Access Type

Open Access Thesis

Date of Award

January 2017

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

David R. Evans

Abstract

FAM129B/Minerva, a protein implicated in melanoma cell invasion, has been shown to suppress TNFα induced apoptosis in cancer cells (Song, Evans & Evans (2010), JBC 286, 10201-09), thus contributing to the survival of metastatic cells. KEAP1, a substrate recognition molecule for the CUL3 E3 ubiquitin ligase, plays a central role in the activation of the TNFα pathway. We wish to test the hypothesis that the up-regulation of FAM129B in cancer cells suppresses apoptosis by sequestering KEAP1, thus preventing its participation in the apoptotic pathway. The first step is to demonstrate that FAM129B and KEAP1 form a complex. We have cloned and expressed the proteins in E. coli and purified both to homogeneity. Pull downs and immunoprecipitation clearly showed that the two proteins form a high affinity, stoichiometric complex. Using individually cloned domains, as well as mutations of the ETGE motif in FAM129B, the binding sites on each protein have been located. The proline rich region of FAM129B was shown elsewhere (Old et. al. (2009) Mol Cell.34,115) to be phosphorylated by MAP kinase (Erk1/2) at four sites. The effect of MAP kinase, which is upregulated in cancer cells, and PKC phosphorylation on the stability and formation of the complex has also been investigated.

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