COP9 signalosome subunit knockdown in K562 provides novel insight into the function and potential regulation of the CSN

Abstract

The co-ordinated degradation of proteins is vital to all aspects of cellular activity. The main mechanism of intracellular protein degradation is the ubiquitin proteasome system (UPS) which labels target proteins with ubiquitin, thereby marking them for degradation by the 26S proteasome. Protein ubiquitination is mediated by three enzymes; E1, E2, E3. The largest family of E3’s is the cullin-RING E3 ubiquitin ligases (CRLs). CRLs require the cyclic addition and removal of a ubiquitin-like protein called NEDD8 to and from the cullin subunit. Removal of NEDD8 (deneddylation) is mediated by the eight subunit COP9 signalosome (CSN; CSN1-8). Cullin deneddylation by the CSN has been demonstrated to prevent the autocatalytic degradation of the substrate recognition subunit (SRS) of CRLs. The CSN has also been shown to associate with deubiquitinase and kinase activity and has thus been identified as a highly conserved key regulator of protein degradation. CSN subcomplexes have also been identified which function in protein degradation, and a direct role for the CSN complex in transcriptional regulation has been posited. Although the COP9 signalosome (CSN) has been studied in human cells, little is known of its role in haematopoietic cells or of any potential contribution to leukaemogenesis. In this study the deneddylase catalytic subunit CSN5 and the non-catalytic subunit CSN2 were knocked down in the human haematopoietic cell line and chronic myeloid leukemia model, K562. Both knockdowns had similar consequences for CRL activity whilst having divergent effects on the levels of SRS mRNA. Knockdown of either subunit also resulted in a common sequential proteasome-dependent loss of SRS proteins, an observation that had not been previously described. Although both knockdowns resulted in reduced cell proliferation followed by significant cell death, the cellular phenotypes and mechanisms of cell death were distinct. CSN5 knockdown was associated with mitotic defects, G2/M arrest, and culminated in apoptosis. In contrast, CSN2 knockdown resulted in autophagy inhibition and non-apoptotic cell death. This is the first time the CSN has been associated with autophagy. CSN2 and CSN5 knockdowns also had divergent effects on the intact CSN complex. CSN2 loss resulted in significant reduction of the intact CSN whilst, for the first time, the intact CSN complex was shown to be retained in CSN5 knockdown cells with loss of only monomeric CSN5. The common effect on CRL activity by either knockdown suggests a common loss of deneddylase activity, which was explained in CSN2 knockdowns with the loss of the intact CSN complex. However, in the case of CSN5 knockdown, in which the intact complex remained, the reason for loss of deneddylase activity is less easy to explain. The results of this study may indicate for the first time that sustained deneddylase activity is dependent on a novel mechanism requiring a pool of CSN5 monomer. Finally, the significance of monomeric CSN5 function loss to the differential phenotype of CSN5 knockdown cells to cells lacking CSN2 was tested by re-expression of both wild type and deneddylase dead CSN5 in a CSN5 knockdown background. Importantly, both approaches rescued the cellular phenotype to the same extent. Overall, the findings of this study provide novel insight into both the function and potential regulation of the CSN complex, whilst further suggesting that the CSN may be a target worthy of investigation in the treatment of chronic myeloid leukaemia