Neoplastic transformation is usually caused by multiple independent lesions that interfere with normal cell proliferation and differentiation. Abnormalities in tyrosine kinase genes are often implicated in the generation of malignant phenotypes. The broad long term objective of this proposal is to characterize a novel receptor-type tyrosine kinase (Cek5) that we have recently identified as part of a systematic survey of developmentally expressed protein tyrosine kinases. Its amino acid sequence indicates that Cek5 is a member of the Eph family, which at present represents the least characterized of the major families of integral membrane tyrosine kinases. Cek5 is expressed primarily in the adult central nervous system and during embryonic development. The cellular and subcellular distribution of Cek5 in the central nervous and in other tissues will be investigated using immunoelectron microscopy and in situ hybridization techniques. The normal and pathological functions of Cek5 as an intermediate signal transduction pathways will be investigated by characterizing its ligand and substrates. The ligand will be partially purified using conventional chromatography and its activity assayed using cells transfected with Cek5 cDNA. Substrates will be studied by taking advantage of their expected association with the activated Cek5 kinase and phosphorylation on tyrosine. To gain insight into the regulation of Cek5 gene expression, the genomic organization of the Cek5 locus will be determined. The oncogenic potential of Cek5 will be examined by over expressing normal and modified Cek5 cDNAs in eukaryotic cells and by investigating the expression of Cek5 in various tumor cell lines. Cek5 produced in insect cells and monoclonal antibodies specific for Cek5 will represent useful reagents to accomplish the studies proposed. Since tyrosine kinase genes are highly conserved during evolution, the characterization of Cek5 and the preparation of Cek5-specific reagents will also be useful for future studies focused on the possible role of Cek5 in: (i) human cancers and other pathological conditions, (ii) embryonic development, and (iii) nerve regeneration.
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