The Drosophila RING-H2 protein Roc1a is involved in recognition and processing of the Hh signal transducer Ci by the SCF E3 Ubiquitin Ligase. M.A. Noureddine 1, T.D. Donaldson 2, S.A. Thacker 1, R.J. Duronio 1,2,3. 1) Dept. of Biology; 2) Lineberger Comprehensive Cancer Center; 3) PMBB, Univ North Carolina at Chapel Hill, NC 27511.
The Skp/Cullin/F-box (SCF) class of E3 ubiquitin ligases mediates the ubiquitination and subsequent destruction of proteins involved in cell cycle control, signal transduction, and development. The Roc1/Rbx1/Hrt1 family of genes encodes a RING-H2 protein that comprises an essential part of the core ubiquitin ubiquitin ligase activity of SCF. Drosophila contains three Roc-like genes. Each of these encodes a protein that together with E1 and UbcH5 can stimulate the formation of polyubiquitin chains in vitro in the absence of any other SCF subunits. A null allele of Roc1a, the ortholog of human and yeast ROC1, causes early larval lethality, indicating that Roc1a provides a non-redundant function during development. Clonal analysis indicates that dRoc1a mutant imaginal cells fail to proliferate normally, but can nevetheless survive and contribute to adult structures. Current data indicate that F-box proteins confer the substrate recognition capability to SCF complexes. Drosophila slimb encodes an F-box protein that was previoulsy shown to be required for Ci and Arm/B-cat proteolysis in the absence of Hh and Wg signaling, respectively. Whereas cells within slimb null mutant clones inappropriately accumulate both Ci and Arm, Roc1a mutant cells accumulate Ci but not Arm. The accumulation of Ci in Roc1a mutant cells results in ectopic activation of Hh signaling as monitored by dpp-lacZ expression, but only in cells far from the source of endogenous Hh signal at the A/P axis. We conclude from these results that slimb and Roc1a have both mutual and distinct targets in vivo, suggesting that individual F-box proteins can assembly into SCF complexes containing different RING-H2 subunits. Consequently, substrate recognition by Drosophila SCF complexes may not be dictated solely by F-box proteins, and may require a contribution from the RING-H2 subunit.