Role of mTwist1 Phosphorylation on S144 and S148in Promoting the Epithelial-Mesenchymal Transition
Metastasis is responsible for approximately 90% of cancer-related deaths. It is a multi-step process of cancer cells dissociating from a primary tumor and reseeding new growths in distant tissues, during which cells undergo a distinct phenotypic change known as the epithelial-mesenchymal transition (EMT). The EMT is driven by abnormal expression of early embryonic transcription factors, including the transcription factor Twist1. Twist1 has been found to play a role in tumor invasion in cancer patients, and it has several potential phosphorylation sites. The aim of this research is to examine how specific changes to phosphorylatable amino acids within the Twist1 protein will affect its functionality. The role of two neighboring Twist1 phosphorylation sites, S140 and S144 in the human (hTwist1) and S144 and S148 in the mouse (mTwist1) proteins, respectively, remains largely unknown. We hypothesize, regarding the Serine-144 and 148 phosphorylatable sites of mTwist1, that site directed changes to the amino acid sequence could affect the functionality of Twist in inducing the EMT. Our goal is to create plasmids that will express double mutant combinations (S144A/S148A; S144E/S148D; S144A/S148D; S144E/S148A) fused directly to green fluorescent protein (GFP) for subsequent transfection into an epithelial cell line. Site directed mutagenesis will be used to alter the amino acid sequence to substitute non-phosphorylatable and phosphorylation-mimicking residues. Following transfection, the Twist mutants will be analyzed for their ability to induce the EMT by use of various activity assays, morphological observations, fluorescence microscopy, and western blotting.
Research Area | Presenter | Title | Keywords |
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Cancer Studies | Spielberger, Sophie A. | Epithelial to Mesenchymal Transition | |
Psychology and Behavioral Sciences | Cisowski , Dana E. | prevention |