Ligand-mediated phosphorylation of EPHA2 activates the MAP /ERK pathway: Signal transduction and its contributions to cellular adhesion and EPHA2 gene expression

Intracellular signaling by receptor tyrosine kinases regulates many different aspects of cell behavior. Recent studies in our laboratory and others have demonstrated that the EphA2 receptor tyrosine kinase critically regulates tumor cell growth, migration and invasiveness. Although the cellular consequences of EphA2 signaling have been the focus of recent attention, the biochemical changes that are triggered by ligand-mediated activation of EphA2 remain largely unknown. Herein, we demonstrate that ligand stimulation of EphA2 promotes the nuclear translocation and phosphorylation of ERKs, followed by an increase in nuclear induction of the Elk-1 transcription factor. Ligand-mediated activation allows EphA2 to form a molecular complex with the Shc and Grb2 adaptor proteins. Specifically, we demonstrate that tyrosine phosphorylated EphA2 interacts with the PTB and SH2 domains of Shc. We also show that the interaction of EphA2 with Grb2 is indirect and mediated by Shc and that this complex is necessary for EphA2-mediated activation of ERKs and subsequent stimulation of the Elk-1 transcription factor. These studies provide a novel mechanism to demonstrate how EphA2 can convey information from the cell exterior to the nucleus. Another aspect of the ephrin-A1/EphA2 interaction is its influence on the mechanisms that govern EphA2 gene expression. Our present studies demonstrate that EphA2 gene expression is positively regulated by its own ability to bind ligand (ephrin-A1). Treatment of aggressive (MDA-MB-231) or non-aggressive (MCF10A) breast epithelial lines with artificial ligands induced EphA2 gene expression whereas antagonists of EphA2-ligand binding decrease EphA2 mRNA levels. We also demonstrate that ephrin-A1-mediated induction of EphA2 gene expression requires intracellular signaling through the MAP/ERK pathway. These findings provide intriguing evidence that EphA2 expression and function are intimately linked in both non-transformed and malignant epithelial cells. Ultimately, the results in this thesis will help to understand the mechanisms that cause the increased expression of this important and influential oncogene in human cancer