Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

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. Immunoprecipitation 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 DeltaCSN3(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.     

Degradation of the Id2 developmental regulator: targeting via N-terminal ubiquitination

Degradation of cellular proteins via the ubiquitin-proteasome system (UPS) involves: (i) generation of a substrate-anchored polyubiquitin degradation signal and (ii) destruction of the tagged protein by the 26S proteasome with release of free and reusable ubiquitin. For most substrates, it is believed that the first ubiquitin moiety is conjugated to a epsilon-NH(2) group of an internal Lys residue. Recent findings indicate that for several proteins, the first ubiquitin moiety is fused, in a linear manner, to the free alpha-NH(2) group of the protein. Here, we demonstrate that the inhibitor of differentiation (or inhibitor of DNA binding) 2, Id2, that downregulates gene expression in undifferentiated and self-renewing cells, is degraded by the UPS following ubiquitination at its N-terminal residue. Lysine-less (LL) Id2 is degraded efficiently by the proteasome following ubiquitination. Fusion of a Myc tag to the N-terminal but not to the C-terminal residue of Id2 stabilizes the protein. Furthermore, deletion of the first 15 N-terminal residues of Id2 stabilizes the protein, suggesting that this domain serves as a recognition element, possibly for the ubiquitin ligase, E3. The mechanisms and structural motives that govern Id2 stability may have important implications to the regulation of the protein during normal differentiation and malignant transformation.     

The COP9 signalosome inhibits p27(kip1) degradation and impedes G1-S phase progression via deneddylation of SCF Cul1

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.     

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.     

FHL2通过相互作用抑制Id2的功能活性

分化抑制蛋白2（Id2）通过抑制碱性螺旋-环-螺旋（bHLH）类转录因子的功能活性调控多种组织细胞的分化发育,并参与人类多种肿瘤的发生与进展.Id2相互作用蛋白可能调控其翻译后的功能活性．本研究以HLH结构域缺失的Id2作为诱饵蛋白,采用酵母双杂交方法对MCF-7 cDNA文库进行筛选,识别了1个新的Id2相互作用蛋白FHL2 (属于LIM蛋白家族的一员),哺乳动物双杂交实验系统验证了Id2与FHL2之间的相互作用,同时证实,该作用不依赖于Id2中的HLH结构域;GST-pulldown、免疫共沉淀方法,进一步证实FHL2/Id2之间的相互作用;免疫荧光共定位实验结果证实,FHL2/Id2相互作用主要发生在细胞核内;共转染实验结果发现,FHL2通过相互作用阻抑了Id2对bHLH类转录因子E47的功能抑制活性．总之,本研究识别了1个新的Id2相互作用蛋白FHL2,通过直接的相互作用,FHL2抑制了Id2的功能活性,FHL2可能参与调控Id2介导的细胞分化与发育过程,并可能参与肿瘤的发生与进展．     

Id 基因在多种肺癌细胞中的表达及意义

目的：研究Id基因在肺癌和永生化支气管上皮细胞中的表达,探讨其在肺癌细胞中表达的意义。方法：利用半定量RT—PCR和Western blot方法检测多种肺癌和永生化支气管上皮细胞中Id1—Id4 mRNA和Id1—Id4蛋白的表达。结果：A549、NCI—H460、NCI—H446、SK—MES—1、Anip973中Id1-Id3 mRNA均高表达,Id1相对表达较强;而AGZY和MP-184中未表达Id1-Id3 mRNA;腺癌细胞均表达了Id4 mRNA,而NCI-H446、SK—MES—1未表达Id4mRNA。A549,NCA—H460,NCA—H446,SK—MES—1,Anip973中Id1,Id2,Id3蛋白均高表达,A549,NCA—H446,Anip973中Id2的表达高于NCA—H460,SK—MES—1;A549,NCA—H460,Anip973有出的高表达,NCI—H446,SK—MES—1无Id4的表达,Id1-Id4在AGZY和MP-184中均耒表达。结论：4种Id基因均作为癌基因在肺癌的发生发展中发挥作用,Id1,Id2,Id3与肺癌细胞的恶性程度以及增殖和转移密切相关,Id4可做为肺腺癌的检测标志物。     

Ubiquitin-dependent proteolysis of the microtubule end-binding protein 1, EB1, is controlled by the COP9 signalosome: possible consequences for microtubule filament stability

