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

Tanya Bondar,^1,, Anna Kalinina,^2, Lyne Khair,¹ Dragana Kopanja,¹ Alo Nag,^1,¶ Srilata Bagchi,^2* and Pradip Raychaudhuri^1*

Department of Biochemistry and Molecular Genetics (M/C 669), University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, Illinois 60607,¹ Center of Molecular Biology of Oral Diseases (M/C 860), College of Dentistry, University of Illinois at Chicago, 801 S. Paulina Ave., Chicago, Illinois 60612²

Received 6 July 2005/ Returned for modification 18 October 2005/ Accepted 6 January 2006

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.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Section of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520.

These two authors made equal contributions to this work.

^¶ Present address: Department of Medicine, Evanston Northwestern Healthcare Research Institute, Evanston, IL 60201.

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