-[Home]-

>>[Research]<<

-[Publications]-

-[Lab Members]-

-[PhotoGallery]-

-[Protocols]-

Project III
Genetic Analysis of RING Finger Subunits of E3 Ubiquitin Ligases

Regulated protein destruction is an important way to control progress through the cell cycle. Both positive (e.g. cyclins and E2F) and negative (e.g. cdk inhibitors) effectors of the G1-S transition are subject to this type of regulation. Protein destruction occurs via the ubiquitin-mediated proteolytic pathway in which ubiquitin-conjugated substrate proteins are targeted for destruction by the 26Sproteosome. Addition of ubiquitin to the target protein involves the activity of three specific enzymes, the E1 ubiquitin activating enzyme, the E2 ubiquitin-conjugating enzyme and the E3 ubiquitin ligase. The major role of the E3 is to generate substrate specificity by interacting with specific target proteins. One class of E3 that plays an especially important role at the G1-S transition is the SCF complex, which is composed of at least four proteins: Skp1, Cul1 (Cdc53), an F-box-containing protein, and Roc1 (Rbx1, Hrt1). SCF complexes are evolutionarily conserved among eukaryotes; they are found in organisms from yeast to humans. Roc1 contains a RING finger, a highly conserved domain that binds to two zinc ions and is believed to mediate protein-protein interactions. Roc1 interacts with both the C terminus of Cul1 and with the E2 Cdc34, and this interaction stimulates the ubiquitin ligase activity of the complex. Roc1 is also believed to interact with F-box proteins. The N-terminus of Cul1 contains sequences that interact with Skp1 and Skp1 in turn binds to an F-box protein. F-box proteins contain at least two domains: an F-box responsible for binding to Skp1 and another protein-protein interaction domain (such as WD-40 repeats or leucine rich regions) which is believed to interact with the substrate protein. Numerous F-box proteins exist and thus SCF complexes (which are named based upon their F-box protein, i.e. SCFCdc4) potentially regulate the degradation of many different proteins. Several different E3 ubiquitin ligase complexes (e.g. SCF, APC, and VHL) rely on a RING domain protein. Recent evidence suggests that the Roc1 subunit is an essential part of the core ubiquitin ligase activity, possibly serving to stimulate the activity of E2. We wish to understand the function of these proteins in vivo, and whether they contribute to cell cycle control in Drosophila.
The Drosophila genome encodes 3 Roc-like genes (Roc1a, Roc1b, and Roc2), two of which (a and b) are very similar in amino acid sequence. One important question that can be addressed genetically is why flies possess two similar Roc1 proteins? Two possibilities exist. First, Roc1a and Roc1b may have functional redundancy. The alternative is that these Roc1 proteins may have distinct functions. Perhaps Roc1A and Roc1B have different temporal and/or spatial expression patterns which dictate their activity. Indeed, Roc1a seems to be expressed at constant levels throughout fly development whereas Roc1b expression is more tightly regulated. Another possibility is that Roc1a and Roc1b may interact with different molecules and therefore control the degradation of different sets of substrates. This may be a way of generating an additional level of substrate specificity. Therefore, determining the relative roles of the various Drosophila Roc genes may shed light on the mechanism of action of what can potentially be a large family of related E3 ubiquitin ligases. We plan to address this by comparing/contrasting the phenotypes caused by mutation of Roc1a and Roc1b, the latter which we have recently generated by a novel gene targeting technology.