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

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.

Electron microscopy and subunit-subunit interaction studies reveal a first architecture of COP9 signalosome.

The COP9 signalosome is involved in signal transduction, whereas the 26 S proteasome lid is a regulatory subcomplex of the 26 S proteasome responsible for degradation of ubiquitinated proteins. COP9 signalosome and lid possess significant sequence homologies among their eight core subunits and are likely derived from a common ancestor. Surprisingly, from our two-dimensional electron microscopy data, a common architectural plan for the two complexes could not be deduced. None-the-less, the two particles have structural features in common. Both COP9 signalosome and lid lack any symmetry in subunit arrangement and exhibit a central groove, possibly qualified for scaffolding functions.Filter-binding assays with recombinant COP9 signalosome components revealed a multitude of subunit-subunit interactions, supporting the asymmetrical appearance of the complex in electron microscopy. On the basis of two-dimensional images and subunit interaction studies, a first architectural model of COP9 signalosome was created. The fact that four distinct classes of particle views were identified and that only 50 % of the selected particles could be classified indicates a high degree of heterogeneity in electron microscopic images. Different orientations with respect to the viewing axis and conformational variety, presumably due to different grades of phosphorylation, are possible reasons for the heterogeneous appearance of the complex. Our biochemical data show that recombinant COP9 signalosome subunits 2 and 7 are phosphorylated by the associated kinase activity. The modification of COP9 signalosome subunit 2 might be essential for c-Jun phosphorylation. Dephosphorylation does not inactivate the associated kinase activity. Although substrate phosphorylation by COP9 signalosome is significantly decreased by lambda protein phosphatase treatment, "autophosphorylation" is increased.

The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme.

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.