Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein

Abstract

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins-ySKP1, CDC53 (Cullin), and the F-box protein CDC4-that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53 ts mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators͞components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.The irreversible nature of proteolysis makes it well suited to serve as a regulatory switch for controlling unidirectional processes. This principle is clearly evident in the organization of the cell division cycle, where initiation of DNA replication, chromosome segregation, and exit from mitosis are triggered by the destruction of key regulatory proteins (1-3).Proteins typically are marked for proteolytic degradation by attachment of multiubiquitin chains. This process is initiated by a ubiquitin-activating enzyme (E1), which activates ubiquitin by adenylation and becomes linked to it via a thiolester bond. Ubiquitin then is transferred to a ubiquitin-conjugating enzyme, E2. Whereas E2s can attach ubiquitin directly to lysine residues in a substrate, most physiological ubiquitination reactions probably require a ubiquitin ligase, or E3 (4). E3s have been implicated in substrate recognition and, in one case, transfer of ubiquitin from E2 to a substrate via an E3-ubiquitin-thiolester intermediate (5). Once the substrate is multiubiquitinated, it then is recognized and degraded by the 26S proteasome.

The Giardia cell cycle progresses independently of the Anaphase Promoting Complex

“…Indeed, cyclin degradation may be independent of ubiquitination. Our analysis suggests that the SCF complex (Skp1, Cullin, Fbox), a separate multi-enzyme ubiquitin conjugation complex that coordinates cell cycle protein destruction (Skowyra et al, 1997;Lyapina et al, 1998), is also missing from Giardia. The proteasome inhibitor MG262 did not cause cell cycle arrest or appearance of higher molecular weight bands suggesting that no ubiquitin ligases (APC or SCF) are involved in cell cycle regulation in Giardia.…”

Two F‐box/WD‐repeat proteins Pop1 and Pop2 form hetero‐ and homo‐complexes together with cullin‐1 in the fission yeast SCF (Skp1‐Cullin‐1‐F‐box) ubiquitin ligase

“…Recent analysis in higher eukaryotes has identified proteins which have a structure similar to Cdc4, the F-box/WD-repeat proteins including Drosophila Slimb, Caenorhabditis SEL-10 and human ␤TrCP (Hubbard et al 1997;Jiang & Struhl 1998;Margottin et al 1998), although whether these proteins function as SCF to regulate protein degradation remains to be seen. In addition to the F-box/WD-repeat proteins, other components of the SCF complex in higher eukaryotes are beginning to be identified (Ingram et al 1997;Lisztwan et al 1998;Lyapina et al 1998;Ruegger et al 1998).…”

Covalent modification of all members of human cullin family proteins by NEDD8

“…For example, human Cul-1 was found to be a component of an SCF Ub-ligase that regulates multiple cellular functions, such as the G1/S progression of the cell cycle (Lisztwan et al, 1998;Lyapina et al, 1998). Indeed, its yeast homolog Cdc53 was found to be involved not only in the degradation of the CDK (cyclin-dependent kinase) inhibitor Sic1 (Skowyra et al, 1997;Feldman et al, 1997) and the S phase initiator Cdc6 (Drury et al, 1997) but also in gene expression (Li and Johnston, 1997) and methionine biosynthesis (Patton et al, 1998b).…”