The long range goal of this research is to elucidate the cellular function of the c-abl proto-oncogene. C-abl is ubiquitously expressed and two proteins with N-terminal heterogeneity are produced from this proto-oncogene. The c-abl proteins contain a tyrosine kinase domain which is essential to the transforming function of c-abl derived oncogenes. The ubiquitous expression of the c-abl gene suggests that it may serve a function common to all cells. Our working hypothesis is that c-abl tyrosine kinase is a component of cellular signal transduction pathways. Cellular signals regulate c-abl tyrosine kinase which in turn regulates key functions by the phosphorylation of appropriate proteins. The goal of this proposal is to elucidate the mechanism of regulation of the c-abl tyrosine kinase. We have demonstrated that c-abl tyrosine kinase is normally inhibited in cells and release from that inhibition is required for the oncogenic conversion of c-abl. Our results are consistent with the existence of a mammalian cell-specific inhibitor which inhibits c-abl tyrosine kinase through interaction with defined regions of the c-abl protein. The first specific aim is to define the c-abl amino acid sequences which are required for the inhibition of the kinase activity and to determine the mechanism of inhibition mediated by those amino acid sequences. The second specific aim is to determine the relationship between the subcellar location and the inhibition of the c-abl tyrosine kinase. Preliminary results indicate that nuclear localization may be correlated with inhibition. Subcellular distribution of the inhibited wild type and the uninhibited mutant c-abl proteins will be determined. The effect of enforced unclear localization on the activity of the uninhibited mutant c-abl proteins will be investigated.
The third aim i s to purify active c-abl tyrosine kinase. The purified c-abl protein will be used to develop an in vitro system to study the regulation of tyrosine kinase.
The fourth aim i s to develop in vitro methods to characterize the inhibitor. If the inhibitor is a protein, cDNA clone for the inhibitor will be isolated to explore the possibility that the inhibitor gene may act as a recessive oncogene. C-abl is consistently mutated by chromosomal translocation in human chronic myelogenous leukemia and a subset of acute lymphocytic leukemia. We have shown that the mutated bcr/abl protein in leukemic cells have altered subcellular location and its kinase activity is deregulated. The proposed research will investigate the role of subcellular location on the regulation of the c-abl tyrosine kinase and may shed light on the pathogenic mechanism induced by an deregulated tyrosine kinase.
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