Conservation and divergence in the regulation of the RING finger based E3 ubiquitin ligases

Shortly after the appearance of the first eukaryotic organisms approximately 1.5 billion years ago, the cell developed a mechanism for controlling cellular division through the timely destruction of proteins that promote or hinder cell cycle progression. Utilizing a structural motif called the RING finger, a mechanism was perfected that identified target proteins for destruction and then attached a polymeric chain of ubiquitin molecules to serve as the signal for proteolysis by the proteasome. In humans a variant of this mechanism exists as the SCF-ROC1 ubiquitin ligase complex, composed of SKP1, CUL1, an interchangeable F-box protein, and ROC1. I show here that ROC1, a small RING finger protein, is the catalytic component in the human SCF-ROC1 complex. ROC1-CUL1 association is necessary for the ubiquitination catalytic activity of the holoenzyme, and it is responsible for NEDD8 modification of CUL1 and for CUL1 translocation to the nucleus, two steps in the maturation of the enzyme. Remarkably, the same ROC1 protein that functions in the human SCF-ROC1 complex is present in almost identical form in the yeast Saccharomyces cerevisiae. Yeast Roc1 protein associates not only with the homolog of CUL1, but also with the other two yeast cullin family members, Cul3 and Cul8. I report that yeast deleted for cul8 grow slower than wild type, delaying the yeast in anaphase as determined by an increase in large-budded cells, with a disproportionately large number of those cells containing elongated mitotic spindles. Cul8 in association with Roc1 has ubiquitin ligase activity, and ectopic expression of Cul8 rescues the cul8Delta phenotype, but cul8 deficient in binding Roc1 is unable to rescue the cul8Delta phenotype. RING fingers are also present in ubiquitin ligases that combine the substrate-targeting and ubiquitination catalysis functions in a single protein. An example is HDM2, the ubiquitin ligase of p53 tumor suppressor. HDM2 is usually constitutively active ubiquitinating and degrading p53, but it has protein binding partners that modulate its effect on p53 stability. L11 and ARF proteins bind HDM2 to prevent p53 ubiquitination. HDMX binds HDM2 and stabilizes p53 not by preventing its ubiquitination, but by retaining the HDM2-HDMX-p53 complex in the nucleus.