Approaches for examining relevant protein-protein interactions essential for the function of thev-Myc oncoprotein

Nadja Therese Mascarenhas, Purdue University

Abstract

To elucidate and functionally characterize the complex biological role of the Myc oncoprotein in molecular terms, the MC29 v-Myc oncoprotein was examined for relevant protein-protein interactions that are essential for its function. Specific antisera were generated to the Gag region, the carboxy terminal HLH/LZ domain of the v-Myc protein, to Max, a Myc interacting protein, and to B-ATF, a potential Myc influencing protein. These immunological reagents helped study and confirm properties of the v-Myc protein such as size, abundance and cellular location. Immunoprecipitation assays employing in vitro transcribed and translated v-Myc and Max proteins demonstrated a definite v-Myc/Max interaction in vitro. Additional immunoprecipitation assays with a series of v-Myc mutants demonstrated that the integrity of the b/HLH/LZ motif was important in mediating the v-Myc/Max interaction. Interestingly, a result of this study also indicated that subtle changes within Helix 1 could be tolerated in the v-Myc/Max interaction. Studies on the Max protein revealed phosphorylation of this protein occurred in vitro and that such modification influenced the ability of the Max protein to bind Myc variant forms. Analysis of the importance of the v-Myc/Max interaction in terms of DNA binding was carried out. Results indicated that v-Myc, unlike c-Myc, bound DNA efficiently even in the absence of Max, suggesting this could account for the more effective and aggressive transforming ability of the v-Myc oncoprotein. The importance of protein interactions with another functional region of the v-Myc protein, namely, the amino terminal transcription activation domain (TAD) was addressed. The transcription activation potential of several v-Myc TAD deletion mutants were studied using the yeast GAL4 system. Results revealed the inability of the Gag moiety to activate transcription and also documented that amino acids 12-27 within the v-Myc TAD protein had a distinct capacity to activate transcription. Surprisingly, these results demonstrated that a region within the v-Myc TAD (a.a.112-244) negatively regulated the transcription activation potential of the v-Myc protein. Intrigued by this observation, a quest to identify potential Myc interacting, repressor proteins was undertaken. Additional transcription activation studies revealed down regulation of the v-Myc TAD by the p107 protein. The residues potentially targeted within the v-Myc TAD were a.a.1-42 and a.a.90-219. The importance and relevance of such regulation in v-Myc mediated transformation needs to be ascertained. This study addressed questions regarding the v-Myc oncoprotein and analyzed relevant protein-protein interactions that are essential for the function of the v-Myc oncoprotein, thus, characterizing biological interactions involved in cellular paths that lead to and away from the Myc oncoprotein.

Degree

Ph.D.

Advisors

Taparowsky, Purdue University.

Subject Area

Molecular biology|Biology

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