I. previously established, at 48h after 30J and 60J UV irradiation, we got the extracts and added 30mM ATP to aliquots of the extracts prior to incubation at 30C for 1 hour. CUL1 complexes were then immunoprecipitated overnight using an anti-CUL1 antibody, followed by SDS-PAGE and Western blot analysis. Both neddylated and unneddylated CUL1 were detected by the anti-CUL1 antibody. The neddylated CUL1 which migrated slower than the unneddylated CUL1, was produced in an ATP-dependent manner in the extracts and was also recognized by an anti-NEDD8 antibody on Western blot analysis. We found that extracts prepared from cells transfected with DCUN1D3 shRNA were less potent in the cullin1 neddylation reaction than extracts from control cells after UV irradiation (Physique ?(Physique4C).4C). The extracts prepared from cells cotransfected with DCUN1D3 shRNA and CAND1 were further less potent in the CUL1 neddylation reaction (Physique ?(Physique4C).4C). The CUL1-NEDD8 levels were also analyzed by Image J and summarized in the line gragh. DISCUSSION In previous study, we have exhibited that knockdown endogenous expression of DCUN1D3 blocked the S phase progression after UV irradiation [1]. We show that this silence of DCUN1D3 can increase the cyclin-dependent kinase inhibitor p27 protein levels in HeLa cell line after UV irradiation. It has been well that p27 is usually a tumor suppressor, not only because of its activity as a CKI, but also because of evidence from mouse models. In mouse models, p27 functions as a haploinsufficient tumor suppressor and in human cancer, a low level of p27 is usually correlative with higher tumor grade and poor survival [25, 26, 27]. It is also well established that p27 specifically inhibits the activity of Cdk2 in G1-to-S preventing prmature onset of DNA replication [18]. So the accumulation of p27 can cause the G1-to-S phase block in cells. In our obtaining, inhibition of the endogenous DCUN1D3 caused the accumulation of p27 and a significant block of the S phase progression under UV damage. So this result is usually well concerted with the p27’s role in cell cycle progression. p27 is the primary target of SCFSKP2 complex. And SKP2 was originally discovered as a protein that associates with cyclin A-CDK2 in transformed cells, and is now known to be an F-box protein of the SCF complex. In conjunction with SKP2, the SCF complex targets p27 CKIs for degradation [28, 29, 30]. However, in the time since it became widely that SKP2 mediates p27 degradation in G1 phase, several discrepancies have emerged. First, SKP2 is not expressed until early S phase, unequivocally later than the degradation of p27 apparent at G1 [31]. Second, p27 is usually exported from the nucleus to the cytoplasm at G1, whereas SKP2 is restricted to the nucleus. Third, the downregulation of p27 at the G0-G1 transition occurs normally in SKP2?/? cells and is sensitive to proteasome inhibitors [29]. These temporal, spatial and genetic discrepancies indicate that p27 is usually CDK2-IN-4 degraded at G1 in the cytoplasm by a proteasome-dependent, but SKP2-impartial, mechanism. Recently, an E3 enzyme, designated KIP1 ubiquitylation-promoting complex (KPC), that binds with and ubiquitylates p27 in G1 phase and is localized to the cytoplasm of mammalian cells [32]. KPC consists of two subunits, KPC1 and KPC2. KPC1 contains a RING-finger domain name near its C terminus, and functions as the catalytic subunit [33]. KPC2 is usually a member of the UBL-UBA family of proteins. KPC2 stabilizes KPC1, recruits polyubiquitylated p27 and binds with the 26S proteasome, thereby promoting the degradation of p27 [34]. Inhibition of either KPC1 or KPC2 by RNA interference or with dominant-negative mutants delays p27 degradation at the G0-G1 transition. These results RAD21 indicate that p27 is usually degraded by two distinct mechanisms: translocation-coupled cytoplasmic ubiquitylation by KPC at the G0-G1 transition and nuclear ubiquitylation by SKP2 during S and G2 phases. Given that, in a subset of breast cancers (32 out of 84 samples) [35], p27 levels were low despite SKP2 not being expressed, it remains possible that this CDK2-IN-4 expression of KPC might be elevated in such cases. This dynamic degradation pattern of p27 is also in agreement with our previous findings of the dynamic subcellular localization of DCUN1D3. Since the DCUN1D3 protein is usually translocated from the cytoplasm to nucleus after UV damage, it is possible that DCUN1D3 participates the SKP2-mediated proteolysis of p27 in nucleus. In light of the cytoplasmic degradation of p27, more experiments are required to elucidate the role of DCUN1D3 in cytoplasm or whether DCUN1D3 is usually associated with the KPC complex. Through the CoIP experiments, we found DCUN1D3 bound to CAND1, which accords with the previous report [36]. CDK2-IN-4 And CAND1 can augment the S phase CDK2-IN-4 block induced by inhibition.