Fig. 1. CK2 and PKD co-purify with the CSN during its preparation from human erythrocytes. (A) Western blots with fractions 21–47 from DEAE column (Seeger et al., 1998). The blots were probed with anti-PKD, anti-5/6-kinase, anti-CK2α and anti-CSN5 antibodies. Fractions 30–40 indicated by arrows were pooled for determination of specific curcumin-sensitive kinase activity [see (E) and Materials and methods] and for further purification. (B) Western blots with fractions 3–18 from a 10 to 40% glycerol gradient using same antibodies as in (A). Fractions 11–14 (arrows) were pooled for further use. (C) Western blots with fractions 28–45 obtained from a RecourceQ column using the same antibodies as in (A). Fractions 36–43 were pooled. (D) SDS–PAGE of 20-µl aliquots of fractions 11 and 12 after MonoQ ion exchange chromatography. Core CSN subunits are indicated. Western blots with the same fractions using anti-PKD, anti-5/6-kinase and anti-CK2α antibodies revealed the presence of CK2α and PKD, but not of 5/6-kinase, in the final CSN preparation. (E) Curcumin-sensitive kinase activity co-purifies with the CSN. Fixing the kinase activity in the lysate to 1, a 18 000-fold co-purification of CSN-associated kinase activity was achieved by the final preparation steps.

Fig. 2. CK2 and PKD are associated with the CSN. (A) Electron microscopy of CSN particles that were incubated with gold-labeled uncleav able ATP analog. The gallery (right panel) shows typical positions of gold clusters mediated by kinases associated with CSN complexes. (B) Immunoprecipitation of the purified CSN was performed with anti-CSN7 antibody. In the left panel purified CSN and recombinant Myc-tagged PKD were mixed and the mixture was immunoprecipitated (IP: anti-CSN7). The western blot revealed the presence of CSN5 (anti-CSN5) and of Myc-tagged PKD in the precipitate. Myc-PKD is the recombinant PKD alone and for control immunoprecipitation protocol was performed without anti-CSN7 antibody, indicating unspecific binding to protein A–Sepharose. In the right panel, purified CSN shown in Figure 1D was immunoprecipitated (IP: anti-CSN7). The two lanes of western blots show two different immunoprecipitations and indicate the presence of CSN5 and of CK2α in the precipitate. The control was performed without anti-CSN7 antibody. (C) Immunoprecipitation of the CSN from HeLa cell lysate using the anti-CSN7 antibody. The left panel shows immunoblots of the precipitates (IP: anti-CSN7). The blot was probed with anti-PKD, anti-Cul1, anti-CK2α and anti-CSN5 antibodies. Control experiments were performed as in (B). Immuno precipitation with pre-immune serum (IP: pre-immune) indicates the specificity of immunoprecipitations. The right panel shows autoradiography of an SDS–PAGE carried out with the reaction mixtures of kinase assays. The anti-CSN7 immunoprecipitate was used as a source of kinase activity and was incubated in the presence of IκBα or c-Jun as a substrate, [γ-³²P]ATP, and with or without curcumin. To demonstrate the specificity of the kinase reaction, the ability of anti-CSN7 immunoprecipitate to phosphorylate recombinant CSN7 or CSN6 was tested. Depending on the exposure time, phosphorylation of heavy chain rabbit IgG was seen (IgG).

Fig. 3. Far-western blots indicate that CK2 and PKD bind to subunits of the CSN. (A) The Ponceau stain of nitrocellulose shows selected recombinant CSN subunits used in far-western blots. A Coomassie Blue-stained SDS–PAGE demonstrates the purified CSN, which was immobilized on nitrocellulose. (B) Recombinant N-terminal Myc-tagged PKD binds to full-length CSN3 of the purified CSN and very weak to ΔCSN3(111–403), as indicated by the anti-Myc antibody. Immobilized recombinant CSN7 is shown as negative control. (C) Immobilized recombinant CSN subunits were incubated with recombinant CK2 consisting of 2α2β. After washing the blots were probed with an anti-CK2α (left panel) or an anti-CK2β antibody (right panel). CSN6 is shown as a negative control.

