Abstract 1348 Analyzing the Expression and Tyrosine Phosphatase Activity of TENSIN1

TENSIN1(TNS1), a homolog of the PTEN tumor suppressor, is a pseudophosphatase that lacks the ability to dephosphorylate proteins. Despite an inability to dephosphorylate phosphorylated residues, TNS1 helps regulate cell migration, proliferation, and adhesion, which may lead to pathogenesis if mis-regulated. We aim to better understand this protein through application of three research objectives. The first was assessing the expression efficiency of mutated and wild type (WT) TNS1. Mutated constructs contained a N113C point mutation. Usage of the N113C mutation allows for the determination of whether phosphatase activity can be restored in TNS1, since it mimics a protein tyrosine phosphatase catalytic signature active motif (HCX5R). HEK293 cells were transfected with either WT or mutant TENSIN1 and the protein expression of WT and mutated TNS1 was analyzed by Western blotting. Both WT and mutated TNS1 resolved at the predicted molecular weight at 240 kDA and were consistently expressed, indicating that expression efficiency remained in the mutated form of this protein. Following confirmation of consistent protein expression, objective two is to perform immunoprecipitation to isolate TENSIN1 protein and determine differences of binding partners between WT and mutated TNS1. This goal is ongoing. Protein tyrosine phosphatase activity assays will be used to determine whether the N113C mutant activity is 'restored' as the third objective. Both radioactive isotope labeling with 32 P and non-radioactive phosphatase activity assays will be used to determine the level of phosphatase activity in both WT and mutated TNS1 forms. The anticipated outcome of tyrosine phosphatase activity assays should be little to no phosphatase activity in the WT form of TNS1 and reconstituted phosphatase activity in the N113C mutant. Funding for this project was supported by the College of William and Mary's Charles Center Monroe Grant Program and Dr. Shantá Hinton.