Receptor protein tyrosine kinases (RPTKs) are of fundamental importance in processes such as cell proliferation, survival, and differentiation. The Eph family is the largest subfamily of RPTKs with 13 members having been discovered to date. Eck was the first member of this subfamily for which a ligand was characterized. B61, a cytokine-inducible gene product was originally isolated from tumor necrosis factor-alpha stimulated endothelial cells. Endothelial cells were found to express the eck RPTK and it was further shown that B61 could act as an angiogenic factor in vivo and chemoattractant for endothelial cells in vitro. Using the yeast two-hybrid system, the applicant has identified two molecules that are involved in transducing signals from activated eck RPTK. The first was the p85 subunit of PI3-kinase. The second molecule identified was a novel gene product designated Src-like adapter protein (SLAP). The investigator has defined two Specific Aims.
Specific Aim 1 is designed to determine the physiological consequence of activation of PI 3-kinase by the eck RPTK. This includes identifying the phosphotyrosine residue in the eck cytoplasmic domain that is engaged by the p85 subunit of PI-3 kinase. Additionally, by expressing dominant negative versions of the eck RPTK that are no longer able to activate PI-3 kinase, the investigator plans to determine the contribution of PI-3 kinase to known eck functions such as the induction of chemotaxis.
Specific Aim 2 is designed to characterize SLAP, the first adapter molecule to be described in the src family. This will include defining the phosphortyrosine residue it binds to in the eck cytoplasmic domain and the downstream effector molecules it in turn engages using a combination of yeast 2 hybrid screening and traditional biochemical approaches. Further, a panel of monospecific antibodies will be raised against SLAP to determine its subcellular localization, phosphorylation status and whether it binds to the cytoplasmic domains of other cell surface receptors.
