Date of Award

5-2015

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Carlos A. Molina

Committee Member

Sandra D. Adams

Committee Member

Quinn C. Vega

Abstract

Inducible cAMP Early Repressor (ICER) is a negative transcriptional regulator found in eukaryotic cells. ICER has a tumor suppressive activity and is absent in several human cancers. Cancer cells get rid of ICER by a post-translational modification known as ubiquitination. Ubiquitin molecules, which are very small regulatory proteins, attach to lysine residues of a target protein to induce either a proteolytic or a non-proteolytic pathway. Both pathways target ICER levels inside the nucleus. In the case of the proteolytic pathway, ICER gets sent to the proteasome where it gets degraded and in the non-proteolytic pathway, ICER gets sent to the cytoplasm. Thus, cancer cells take advantage of ubiquitination to either degrade ICER or change its subcellular localization to affect its activity. When ICER is reintroduced into cancer cells, it inhibits the transformed phenotype of these cells. Therefore, my research attempted to characterize the ubiquitination site(s) of ICER and determine the effect of its subcellular localization to eventually block ICER ubiquitination.

Ubiquitination occurs at lysine residues. Since ICER has eleven lysine residues, each residue was previously mutated to a relatively similar amino acid, arginine, using site directed mutagenesis to create ICER with no lysine (ICER KO). Previous studies have shown that ICER KO represses growth related genes (1) more efficiently than wt ICER. Upon testing the activity of this mutated form, eleven independent constructs were created where only one lysine residue was reintroduced at a time. All of the constructs were sub-cloned in a mammalian expression vector, transfected into eukaryotic cells and treated with proteasome inhibitor. The proteasome inhibitor blocks the pathway to the proteasome therefore the lysine residue that is being ubiquitinated could potentially be determined by observing ICER with ubiquitin molecules attached. Subsequently, 15 ICER constructs containing hemagglutinin (HA) tag on the N- terminus and C-terminus were constructed. These constructs were made to select the transfected samples, excluding endogenous ICER that may interfere, when performing protein analysis. Determining the function of the expressed ICER is important to identify ways of inhibiting ICER ubiquitination by targeting ubiquitin from attaching to the lysine residue(s). Blocking ubiquitin from attaching to ICER can be used as an alternate cancer therapy that will target specific cancer cells since it will be present in the nucleus.

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