The oncogene of Abelson Leukemia Virus, v-able, encodes an intracellular membrane-associated non-receptor tyrosine kinase. We have shown that the v-abl tyrosine kinase can stimulate the proliferation of established lines of hemopoietic or fibroblastic cells. The v-abl tyrosine kinase activity is required continuously to abrogate the growth factor dependence of these cells. Because normal mitogenic stimulation is activated by receptor-associated tyrosine kinases, it is widely accepted that oncogenic tyrosine kinases transform cells by the continuous stimulation of the mitogenic pathway. This hypothesis is supported by many correlative evidence, however, the precise mechanism by which oncogenic tyrosine kinase transforms cells is not understood. We have been studying the v-abl tyrosine kinase function in mitogenesis and have made an important finding. We have discovered that the stimulation of cell proliferation by the v-abl tyrosine kinase is dependent on the cellular context. We have isolated two stable subtypes of NIH3T3 cells. In the """"""""positive"""""""" 3T3 cells, expression of the v-abl tyrosine kinase leads to the abrogation of serum requirement and v-abl induces the c-fos, c-jun and c-myc genes. In the """"""""negative"""""""" cells, on the other hand, v-abl tyrosine kinase does not activate the serum-inducible genes but causes growth arrest. The """"""""positive"""""""" phenotype is dominant. Our results demonstrate that v-abl tyrosine kinase requires the collaboration of a 3T3 cellular function to transform cells. These two variant cell lines offer an unique system to study the mechanism by which v-abl tyrosine kinase stimulates mitogenesis. We will investigate the molecular basis of the collaborating function by pursuing the following specific aims: 1. Define the cis-elements in the c-fos and c-jun promoters that determine the differential response to v-abl tyrosine kinase in the positive and the negative 3&3 cells. 2. Identify and characterize the protein factors which interact with the differential response elements in the """"""""positive"""""""" and the """"""""negative"""""""" cells. 3. Investigate the responsiveness of the """"""""positive"""""""" and """"""""negative"""""""" cells to other oncogenes which encode components of the mitogenic signal transduction pathway. 4. Determine the role of phosphotyrosine and the SH2 domain of v-abl in the modulation of mitogenesis. 5. Identify and characterize proteins which interact with v-abl in the """"""""positive"""""""" and """"""""negative"""""""" cells. Protein-affinity matrix will be used to isolate proteins that show differential interaction from the two cell types. 6. Cloning of the dominant-acting collaboration gene in the """"""""positive"""""""" cells by a functional selection. DNA transfection as well as micro- injection methods will be employed to introduce genes or mRNA from the positive cell into the negative cells and select for the collaborating function with the appropriate assays.
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