HUMAN CUL1 FORMS AN EVOLUTIONARILY CONSERVED UBIQUITIN LIGASE COMPLEX(SCF) WITH SKP1 AND AN F-BOX PROTEIN

The SCF ubiquitin ligase complex of budding yeast triggers DNA replica tion by catalyzing ubiquitination of the S phase cyclin-dependent kina se inhibitor SIC1. SCF is composed of three proteins-ySKP1, CDC53 (Cul lin), and the F-box protein CDC4-that are conserved from yeast to huma ns. As part of an effort to identify components and substrates of a pu tative human SCF complex, we isolated hSKP1 in a two-hybrid screen wit h hCUL1, the closest human homologue of CDC53, Here, we show that hCUL 1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-bos protein SKP2 in vitro, forming an SCF-like partic le, Moreover, hCUL1 complements the growth defect of yeast cdc53(ts) m utants, associates with ubiquitination-promoting activity in human cel l extracts, and can assemble into functional, chimeric ubiquitin ligas e complexes with yeast SCF components. Taken together, these data sugg est that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to tho se described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 prote ins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of t he hSKP1 and hCUL1 proteins.