Fig. 4. CK2 and PKD phosphorylate subunits of the CSN. (A) In vitro kinase assays were performed with shown recombinant CSN subunits and purified CSN as substrates (Coomassie). (B) Each recombinant CSN subunit or purified CSN were incubated with recombinant CK2. Autoradiography shows phosphorylated recombinant CSN subunits labeled with stars. Autoradiography of the phosphorylated CSN complex reveals that phosphorylated CSN7 and autophosphorylated CK2β co-migrate in SDS–PAGE. Complex-bound subunits migrate faster in SDS–PAGE as compared with recombinant His₆-tagged CSN subunits. (C) Kinase reactions were carried out as in (B) using recombinant PKD. Phosphorylated recombinant subunits are indicated by stars. Different proteins exert different effects on autophosphorylation of recombinant kinases. To demonstrate that equal amounts of PKD were added to each sample, Coomassie Blue-stained PKD is shown (lower panel). (D) Subunit CSN2 of endogenous CSN is phosphorylated in reticulocyte lysate. Reticulocyte lysate was incubated with [γ-³²P]ATP. After incubation endogenous CSN was immunoprecipitated and the precipitate was analyzed by SDS–PAGE and autoradiography. Control (CSN): purified CSN was incubated with [γ-³²P]ATP and then analyzed by SDS–PAGE and autoradiography.

Fig. 5. CK2 and PKD phosphorylate c-Jun and p53. (A) Recombinant c-Jun and p53 shown in the Coomassie Blue-stained gel were used for in vitro kinase assays. CK2: recombinant c-Jun or p53 was incubated with recombinant CK2 in presence of [γ-³²P]ATP. After 1 h at 37°C the reaction mix was separated by SDS–PAGE and the dried gel was autoradiographed. The autoradiography shows phosphorylation of c-Jun and p53 as well as autophosphorylation of CK2α and CK2β. PKD: the kinase reaction was carried out with recombinant PKD as described above. The autoradiography shows phosphorylation of c-Jun and p53 as well as autophosphorylation of PKD. (B) Inhibition of CK2-dependent p53 phosphorylation by phosphorylated Δp53(145–164) peptide. Recombinant p53 was incubated with recombinant CK2 as outlined in (A). Phosphorylated Δp53(145–164) was added to the reaction mix at indicated concentrations. (C) CK2 phosphorylates Thr155 of p53. Three different modifications of Δp53(145–164) shown in the upper panel were tested in kinase assays with recombinant CK2. Stars indicate phosphorylated serine (S*) or threonine (T*). After kinase reaction samples were analyzed by 15% SDS–PAGE and autoradiography. (D) Inhibition of CK2-dependent c-Jun phosphorylation by Δc-Jun(59–78). Recombinant c-Jun was incubated with recombinant CK2 as described in (A) and Δc-Jun(59–78) was added at indicated concentrations. Lower panel: Δc-Jun(59–78) was phosphorylated by CK2.

Fig. 6. Ub-dependent degradation of c-Jun is stimulated by inhibitors of CSN-associated kinases. (A) HeLa cells were transiently transfected with c-Jun cDNA in pcDNA3.1 encoding an N-terminal Flag tag. Twenty-four hours after transfection cells were treated with curcumin, DRB, emodin, resveratrol or MG 132. HeLa cell lysate was immunoblotted using anti-Flag antibody. Control: cells treated with 0.25% DMSO. (B) HeLa cells transiently transfected with c-Jun as in (A) were treated with indicated concentrations of curcumin or curcumin (50 µM) and MG 132 (10 µM). Ten percent SDS–PAGE and immunoblots with anti-Flag antibody revealed formation of HMW-c-Jun species. (C) The same samples as in (B) were separated by 7.5% SDS–PAGE, blotted to nitrocellulose and tested with anti-Ub antibody.