Regulation of the G1 Phase by the Ubiquitin Pathway

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DESCRIPTION (provided by applicant): SCF ubiquitin ligases complexes, composed of three major subunits, Skp1, Cull, and an F-box protein (Fbp), regulate the proteolysis of many regulators of cell proliferation The substrate specificity of SCFs is determined by distinct Fbp subunits that act as substrate recognition factors. It was demonstrated by the principal investigator that the F-box protein Skp2 is a rate-limiting component of the machinery that ubiquitinylates the cell cycle inhibitor p27, and that Skp2 requires its physical association to Cksl to bind p27 at high affinity. The principal investigator's interest in the ubiquitin pathway lead to the identification of a family of 26 human F-box proteins and to the discovery that one member of this family, called beta-Trcp, regulates the stability of the proto-oncogene beta-catenin. In Aim 1 the regulation and the role of SCFskp2 and Cks1 in the control of the mammalian cell division cycle will be studied. In particular, the function of Skp2 phosphorylation on Ser-76 and the role of an alternatively spliced Skp2, recently identified by the principal investigator, will be studied. In Aim 2, a mutant mouse in which the beta-Trcp locus has been inactivated using homologous recombination will be studied. Using this approach the principal investigator will determine the function of beta-Trcp in growth and development as well as its role in specific tissues and organs. The principal investigator will study how SCF-beta-Trcp regulates processes such as cellular proliferation and the immune response. The principal investigator will be able to determine the in vivo effects of lack of beta-Trcp on its putative substrates. Finally, in Aim 3 the principal investigator will investigate the function of another member of the newly identified human Fbp family, namely Fbl3, which he has found to be localized on the centrosome. Under Aim 3, the principal investigator will also clone and characterize three proteins that he has found associated specifically with Fb13 in vivo.