TEK is a newly cloned receptor tyrosine kinase that is expressed exclusively in endothelial precursors and in the endothelium of actively growing embryonic and adult vessels. Disruption of TEK function in transgenic mice results in profound abnormalities of the embryonic vasculature and early embryonic lethality. Taken together, these data strongly suggest that TEK signaling is indispensable for normal vascular development. To define more precisely the role of TEK signaling in vasculature development, the Specific Aims of the present proposal are to: 1. Determine the signaling pathways, known or novel, which are modulated by TEK kinase activation. They have established that the autophosphorylated TEK kinase domain associates with GRB-2 and SH- PTP2. The nature of these interactions will be explored using synthetic peptides and recombinant SH-2 domains. Endothelial libraries will be probed using interaction cloning and 2-hybrid screening in order to identify additional signaling molecules that may interact with the activated TEK kinase. To circumvent the inavailability of a TEK ligand, a chimeric receptor (FTEK) consisting of the c-fms (CSF-1 receptor) ligand binding and transmembrane domain coupled to the TEK kinase domain was constructed. This chimera will be expressed in mammalian cells to confirm the interaction of the activated TEK kinase with GRB-2 and SH-PTP2 and with other known or novel signaling pathways identified by interaction cloning or 2-hybrid screening. 2. Determine the cellular responses mediated by TEK kinase activation. Cells expressing f-TEK will be used to determine whether TEK kinase activation triggers """"""""angiogenic"""""""" cellular responses such as mitogenesis, chemotaxis, morphogenesis, and protease gene regulation. Mutated forms of the TEK kinase will be used to determine the relationship of these cellular responses to the activation of specific downstream signaling molecules. 3. Determine the consequences of blocking TEK signal transduction during blood vessel development in vivo. Mutated forms of TEK designed to block signaling by the endogenous receptor will be expressed during blood vessel development in zebrafish embryos. Because zebrafish embryos are transparent and because they develop outside the mother, the effect of TEK inhibition on vascular growth can be monitored over time and the role of TEK kinase signaling can be determined more precisely than in the transgenic mouse model. Based on current evidence, TEK is likely to be an important mediator of endothelial responses during blood vessel growth and differentiation. Relating the biological events and cellular responses that occur following TEK kinase activation to the intracellular signaling molecules that drive them should lead to further insight into the molecular mechanisms of vascular growth. The findings could in turn form the basis of therapeutic approaches designed to stimulate or inhibit blood vessel growth in a variety of disease states including atherosclerosis, cancer, and diabetes.
