Cul4A and DDB1 Associate with Skp2 To Target p27^Kip1 for Proteolysis Involving the COP9 Signalosome

Abstract

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27 Kip1 . Moreover, expression of DDB1 reduces the level of p27 Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27 Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27 Kip1 .The Cul4A gene is amplified and overexpressed in breast and hepatocellular carcinomas (6, 42). Also, Cul4A is essential for mammalian development (18). It encodes a protein of the cullin family. The cullins are central components of several E3 ubiquitin ligases (11). Cul4A associates with the damaged-DNA binding protein DDB (22,32). DDB consists of two subunits: DDB1 and DDB2. The DDB2 subunit is mutated in xeroderma pigmentosum (complementation group E) (reviewed in reference 35). Cul4A participates in the ubiquitination of the DDB2 subunit of DDB and induces its proteolysis through the ubiquitin-proteasome pathway (22). Recent studies indicated that the DDB1 subunit of DDB functions as an adaptor for substrate binding by Cul4A in a manner similar to how Skp1 functions in the Skp1-cullin1-F-box (SCF) complex (15). However, unlike the case for Skp1, there are instances where DDB1 directly targets a substrate without additional adaptor proteins. For example, Cul4A has been implicated in the proteolysis of the replication licensing protein Cdt1 following DNA damage (14, 44). It was shown that the interaction between Cul4A and Cdt1 is mediated by DDB1 (15). In other examples, Cul4A-DDB1 interacts with additional adaptors to target a specific protein. The DDB1-Cul4A complex associates with hDET1, an ortholog of Arabidopsis De-etiolated-1, and hCOP1, an ortholog of Arabidopsis constitutively photomorphogenic-1 (COP1), to induce proteolysis of the c-Jun protein through the ubiquitin-proteasome pathway (40). In that study, the authors proposed that the hDET1-hCOP1 functioned as the heteromeric substrate adaptor and, in keeping with the SCF E3 ligase,...

DDB1 is essential for genomic stability in developing epidermis

“…DDB1 has been recently appreciated as having functions independent of its direct role in DNA damage repair. The DDB1-Cul4A E3 ubiquitin ligase targets for degradation several proteins that regulate cell-cycle progression, including c-Jun, Cdt1, and p27Kip1 (11,12,16,17). We assessed the levels of these proteins by immunoblotting whole-cell extracts of primary keratinocytes isolated from control and DDB1-deficient E17.5 embryos and found a dramatic elevation in the level of c-Jun, but no significant change in Cdt1 and p27Kip1, in the absence of DDB1 (Fig.…”

“…The DDB1-Cul4A ligase has been shown to target a variety of substrates. These substrates include the DNA replication licensing factor Cdt1 (11,12) via additional adaptors PCNA (13) and Cdt2 (14,15), protooncoprotein c-Jun via hDET1 and Cop1 (16), cell-cycle inhibitor p27Kip1 (17), and several histones (18,19), all highlighting the importance of DDB1 in regulating cell cycle and DNA metabolism. Proteomic studies of proteins that interact with DDB1-Cul4A reveal a family of WD40-repeat proteins as substrate-recruiting adaptors for the E3 ligase (15,(20)(21)(22).…”

Control of the Cdc6 replication licensing factor in metazoa: The role of nuclear export and the CUL4 ubiquitin ligase

“…The finding that CUL-4 orthologs in Drosophila and humans also negatively regulate the stability of CDK inhibitors of the CIP/KIP family provides the possibility that the latter pathway is also conserved. [49][50][51][52] What is the Role of CDKs in Regulating DNA Replication in Metazoa?…”

Knockdown of Cul4A increases chemosensitivity to gemcitabine through upregulation of TGFBI in lung cancer cells

“…Thus, TGFBI is a target of the Cul4A E3 ligase complex. Cul4A has been reported to play a role in the ubiquitination and proteolysis of some well-defined tumor suppressors, including p53 (7), NF2 (8), RASSF1A (9), p27 (19), DDB2 (20) and p21 (10,11). To the best of our knowledge, the association of Cul4A and TGFBI has not been previously reported.…”