Dissection of signaling pathways and cloning of new signal transducers in tyrosine kinase-induced pathways by genetic selection

We used genetic strategies which have been proven valuable to decipher sign aling pathways in comparatively simple organisms such as Drosophila and Cae norhabditis elegans, to dissect signaling network activated by tyrosine kin ases in mammals. The strategy was developed further towards a generally app licable expression cloning system to identify signal transducers in tyrosin e kinase pathways. This system is based on the ability of downstream acting genes to rescue the transformation phenotype of partial loss-of-function m utants of BCR-ABL which still retain tyrosine kinase activity. Using this s trategy we have previously shown that overexpression of c-Myc and Cyclin D1 can rescue a signaling defective SH2 mutant of BCR-ABL for transformation. In an unbiased approach to identify new compensating genes, a cDNA library was introduced by retroviral infection into fibroblasts which express the BCR-ABL SH2 mutant. CDNA clones, capable of rescuing the SH2 mutant for tra nsformation should result in colony formation in soft agar. A PCR approach was used to recover these compensating genes from the genomic DNA of the tr ansformed fibroblasts. Sequencing analysis of the initial cDNAs identified three known genes, the adapter molecule Shc, the kinases SPRK and p38 MAPK. These genes have been found to interact functionally with BCR-ABL for fibr oblast and hematopoietic cell transformation. Currently, we are constructin g and screening new libraries to identify novel genes which complement the BCR-ABL SH2 mutant. Our results demonstrate that this cloning approach is a n effective means of identifying and characterizing signaling molecules tha t function in specific signaling pathways. This in turn may identify specif ic targets for mechanism-based therapeutic intervention to block altered si gnaling.