COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system

Abstract

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.

Fig. 1. Full-length p53wt is phosphorylated at the sequence-specific DNA-binding domain by purified CSN. (A) Recombinant His₆-tagged, full-length p53wt and Δp53(1–154) were incubated in the presence of purified human CSN and [γ-³²P]ATP. Curcumin and Δp53(145–164) were added to assays with p53wt at final concentrations of 50 and 200 µM, respectively. For Δp53(145–164), a K_i of ∼40 µM was estimated. After 60 min, the reaction mixture was separated by SDS–PAGE and stained with Coomassie. The dried gels were analyzed by autoradiography. (B) Competition of CSN-dependent phosphorylation of full-length p53wt with increasing amounts of Δp53(1–154) using kinase assay conditions as in (A). SDS–poly acrylamide gels were stained with Coomassie, dried, and ³²P-labeled p53 was visualized by autoradiography. (C) Functional domains of p53 and the major phospho-peptide identified by mass spectrometry and peptide sequence analysis. Phospho-peptide analysis was carried out as described in Materials and methods. The putative phosphorylation sites Ser149, Thr150 and Thr155 indicated are phosphorylated in Δp53(145–164).

Fig. 2. p53wt binds to the purified CSN complex. (A) Recombinant p53wt tightly associates with purified human CSN complex in glycerol gradients. Purified CSN and His₆-tagged p53 were mixed in a molar ratio of ∼1:1 and the mixture was analyzed by glycerol gradient centrifugation (p53+CSN). In control gradients, p53wt alone was centrifuged. The gradients were fractionated and analyzed by western blotting with an anti-p53 antibody (Anti-p53). After stripping, the same blots were re-probed with a CSN-specific antibody (Anti-CSN2). p53 alone sediments at 200 kDa, indicating that most tumor suppressor is oligomerized. (B) Electron microscopy of gold-labeled p53 alone (p53 alone) and of purified CSN complexes incubated with gold-labeled p53 (p53+CSN). The sections of electron micrographs shown were recorded at 2 µm defocus. About one-third of the CSN complexes are labeled with p53–gold particles (black arrowhead). Free CSN complexes (white arrowhead) show a wide variability in shapes, as well as diffuse boundaries. A gallery of extracted p53–gold-labeled CSN complexes is shown.

Fig. 3. p53wt and Δp53(1–154) bind to the CSN subunit 5/Jab1. (A) Far-western blots performed with immobilized, recombinant CSN subunits. Recombinant subunits used were separated by SDS–PAGE and stained with Coomassie. The same proteins were immobilized on nitrocellulose and incubated with p53wt. After washings, the blots were tested with an anti-p53 antibody (Anti-p53). (B) Far-western blots performed with immobilized p53wt and Δp53(1–154). ΔCSN3(111–403) was used as a negative control. Immobilized proteins were incubated with recombinant CSN5/Jab1. The blots were tested with a specific anti-CSN5 antibody (Anti-CSN5). To avoid false-positive interactions, blots were stripped and re-probed with the same antibody. All specific interactions disappeared after stripping (data not shown). (C) Pull-down assays with p53wt or Δp53(1–154) and in vitro translated, ³⁵S-labeled CSN5/Jab1. CSN5/Jab1 was translated in reticulocyte lysate using a CSN5 cDNA-pcDNA3.1 construct possessing a T7 promotor and coding for an N-terminal Flag tag (translated JAB1). The occurrence of three different bands might be due to internal starts of translation. Recombinant p53wt, Δp53(1–154) or Mdm2 (control) was bound to Ni-NTA magnetic agarose and incubated with ³⁵S-labeled Jab1-containing lysate. After SDS–PAGE, ³⁵S-labeled Jab1 was visualized by autoradiography. Weak bands seen in the control indicate unspecific binding of CSN5/Jab1. Coomassie of JAB1 denotes recombinant His₆-tagged Jab1 separated by SDS–PAGE and stained with Coomassie. (D) Sequence alignment of the regions of c-Jun (Claret et al., 1996) and p27^Kip1 (Tomoda et al., 1999) that bind to CSN5/Jab1 with Δp53(1–154). The region with the highest homology is shown.

