Peth Andreas, Berndt Christoph, Henke Wolfgang, Dubiel Wolfgang
Department of Surgery, Division of Molecular Biology, Charité - Universitätsmedizin Berlin, Monbijoustrasse 2, D-10117 Berlin, Germany.
BMC Biochem. 2007 Dec 19;8:27. doi: 10.1186/1471-2091-8-27.
The COP9 signalosome (CSN) is a conserved protein complex in eukaryotic cells consisting of eight subunits (CSN1 to CSN8). Recent data demonstrate that the CSN is a regulator of the ubiquitin (Ub) proteasome system (UPS). It controls substrate ubiquitination by cullin-RING Ub ligases (CRLs), a process that determines substrate specificity of the UPS. The intrinsic deneddylating activity localized to CSN5 as well as the associated kinases and deubiquitinating activity are involved in the regulatory function of CSN. The exact mechanisms are unclear. In this study we knocked down CSN1 (siCSN1), CSN3 (siCSN3) and CSN5 (siCSN5) by specific siRNA oligos permanently expressed in HeLa cells. The analysis and comparison of siRNA cells revealed differential impact of individual subunits on CSN structure and function.
Permanent knockdowns of CSN1 and CSN3 led to a reduction of the subunits to approximately 40%, which is accompanied by a proportional decrease of the CSN holocomplex. In contrast, downregulation of CSN5 in HeLa cells reduced the CSN5 protein below 20% without significant effects on the remaining complex. The CRL component Rbx1 was characterized by accelerated proteolysis in siCSN1 and siCSN3 and also in siCSN5 cells, however, with lesser extent. Immunoprecipitated CSN complex from siCSN5 cells was less effective in phosphorylating c-Jun and p27. Accelerated degradation of c-Jun in siCSN5 cells was rescued by overexpression of CSN5 as well as of the deneddylation mutant CSN5D151N. Overexpression of CSN5 cannot rescue c-Jun destabilization in siCSN1.
There exists a coordinated downregulation of CSN subunits in the CSN1 and CSN3 knockdowns. The underlying regulatory mechanisms are obscure. CSN5 seems to possess a specific status in HeLa cells. Its reduction is not connected with coordinated downregulation of other subunits. CSN knockdowns confirm that the stabilization of the CRL component Rbx1 is a major CSN function. In addition, downregulation of CSN subunits influences the stability of important cellular regulators such as c-Jun and p27.
COP9信号体（CSN）是真核细胞中一种保守的蛋白质复合体，由八个亚基（CSN1至CSN8）组成。最近的数据表明，CSN是泛素（Ub）蛋白酶体系统（UPS）的调节因子。它通过Cullin-RING Ub连接酶（CRL）控制底物泛素化，这一过程决定了UPS的底物特异性。定位于CSN5的内在去泛素化活性以及相关的激酶和去泛素化活性参与了CSN的调节功能。确切机制尚不清楚。在本研究中，我们通过在HeLa细胞中永久表达的特异性siRNA寡核苷酸敲低了CSN1（siCSN1）、CSN3（siCSN3）和CSN5（siCSN5）。对siRNA细胞的分析和比较揭示了各个亚基对CSN结构和功能的不同影响。
CSN1和CSN3的永久敲低导致亚基减少至约40%，同时CSN全复合体也相应减少。相比之下，HeLa细胞中CSN5的下调使CSN5蛋白降至20%以下，而对其余复合体无显著影响。CRL组分Rbx1在siCSN1和siCSN3以及siCSN5细胞中表现为蛋白水解加速，不过程度较轻。来自siCSN5细胞的免疫沉淀CSN复合体在磷酸化c-Jun和p27方面效果较差。siCSN5细胞中c-Jun的加速降解可通过CSN5以及去泛素化突变体CSN5D151N的过表达得到挽救。CSN5的过表达无法挽救siCSN1中c-Jun的不稳定。
在CSN1和CSN3敲低中存在CSN亚基的协同下调。潜在的调节机制尚不清楚。CSN5在HeLa细胞中似乎具有特殊地位。其减少与其他亚基的协同下调无关。CSN敲低证实了CRL组分Rbx1的稳定是CSN的主要功能。此外，CSN亚基的下调会影响重要细胞调节因子如c-Jun和p27 的稳定性。
