1TK TYROSINE KINASE--NORMAL AND PATHOLOGIC FUNTION

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The receptor-type protein tyrosine kinases (PTKs) are cell surface molecules which bind growth factor ligands, and initiate pleotropic intracellular signaling cascades. In addition, most receptor PTKs are protooncogenes, and some have been implicated in human malignancies. Thus, the study of receptor tyrosine kinases has increased our understanding of neoplasia, cellular growth factors and the molecular dynamics of intracellular signaling. I have recently cloned, sequenced and characterized the full length cDNA of the human ltk gene, a new receptor PTK gene which encodes a 3.1 kB mRNA and a 100 kD protein with demonstrated tyrosine kinase activity. Preliminary evidence indicates that ltk expression is restricted to hematopoietic and neural crest derived cells, suggesting its activity is relatively tissue specific. However, its physiological function, putative ligand and oncogenic potential are all unknown. The broad aim of this proposal is to define the normal and pathologic function of the ltk gene product, with an emphasis on investigating its probable role as a receptor for a cellular growth factor and its possible role as a protooncogene. Specifically, four sets of experiments are proposed to achieve this objective. First, the characterization of the ltk gene and protein will be completed, focusing on the in vivo biosynthesis of the ltk protein and the cloning of the genomic sequences and upstream promoter region. In the second set of experiments, two approaches are proposed to identify the tissue(s) where the ltk gene acts physiologically: an expression survey and gene "knock out" experiments. The temporal and spatial expression of ltk in mouse embryos and its expression in adult tissues will be determined by in situ hybridization, RNAase protection analysis and immunofluorescence. To identify tissue(s) where ltk functions, targeted gene disruption of the ltk gene in mouse embryonic stem cells will be carried out, to produce mice which are homozygously disrupted at the ltk locus. Such mutant mice are expected to provide a powerful genetic model of ltk function. In the third aim, the oncogenic potential of the ltk gene will be investigated by determining whether various ltk derivatives, patterned after the transforming alleles of other PTKs, are able to transform fibroblasts or lymphoid cells, and by screening human tumors for the presence of activating alterations. The ultimate goal is to determine the role of ltk in human malignancy. Finally, two strategies are proposed for isolating the ltk ligand. One exploits the ability of some receptor-ligand pairs to transform cells via an autocrine loop mechanism, and the other involves screening known growth factors for their ability to induce ltk receptor autophosphorylation as an indirect assay of receptor binding. Identification of the ltk ligand would provide additional insights into the mechanism of growth control and may identify a novel growth factor with therapeutic value.