These proposed studies are a direct & logical expansion of Specific Aim 4 in the prior grant. O-GlcNAc glycosylation is a ubiquitous form of post-transnational modification that is found on myriad nuclear and cytoplasmic proteins. It is both as abundant and as dynamic as phosphorylation. O-Glc Nac has thus far, clear pore, cytoskeletal, viral chromatin-, and nuclear oncogene-proteins. O-GlcNAc is often attached to Ser(Thr) residues at sights analogous to those used by the 'growth- factor' or proline-specific kinases'. O-GlcNAc is reciprocal with phosphorylation on those proteins studied thus far, suggesting a """"""""yen- yang"""""""" relationship. Shared properties of O-GlcNAc-bearing proteins suggest the saccharide may mediate/modulate reversible subunit-subunit interactions key to tissue-specific transcriptional control & signal transduction.
Specific Aims Are: 1) Continue to elucidate the function(s) O-GlcNAc on the c-myc, v-erbA, & E1A nuclear oncogene proteins. 2) Continue to investigate the role(s) of O-GlcNAc on estrogen receptors in both normal and breast cancer cells. 3) Collaborate on the functions of O-GlcNAc on the Retinoblastoma (Rb) tumor suppressor Protein. 4) Continue to investigate the Role(s) of O-GlcNAc in the infection of cells by human Herpes and Cytomegaloviruses. 5) Determine the Effects of ras Transformation of NIH/3T3 Cells on O-GlcNAc Levels, Distribution & Metabolism. Sites of attachment, stoichiometry, and rates of turnover of O-GlcNAc on each protein will be determined in cancer cells, normal cells and in baculoviral expression systems. Site-directed mutagenesis, enzymatic removal or capping, and inhibitor of O-GlcNAcase or Transferase will be employed to evaluate the roles(s) of O-GlcNAc, using in vitro system that have already been used to study the role(s) of phosphorylation of these same molecules. The involvement of the saccharide in the multimeric associations and transcriptional activities of these oncogenes/suppressor will be evaluated. Using overexpressed glycoproteins, the protein structural consequences of O-GlcNAc addition will be evaluated. Finally, the effects of ras transformation on overall O-GlcNAc metabolism will be systemically evaluated. O-GlcNAc is as abundant and as dynamic as phosphorylation. O-GlcNAc likely provides another (perhaps opposite) level of functional control of important regulatory proteins, and in some cases may be an """"""""off- switch"""""""" for phosphorylation sites. Since phosphorylation is of major importance to the functions of both nuclear oncogenes and tumor suppressor, it is likely that an understanding of O-GlcNAc metabolism may provide an entirely untapped and unanticipated avenue for targeting cancer therapy. Estrogen receptor glycosylation may be particularly relevant to breast cancer etiology.
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