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

Abstract

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 p27 Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27 Kip1 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 p27 Kip1 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 .