During formation and growth lens cells must execute a program of proliferation, mitotic withdrawal and differentiation. In most cells, in order to progress from proliferative to differentiated phenotypes, many regulatory and structural proteins (i.e. p57) are removed via the ubiquitin (Ub) proteolytic pathway. Function of the Ub proteolytic pathway is found in all eucaryotic cells and is implicated in virtually every facet of cellular regulation, including cell cycle progression and differentiation, the stress response, DNA repair, signal transduction, gene regulation, control of protooncogenes, cellular remodeling, antigen presentation, and removal of damaged proteins. We have demonstrated the presence of a functional Ub pathway in lens cells and gathered preliminary data which is consistent with the Ub pathway regulating progress from a mitotic to a differentiated phenotype. However, the role of the Ub pathway in regulation of cell cycle and differentiation in lens has been the focus of only three publications, and no specific substrate for the Ub dependent pathway has been identified in a whole lens cell system. Based on our initial publications, preliminary data and data from other cell types, we hypothesize that the Ub proteolytic pathway, particularly ubiquitin conjugating enzymes Ubc2,3,and 4, are required to allow transition of lens cells from a proliferative to a differentiated cell type. We will test this hypothesis using lens explants and in vivo. Our long-term goals are to identify and demonstrate the function of components of the Ub proteolytic pathway, including substrates, which regulate lens cell proliferation and differentiation. The investigators have experience in all of the techniques which are required for this research. In addition, they will have 1) the guidance of Drs. Borras and Zelenka for infection of lens cells with adenovirus, and 2) the contributions of dominant negative constructs of all available Ubc and Ubc knockout mice from Drs. Pagano and Wing. Since there is only a limited literature regarding functions of the Ub pathway in mammalian systems, this work will be of interest to all scholars of mammalian development and differentiation. Dr. Taylor's group is a major contributor to the literature regarding Ub pathway functions in response to cellular stress.
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