Ubiquitin-mediated Proteolysis

Protein degradation is a commonly employed mechanism for the control of protein abundance. It is a particularly effective method for promoting unidirectional cell cycle transitions because of its rapidity and irreversibility. Three major cell cycle transitions, entry into S phase, separation of sister chromatids, and exit from mitosis, require the degradation of specific proteins via the ubiquitin-26S proteasome pathway. The formation of ubiquitin-protein conjugates involves three components that participate in a cascade of ubiquitin transfer reactions: a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a specificity factor (E3). Ubiquitin is activated as a thiol-ester on E1 in an ATP dependent reaction, transferred to an E2 as a thiol ester and, ultimately, conjunction with an E3, which functions in substrate recognition and in some instances may serve as a thiol-ubiquitin carrier. Together, these enzymes polyubiquitinate lysine residues in target proteins through formation of isopeptide bonds with ubiquitin, leading to recognition by the 26S proteasome and protein destruction.

We together with Dr. Wade Harper's lab, have identified a new component of the machinery cells use to destroy proteins in a regulated manner, the SCF (Skp-Cullin-F-box). The SCF complex is an E3 ubiquitin ligase that targets proteins for ubiquitin-mediated proteolysis. The SCF contains a scaffolding protein Cdc53 (or Cullin) that acts as an adaptor to recruit an E2 ubiquitin conjugating enzyme. Cdc53 also binds to Skp1 which recruits F-box proteins into the complex. Proteins containing the F-box motif are the specificity factors that recognize substrates, many in a phosphorylation dependent manner. We have shown that in vitro the SCF is capable of ubiquitinating phosphorylated Sic1, a Cdk inhibitor. SIc1 is phosphorylated by Cln/Cdc28 complexes and phosphorylated Sic1 can now bind to the F-box protein Cdc4 and become ubiquitinated. There are a large number of F-box proteins and therefore many different SCF complexes, and they have been implicated in a wide variety of pathways including cell cycle progression (Sic1, Far1, Cln, Cdc6 degradation), catabolite repression, sulfur metabolism, NF-kB regulation, Hedgehog signaling, Notch signaling, Wnt signaling, floral development, etc. The SCF pathway may be the central pathway through which protein kinases control substrate stability and may also regulate ubiquitination of certain proteins in a phosphorylation-independent manner.