The COP9 signalosome (CSN) is a regulatory particle of the ubiquitin (Ub) proteasome system (UPS) consisting of eight subunits (CSN1-CSN8). We show that the CSN stabilizes the microtubule end-binding protein 1 (EB1) towards degradation by the UPS. EB1, the master regulator of microtubule plus ends, controls microtubule growth and dynamics. Therefore, regulation of EB1 stability by the CSN has consequences for microtubule function. EB1 binds the CSN via subunit CSN5. The C terminus of EB1 is sufficient for interaction with the CSN. Dimerization of EB1 is a prerequisite for complex association and subsequent CSN-mediated phosphorylation, as revealed by studies with the EB1I224A mutant, which is unable to dimerize. In cells, EB1 and CSN co-localize to the centrosome, as demonstrated by confocal fluorescence microscopy. EB1 is ubiquitinated and its proteolysis can be inhibited by MG132, demonstrating that it is a substrate of the UPS. Its degradation is accelerated by inhibition of CSN-associated kinases. HeLa cells permanently expressing siRNAs against CSN1 (siCSN1) or CSN3 (siCSN3) exhibit reduced levels of the CSN complex accompanied by lower steady-state concentrations of EB1. In siCSN1 cells, EB1 is less phosphorylated as compared with control cells, demonstrating that the protein is most likely protected towards the UPS by CSN-mediated phosphorylation. The CSN-dependent EB1 stabilization is not due to the CSN-associated deubiquitinating enzyme USP15. Treatment with nocodazole revealed a significantly increased sensitivity of siCSN1 and siCSN3 cells towards the microtubule depolymerizing drug accompanied by a collapse of microtubule filaments. A nocodazole-induced cell-cycle arrest was partially rescued by CSN1 or EB1. These data demonstrate that the CSN-dependent protection of EB1 is important for microtubule function.     

Dysregulation of the ubiquitin-proteasome system by curcumin suppresses coxsackievirus B3 replication

Curcumin (diferuloylmethane), a natural polyphenolic compound extracted from the spice turmeric, has been reported to have anti-inflammatory, antioxidant, and antiproliferative properties by modulating multiple cellular machineries. It inhibits several intracellular signaling pathways, including the mitogen-activated protein kinases (MAPKs), casein kinase II (CKII), and the COP9 signalosome (CSN), in various cell types. It has also been recently demonstrated that exposure to curcumin leads to the dysregulation of the ubiquitin-proteasome system (UPS). Coxsackievirus infection is associated with various diseases, including myocarditis and dilated cardiomyopathy. In searching for new antiviral agents against coxsackievirus, we found that treatment with curcumin significantly reduced viral RNA expression, protein synthesis, and virus titer and protected cells from virus-induced cytopathic effect and apoptosis. We further demonstrated that reduction of viral infection by curcumin was unlikely due to inhibition of CVB3 binding to its receptors or CVB3-induced activation of MAPKs. Moreover, gene silencing of CKII and Jab1, a component of CSN, by small interfering RNAs did not inhibit the replication of coxsackievirus, suggesting that the antiviral action of curcumin is independent of these pathways. Finally, we showed that curcumin treatment reduced both the 20S proteasome proteolytic activities and the cellular deubiquitinating activities, leading to increased accumulation of ubiquitinated proteins and decreased protein levels of free ubiquitin. We have recently demonstrated that the UPS-mediated protein degradation and/or modification plays a critical role in the regulation of coxsackievirus replication. Thus, our results suggest an important antiviral effect of curcumin wherein it potently inhibits coxsackievirus replication through dysregulation of the UPS.     

Intracellular IL-1 receptor antagonist type 1 inhibits IL-1-induced cytokine production in keratinocytes through binding to the third component of the COP9 signalosome