Fig. 4. CSN-specific phosphorylation of p53wt and p53 mutants. The kinase assays with purified CSN and recombinant p53wt or p53 mutants were carried out as outlined in Materials and methods. The putative CSN-specific phosphorylation sites Ser149, Thr150 and Thr155 were mutated to alanine or valine as indicated by site-directed mutagenesis (see Materials and methods). The intensity of p53S149A, T150V, T155V phosphorylation was ∼25% of that with p53wt as estimated by densitometry using Aida 2.1.

Fig. 5. Impact of CSN-specific phosphorylation on Ub–26S proteasome-dependent p53 degradation in reticulocyte lysate. (A) E6-dependent degradation of p53wt is inhibited by lactacystin (20 µM), curcumin (50 µM) and Δp53(145–164) (200 µM). p53T155V, p53S149A, T150V, T155V and Δp53(1–154) mutants are stabilized against HPV E6- and proteasome-dependent degradation. p53wt and p53 mutant cDNAs were in vitro translated in the presence of [³⁵S]methionine and degradation assays were performed as outlined in Materials and methods. ³⁵S-labeled p53wt or p53 mutant proteins were visualized by autoradiography. (B) Binding to Mdm2 is reduced with p53 mutants possessing a substitution of Thr155 to valine. Pull-down assays were performed with in vitro translated, ³⁵S-labeled p53wt or p53 mutants and with recombinant, unlabeled Mdm2 as described in Materials and methods. As control, recombinant Mdm2 and p53wt were separated on an SDS–polyacrylamide gel, which was stained with Coomassie (Coomassie of Mdm2 and of p53). The negative control without Mdm2 (p53 without Mdm2) was obtained by incubating ³⁵S-labeled, in vitro translated p53wt with magnetic beads in the absence of recombinant Mdm2. Radioactive p53wt or p53 mutants bound to Mdm2 were visualized by autoradiography. Binding of p53T155V to Mdm2 is approximately four times less than that of p53wt as estimated by densitometry using Aida 2.1.

Fig. 6. p53T155V and p53S149A, T150V, T155V are stabilized in HeLa and HL 60 cells and induce p21. (A) HeLa cells and HL 60 cells were transiently transfected with vector alone (control), p53wt, the double mutant p53S149A, T150V, the mutant p53T155V or the triple mutant p53S149A, T150V, T155V expression constructs. Lysates of HeLa and HL 60 cells were tested 24 h after transfection by western blotting using an anti-p53 antibody (Anti-p53). The same protein amounts were loaded as estimated by Bradford assay. As expected, there is no p53 in HL 60 cell lysates transfected with the vector alone and the endogenous p53 levels in HeLa cells are not detectable. As estimated by densitometry using Aida 2.1, p53T155V and p53S149A, T150V, T155V proteins accumulate up to five times in HeLa and more than twice in HL 60 cells as compared with p53wt or p53S149A, T150V proteins. (B) p53wt and p53 mutants transfected into HL 60 cells induce the cyclin-dependent kinase inhibitor p21. Lysates of HL 60 cells obtained 24 h after transfection were tested by western blotting using an anti-p21 antibody (Anti-p21). There is no p21 seen after transfecting HL 60 cells with the vector alone (control). (C) Immunoprecipitation of in vitro translated ³⁵S-labeled p53wt in the absence (p53wt) and presence of curcumin (+Curcumin) and of p53 mutants by the conformation-specific anti-p53 antibodies PAb 1620 and PAb 240. The immunoprecipitates were analyzed by SDS–PAGE and autoradiography.

Fig. 7. Inhibition of CSN-specific phosphorylation leads to accumulation of endogenous p53 in HeLa and MCF-7 cells. At 6 h after curcumin treatment (+Curcumin), endogenous p53 increased dramatically in HeLa cells as compared with cells incubated without curcumin (–Curcumin). At the same time, the transcription factor c-Jun (Anti-c-Jun) was degraded, indicating an intact Ub–26S proteasome system. In MCF-7 cells, endogenous p53 is stabilized in the presence of curcumin or Δp53(145–164) as compared with untreated cells (control).