Figure 4

Phosphorylation of c-Jun and p27 by the CSN immunoprecipitated from siCSN5 cells. (a) Autophosphorylation of CSN subunits CSN2 and CSN7 with CSN immunoprecipitates from control cells (siGFP) and from siCSN5 cells. The CSN was precipitated with the anti-CSN7 antibody. The immunoprecipitate was incubated with ³²P-γ-ATP, separated by SDS-PAGE and the dried gel was exposed to X-ray films. The autoradiography shows phosphorylation of CSN2 and CSN7 in the precipitate from siGFP as well as siCSN5 cells. Although same numbers of cells were used and the amount of the CSN complex is almost identical in control and siCSN cells, there was a slight increase of CSN2 and a decrease of CSN7 and overall phosphorylation with the CSN from siCSN5 cells. The exact reasons for these differences are not known at the moment. (b) Immunoprecipitates from siGFP or siCSN5 cells were used to phosphorylate c-Jun. The upper panel shows the phosphorylation of c-Jun by autoradiography demonstrating a decrease of c-Jun phosphorylation by more than 50% as estimated by densitometry. In the middle panel aliquots were analyzed by Western blotting using the anti-CSN3 antibody to control that equal amounts of the CSN were immunoprecipitated. The lower panel shows the Coomassie stain of c-Jun indicating that the same amounts of the protein were used for kinase assays. (c) The same experiments as performed with c-Jun (see b) were carried out with p27. As seen in the autoradiography the phosphorylation of p27 is significantly reduced with the CSN from siCSN5 cells as compared to the control.