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

Abstract

We used genetic strategies which have been proven valuable to decipher signaling pathways in comparatively simple organisms such as Drosophila and Caenorhabditis elegans, to dissect signaling network activated by tyrosine kinases in mammals. The strategy was developed further towards a generally applicable expression cloning system to identify signal transducers in tyrosine kinase pathways. This system is based on the ability of downstream acting genes to rescue the transformation phenotype of partial loss-of-function mutants of BCR-ABL which still retain tyrosine kinase activity. Using this strategy 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 transformation should result in colony formation in soft agar. A PCR approach was used to recover these compensating genes from the genomic DNA of the transformed 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 fibroblast and hematopoietic cell transformation. Currently, we are constructing and screening new libraries to identify novel genes which complement the BCR-ABL SH2 mutant. Our results demonstrate that this cloning approach is an effective means of identifying and characterizing signaling molecules that function in specific signaling pathways. This in turn may identify specific targets for mechanism-based therapeutic intervention to block altered signaling.

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This paper is based on and is an extension of data presented as a Poster at the XIXth Symposium of the IACRLRD, Mannheim, Germany, July 1997, President: Professor Dr Rüdiger Hehlman. It received the 'Professor Sven-Aage Killmann Leukemia Poster Prize'. The paper has been through the normal peer-review process

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Mahlmann, S., McLaughlin, J., Afar, D. et al. Dissection of signaling pathways and cloning of new signal transducers in tyrosine kinase-induced pathways by genetic selection. Leukemia 12, 1858–1865 (1998). https://doi.org/10.1038/sj.leu.2401231

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  • DOI: https://doi.org/10.1038/sj.leu.2401231

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