Molecular control of the NEMO family of ubiquitin-binding proteins

Molecular control of the NEMO family of ubiquitin-binding proteins. and pro-inflammatory cytokine production. Comparison of tAg binding partners from other human polyomavirus demonstrates that interactions with NEMO and PP4R1 are unique to MCPyV. Collectively, these data identify PP4R1 as a novel target for computer virus subversion of the host anti-viral response. coupled transcription/translation (ITT), and used in GST Retapamulin (SB-275833) pull-down experiments with bacterial expressed GST-tAg [23]. Analysis showed that, in contrast to PP4c, GST-tAg did not bind directly to NEMO (Physique ?(Figure2A).2A). It was possible that tAg might recruit NEMO in a PP4c-dependent manner; therefore, we next decided whether co-incubation with NEMO and PP4c would recapitulate the tAg-NEMO conversation we had observed in cells. GST pull-downs were performed in mixed reactions made up of ITT produced NEMO and PP4c. Even with the presence of PP4c, GST-tAg was not able to bind to NEMO but that this interaction between tAg and NEMO must be mediated through an additional conversation beyond PP4c. Open in a separate window Physique 2 MCPyV tAg interacts with PP4c but not NEMO suggested that a further host protein partner was necessary to allow tAg to complex with NEMO. PP4c has been shown to associate with the non-catalytic protein phosphatase 4 regulatory sub-unit 1 (PP4R1) in the cytoplasm of cells [24]. Given the crucial role of these scaffolding sub-units in phosphatase Retapamulin (SB-275833) function, we set out to determine whether PP4R1 might be required for tAg-mediated inhibition of NF-B. Initially, we evaluated whether endogenous PP4R1 was able to interact with PP4c and NEMO in MCC13 cells. FLAG-tagged versions of PP4c and NEMO were precipitated from MCC13 cells and western blot analysis showed that endogenous PP4R1 interacted with both proteins (Physique ?(Physique3A3A and ?and3B).3B). Next, we explored whether PP4R1 interacts with tAg. Firstly, MCC13 cells were transfected with GFP or GFP-tAg and precipitations performed using GFP-TRAP beads. Results show that endogenous PP4R1 bound to GFP-tAg but not GFP alone (Physique ?(Figure4A).4A). To ensure that the conversation observed was not a result of tAg over-expression, lysates were generated from MKL1 cells, a MCPyV Retapamulin (SB-275833) positive MCC tumour cell line. Lysates were next precipitated with an anti-PP4R1 antibody or a pre-immune IgG control. Tumour expressed tAg was successfully precipitated with endogenous PP4R1 but not with the pre-immune IgG control (Physique ?(Physique4B).4B). These data demonstrate that tAg interacts with PP4R1 in a MCPyV positive MCC cell line. Together, these data provide the first evidence of a viral protein associating with PP4R1. Open in a separate window Physique 3 PP4R1 interacts with PP4c and NEMO in MCC13 cellsMCC13 cells were transfected with vacant plasmid or FLAG-tagged (A) PP4c or (B) NEMO. Immunoprecipitations were performed using FLAG-agarose beads and analyzed by western blot with antibodies against FLAG or endogenous PP4R1. Total cell lysates served as a positive control for expression. Western blots shown are representative of at least three impartial experiments. Open in a separate window Physique 4 PP4R1 is usually a novel tAg binding partner required for NEMO binding(A) GFP-TRAP co-immunoprecipitations were performed on lysates from MCC13 cells transfected with plasmids expressing GFP or GFP-tAg and analyzed by western blot with antibodies against GFP and endogenous PP4R1. Total cell lysates served as a positive control for protein expression and GAPDH as a loading control. (B) MKL1 cell lysates were precipitated with an anti-PP4R1 antibody or a pre-immune IgG control and analyzed by western blot. Samples were probed with antibodies against PP4R1, tAg (2T2) and GAPDH served as a loading control. (C) Equal amounts of bacterially expressed GST and GST-tAg were bound to glutathione-agarose beads and incubated with ITT produced HA-PP4R1 and FLAG-PP4c/NEMO alone or in combination. Following washes, bound proteins were separated by SDS PAGE and probed with antibodies against GST, HA and FLAG. A sample of the ITT input was analyzed to confirm appropriate expression of the epitope-tagged proteins. (D) Equal amounts of bacterially expressed GST and GST-NEMO were bound to glutathione-agarose beads and incubated with ITT produced HA-PP4R1 and FLAG-PP4c/tAg alone or in combination. Following washes, bound proteins were separated by SDS PAGE and probed with antibodies against GST, HA and FLAG. A sample of the ITT input was analyzed to confirm appropriate expression of the epitope-tagged proteins. (E) MCC13 cells were transfected with plasmids expressing GST, GST-NEMO or GST-NEMO D311N in combination with GFP-tAg. Cell lysates were incubated with glutathione-agarose beads and precipitates probed with antibodies against MAP2K7 GST, GFP, PP4c and PP4R1. Total cell lysates served as an expression control and GADPH as a loading control. Western blots shown are representative of.