dominant-negative RdxA were harvested 24 and 48 hours post-infection and autophosphorylation of endogenous PKC at T655 was measured by western blotting with an antibody against PKC:phosphoT655 (Fig. 2B). A cell line expressing dominant-negative PKC (TA: PKCT512A) served as control. Both the control cells and the RdxA-expressing cells showed a strongly reduced level of PKC:phosphoT655, indicating that the Rdx-PKC interaction controls the activity of PKC. Next, to see if Rdx binding to PKC might influence the substrate specificity of the kinase, we performed phosphorylation assays followed by tryptic phosphopeptide profiling. For this, a purified non-phosphorylated recombinant peptide, either PDK1N446 (aa 1C446) or NS1C (aa 545C672) used as control, was incubated with PKC and 32P-ATP in the presence or absence of purified functionally active Rdx (Fig. 2C). Whichever fragment was used, some 32P-labeled peptides appeared only when Rdx was included in the reaction. Taken together, these results suggest that Rdx acts as an adaptor to control PKC activity and substrate specificity and further support our hypothesis that in the perinuclear area, a PKC/Rdx complex mediates PDK1 phosphorylation and upregulation. Open in a separate window Fig 2 Rdx interacts with PKC and controls its activity and substrate specificity.(A, B) A9 cells and derivatives expressing the gene encoding the indicated variant protein under the control of the NS1-inducible P38 promoter were infected with MVM (30 pfu/cell) and analyzed at the indicated times p.i. (A) Rdx interacts physically with PKC inside cells. Left panel: Cell lines expressing MycPKC (PKC) alone or together with Flag-tagged CKIIE81A (CKII), RdxT564A(Rdxa), RdxT564E (RdxE), EzT566A (EzA), EzT566E (EzE), or MoeT547A (MoeA), were harvested 36 h p.i. Co-immunoprecipitation assays were performed under non-denaturing conditions with mouse monoclonal Flag-tag-specific M2 antibodies. Immunoprecipitates (IPFlag) and, for comparison, whole-cell lysates (Lys) were analyzed by western blotting with rabbit anti-Myc antibodies to detect MycPKC. The percentage of Flag-positive cells in these lines was determined by immunofluorescence with M2 antibodies (% Flag+ cells). Arrows indicate the position of MycPKC in CoIPs. n.d. stands for not determined. Right panel: A9, and cell lines expressing MycPKC or MycCKII were harvested 36 h p.i. Co-immunoprecipitation assays were performed under non-denaturing conditions with anti-Myc antibodies. Immunoprecipitates (IPMyc) and, for comparison, whole-cell lysates (Lys) were analyzed by western blotting with goat anti-Rdx antibodies to detect endogenous radixin. The percentage of Myc-positive cells in these lines was determined by immunofluorescence with anti-Myc antibodies (% Myc+ cells). Arrows indicate the position of Rdx in CoIPs (B) Rdx controls the activity of PKC in MVM-infected A9 cells. A9 cells and derivatives expressing dominant-negative PKCT512A (TA) or RdxT564A (RdxA) were harvested at the indicated times p.i. and analyzed by western blotting. As a measure of endogenous PKC activity, the amount of PKC auto-phosphorylated at T655 (P655) was estimated as compared to the total amount of the kinase (PKC). The loading control was -tubulin (Tub). (C) Radixin controls the substrate specificity of PKC..