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, Δp53(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 Δp53(145–164) results in accumulation of endogenous p53.

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNFα-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.

The COP9 signalosome (CSN) is a conserved protein complex that regulates assembly and activity of cullin‐RING ubiquitin ligases (CRLs). Ubiquitin‐dependent degradation of the NF‐κB inhibitor IκBα preceeds nuclear translocation of NF‐κB. For the first time, we show here an inducible interaction of the CSN with IκBα and that the CSN controls IκBα and NF‐κB activity. Strikingly, disruption of the CSN by a small interfering RNA‐mediated knockdown of single CSN subunits results in a reduced re‐accumulation of IκBα and prolonged nuclear translocation of NF‐κB in TNFα‐stimulated cells. The control of IκBα by the CSN is regulated by deubiquitinylation of IκBα conferred by the CSN‐associated deubiquitinylase USP15. Protein expression levels of cullin1 and the CRL substrate adapter β‐TrCP are reduced in nonstimulated cells with a disrupted function of the CSN, which might account for an impaired basal turnover of IκBα. We propose that the CSN controls both CRL activity and stability of the CRL substrate IκBα. In consequence, basal and signal‐induced CRL‐dependent turnover of IκBα is precisely adapted to specific cellular needs.

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c‐Jun and p53 with consequence for their ubiquitin (Ub)‐dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co‐purify with CSN. Immunoprecipi tation and far‐western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold‐labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co‐immuno precipitates with CSN from HeLa cells. CK2 binds to ΔCSN3(111–403) and CSN7, whereas PKD interacts with full‐length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c‐Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN‐associated kinases and induce degradation of c‐Jun in HeLa cells. Curcumin treatment results in elevated amounts of c‐Jun–Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.