Studying the ERK phosphorylation of the BCAT protein

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The cytosolic and mitochondrial isoforms of human branched chain aminotransferase (hBCATc and hBCATm, respectively) catalyze the transamination of the branched chain amino acids (BCAA) to their respective α-keto acids and glutamate. In addition, we recently found that hBCAT is phosphorylated by the canonical mitogen- activated protein kinase (MAPK) family member, extracellular regulated kinase 1/2 (ERK1/2). Our research team has shown direct evidence that ERK1/2 protein interacts with hBCATc in vitro and in breast cancer cells. More specifically, we found in breast cancer cells the overexpression of BCAT1 (gene encoding hBCATc) resulted in decreased levels of ERK1/2 activation/phosphorylation, and our in vitro experiments showed activated/phosphorylated ERK1/2 phosphorylates hBCATc regulated by the redox center of hBCAT. Together, the physiological implications of these observations are considerable to better understand the cellular and molecular mechanisms of how hBCATc interacts with ERK1/2. In this proposal we will focus on the protein- protein interaction of ERK1/2-mediated phosphorylation of the hBCATc. Based on preliminary studies, we hypothesize that the redox center regulated phosphorylation of hBCATc by ERK1/2 signals this metabolic protein to play additional functional roles in the cell, with the redox center of hBCAT playing an important role in protein-protein interactions between hBCAT and ERK1/2. We hypothesize that hBCAT is differentially phosphorylated by various kinases, which causes a conformational change of hBCAT that potentially confers p-hBCAT with new functions. In the proposed project, we will study the regulatory effects of phosphorylation of hBCAT by ERK1/2 using complementary biochemical and biophysical strategies, including kinetics analysis, mass spectrometry and X-ray crystallography. Likewise, redox regulation of ERK1/2 is also a theme to be developed in this project. Together, these experiments are expected to provide a comprehensive basis for understanding the mechanism of the redox-regulated phosphorylation of BCAT by ERK1/2, and the structural effects of phosphorylation on hBCAT. Moreover, these studies will establish a workflow to examine various hBCAT-kinase interactions and help us further study the phosphorylation-mediated conformational changes of hBCAT by other kinases that will help us to better understand the functional consequences of intracellular phosphorylation of hBCAT. This project will provide for the first-time unique insights into the molecular mechanisms underlying the redox regulation of hBCAT-ERK1/2 interactions, which will fundamentally inform its role in disease conditions such as breast cancer and Alzheimer’s disease.