Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21CIP1/WAF1 and cyclin D proteins

We have identified the yeast and human homologs of the SKP1 gene as a suppressor of cdc4 mutants and as a cyclin F-binding protein. Skp1p indirectly binds cyclin A/Cdk2 through Skp2p, and directly binds Skp2p, cyclin F, and Cdc4p through a novel structural motif called the F-box. SKP1 is required for ubiquitin-mediated proteolysis of Cin2p, Clb5p, and the Cdk inhibitor Sic1p, and provides a link between these molecules and the proteolysis machinery. A large number of proteins contain the F-box motif and are thereby implicated in the ubiquitin pathway. Different skp1 mutants arrest cells in either G1 or G2, suggesting a connection between regulation of proteolysis in different stages of the cycle.

We have reconstituted the ubiquitination pathway for the Cdk inhibitor Sic1 using recombinant proteins. Skp1, Cdc53, and the F-box protein Cdc4 form a complex, SCFCdc4, which functions as a Sic1 ubiquitin-ligase (E3) in combination with the ubiquitin conjugating enzyme (E2) Cdc34 and E1. Cdc4 assembled with Skp1 functions as the receptor that selectively binds phosphorylated Sic1. Grr1, an F-box protein involved in Cln destruction, forms complexes with Skp1 and Cdc53 and binds phosphorylated Cln1 and Cln2, but not Sic1. Because the constituents of the SCF complex are members of protein families, SCFCdc4 is likely to serve as the prototype for a large class of E3s formed by combinatorial interactions of related family members. SCF complexes couple protein kinase signaling pathways to the control of protein abundance.