Biochemical and biophysical studies of the human ephrin receptor protein tyrosine kinase, EphA2
Abstract
The focus of this work is EphA2, a member of the Eph family of receptor tyrosine kinases that has been implicated in cellular transformation and metastasis. It is a 110 kDa protein found primarily in adult epithelial cells where is mediates cell-cell contacts. In normal cells, EphA2 is found at low concentrations, but with high levels of tyrosine phosphorylation. This situation is reversed in aggressive epithelial tumor cells, here there is an increase in EphA2 protein levels, and a loss of tyrosine phosphorylation. Use of antibodies to cause EphA2 to dimerize on turmor cells has been found to restore normal protein and phosphorylation levels and cause the cells to revert to a normal phenotype. These observations show EphA2 to be a possible target for cancer therapies. In order to understand how EphA2 functions, we have overexpressed several constructs of the cytoplasmic domains of the protein (juxtamembrane linker region, tyrosine kinase domain, and sterile alpha motif) in a bacterial expression system. The resulting proteins were purified and used for biochemical and biophysical characterization. The preferred target substrate sequence was determined to be that of the autoregulatory tyrosines of Eph family receptors, followed by the general sequence Y-I/V, for the kinase using a peptide assay derived from human signaling proteins. Examination of the interaction of EphA2 with the tyrosine phosphatase, LMW-PTP, revealed that the two proteins can function as substrates for each other in vitro. In addition, the interaction is sufficient to allow isolation of each protein by pull-down assay using a tagged version of the binding partner. Finally, the affinity of the cytoplasmic domains for ATP was determined to be 9.2μM. Removal of the juxtamembrane linker or the SAM domain altered the affinity to approximately 300μM, and removal of both to 15μM. The rate of the reaction is seen to be approximately halved by removal of the SAM domain.
Degree
Ph.D.
Advisors
Stauffacher, Purdue University.
Subject Area
Biophysics|Molecular biology
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