Identification of substrates of the Drosophila F-Box protein Skp2 by flow cytometric and biochemical assays

Thomas Rössler
Proteasomal degradation of proteins is one of many ways for a cell to regulate different processes to achieve cell homeostasis and correct functioning. It is especially important for cell cycle regulation, since it allows for timely, rapid degradation of proteins. Key players of proteasomal degradation are E3 ubiquitin ligases that transfer ubiquitin molecules to the respective substrate proteins, which ultimately lead to recognition by the proteasome and degradation of said proteins. A
more » ... proteins. A prominent E3 ubiquitin ligase that also has important tasks in cell cycle regulation is the SCF-complex. One part of the SCF-complex, the F-Box proteins, fulfill the role of substrate recognition subunits. Skp2 is one of the most characterized F-Box proteins in higher eukaryotes, with a broad range of different substrates. Perhaps the most important are p21, p27 and p57, cyclin dependent kinase inhibitors (CKI) that are responsible for inhibition of the activity of the CycE/Cdk2 complex. These CKIs are thereby exerting a regulatory function in the transition from G1- to S-Phase. In the cell cycle, SCFSkp2 does target the three CKIs for ubiquitination and subsequent proteasomal degradation, resulting in CycE/Cdk2 activity and beginning of S-Phase. Other substrates of Skp2 are for example Cdt1, necessary for the licensing of origin of replications and ordered DNA replication, or CycE, which activates the Cyclin dependent kinase 2. Skp2 in Drosophila melanogaster is poorly characterized, in comparison. Only one substrate has been proposed up to date, the p21, p27 and p57 homologue Dacapo, although the data availability is not definite in this case. This thesis centers on the characterization of Skp2 in Drosophila Schneider cells and the search for new Skp2 interaction partners in the fly. This was achieved by analysis of the cell cycle distribution, live cell imaging experiments, biochemical interaction assays, mass-spectrometric analysis of Skp2 binding partners and protein stability analysis by flow cytometry, which was developed i [...]
doi:10.5283/epub.40986 fatcat:xps33mdc7nacnf4ooiqfksm6gu