Investigating the differential availability of SH2/SH3 domains of Src Family Kinases for participation in Oncogenic Pathways

Kinases are enzymes responsible for the phosphorylation, and subsequent activation or inhibition, of proteins in biological systems. Their central role in regulation of cell signaling pathways has led to the identification of many kinases as potential drug targets for cancer therapies. Disregulation of Src (Sarcoma) Family Kinases (SFKs) has been shown to play an important role in tumor development and metastasis. SFKs are composed of three domains: the catalytic domain, responsible for tyrosine phosphorylation; and the SH3 and SH2 domains, which regulate catalytic activity and play a role in protein-protein interactions. Intramolecular binding of SH2/SH3 domains clamp the kinase into a closed and inactive state. Activation of SFKs requires the disengagement of both the SH2 and SH3 domains generating an extended protein conformation. Current therapeutic approaches target the enzymatic activity of SFKs. However, unbiased screening of kinase mutations in cancer show that catalytically inactive kinases are very prevalent in oncogenic pathways indicating that SFKs may participate in cancers through non-catalytic functions, such as scaffolding and sub-cellular targeting through the SH2/SH3 domains. We hypothesize that Type I and Type II ATP-competitive inhibitors which block enzymatic activity may differentially affect the accessibility of the SH2/SH3 domains for intermolecular interactions.  Thus, therapeutics which inactivate catalytic activity but stabilize an extended kinase conformation may actually stimulate oncogenic pathways. Florescence polarization, limited proteolysis, and pull down assays will be used to probe the accessibility of regulatory domains. Thorough understanding of the non-catalytic functions of SFKs will guide future therapeutic development for cancers involving SFKs.