The IL-1 receptor antagonist (IL-1Ra) exists in four isoforms, three of which lack signal peptides and are primarily intracellular proteins. The biologic roles of the intracellular isoforms of IL-1Ra have remained unknown. The objective of these studies was to determine whether the major intracellular isoform of IL-1Ra 18-kDa type 1 (icIL-1Ra1), mediated unique functions inside cells. A yeast two-hybrid screen with HeLa cell lysates revealed specific binding of icIL-1Ra1, and not of the other IL-1Ra isoforms, to the third component of the COP9 signalosome complex (CSN3). This binding was confirmed by Far Western blot analysis, sedimentation on a glycerol gradient, glutathione pull-down experiments, and coimmunoprecipitation. In addition to binding specifically to CSN3, icIL-1Ra1 inhibited phosphorylation of p53, c-Jun, and IkappaB by the crude CSN-associated kinase and of p53 by recombinant protein kinase CK2 and protein kinase D, both associated with CSN3. The biologic relevance of the interaction between icIL-1Ra1 and CSN3 was demonstrated in the keratinocyte cell lines KB and A431, both possessing abundant CSN3. A431 cells exhibited high levels of icIL-1Ra1 but lacked both detectable IL-1alpha-induced IL-6 and IL-8 production and phosphorylation of p38 MAPK. KB cells displayed the opposite pattern which was reversed after transfection with icIL-1Ra1 mRNA. Inhibition of CSN3 or of icIL-1Ra1 production through gene knockdown with specific small interfering RNA in A431 cells each led to an inhibition of IL-1alpha-induced IL-6 and IL-8 production. Thus, icIL-1Ra1 exhibits unique anti-inflammatory properties inside cells through binding to CSN3 with subsequent inhibition of the p38 MAPK signal transduction pathway.     

The Human COP9 Signalosome Protects Ubiquitin-conjugating Enzyme 3 (UBC3/Cdc34) from β-Transducin Repeat-containing Protein (βTrCP)-mediated Degradation

The COP9 signalosome (CSN) is an essential multisubunit complex that regulates the activity of cullin-RING ubiquitin ligases by removing the ubiquitin-like peptide NEDD8 from cullins. Here, we demonstrate that the CSN can affect other components of the ubiquitination cascade. Down-regulation of human CSN4 or CSN5 induced proteasome-mediated degradation of the ubiquitin-conjugating enzyme UBC3/Cdc34. UBC3 was targeted for ubiquitination by the cullin-RING ubiquitin ligase SCF^βTrCP. This interaction required the acidic C-terminal extension of UBC3, which is absent in ubiquitin-conjugating enzymes of the UBCH5 family. Conversely, the UBC3 acidic domain was sufficient to impart sensitivity to SCF^βTrCP-mediated ubiquitination to UBCH5 enzymes. Our work indicates that the CSN is necessary to ensure the stability of selected ubiquitin-conjugating enzymes and uncovers a novel pathway of regulation of ubiquitination processes.     

Serum regulation of Id1 expression by a BMP pathway and BMP responsive element

Immediate early genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP responsive element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad 4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.     

ATM activates p53 by regulating MDM2 oligomerization and E3 processivity

Rapid activation of p53 by ionizing irradiation is a classic DNA damage response mediated by the ATM kinase. However, the major signalling target and mechanism that lead to p53 stabilization are unknown. We show in this report that ATM induces p53 accumulation by phosphorylating the ubiquitin E3 ligase MDM2. Multiple ATM target sites near the MDM2 RING domain function in a redundant manner to provide robust DNA damage signalling. In the absence of DNA damage, the MDM2 RING domain forms oligomers that mediate p53 poly ubiquitination and proteasomal degradation. Phosphorylation by ATM inhibits RING domain oligomerization, specifically suppressing p53 poly ubiquitination. Blocking MDM2 phosphorylation by alanine substitution of all six phosphorylation sites results in constitutive degradation of p53 after DNA damage. These observations show that ATM controls p53 stability by regulating MDM2 RING domain oligomerization and E3 ligase processivity. Promoting or disrupting E3 oligomerization may be a general mechanism by which signalling kinases regulate ubiquitination reactions, and a potential target for therapeutic intervention.     

CASK inhibits ECV304 cell growth and interacts with Id1

Calcium/calmodulin-dependent serine protein kinase (CASK) is generally known as a scaffold protein. Here we show that overexpression of CASK resulted in a reduced rate of cell growth, while inhibition of expression of endogenous CASK via RNA-mediated interference resulted in an increased rate of cell growth in ECV304 cells. To explore the molecular mechanism, we identified a novel CASK-interacting protein, inhibitor of differentiation 1 (Id1) with a yeast two-hybrid screening. Furthermore, endogenous CASK and Id1 proteins were co-precipitated from the lysates of ECV304 cells by immunoprecipitation. Mammalian two-hybrid protein-protein interaction assays indicated that CASK possessed a different binding activity for Id1 and its alternative splicing variant. It is known that Id proteins play important roles in regulation of cell proliferation and differentiation. Thus, we speculate that the regulation of cell growth mediated by CASK may be involved in Id1. Our findings indicate a novel function of CASK, the mechanism that remains to be further investigated.