Cu14A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome

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(Kipl). Moreover, expression of DDB1 reduces the level of p27(Kipl) by increasing its decay rate. The DDB1-induced proteolysis of p27(Kipl) requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of P27(Kipl) in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p2(Kipl) also involves Skp2, an F-box protein that allows targeting of p27(Kipl) for ubiquitination by the Skpl-Cull-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 Cull-Skpl, utilizes Cu14A-DDB1 to induce proteolysis of p27(Kipl).