Abstract 2377: The AML linked Src family kinase Fgr is uncoupled from SH2 and SH3 domain regulation and drives oncogenic transformation

Abstract

Abstract Fgr is a member of the Src family of non-receptor protein-tyrosine kinases, which are over-expressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and strongly upregulated in a subset of primary human AML bone marrow samples, suggesting that Fgr kinase activity may contribute to AML pathogenesis. To investigate the oncogenic potential of Fgr, we expressed the wild-type kinase in Rat-2 fibroblasts and scored transformation in soft-agar colony-forming assays. Remarkably, Fgr expression resulted in strong transforming activity, suggesting that over-expression of the kinase is sufficient to overcome regulatory control by its SH2 and SH3 domains. To test this idea, we mutated the negative regulatory tail tyrosine of Fgr. Phosphorylation of the homologous tyrosines in c-Src, Hck and other Src-family members by the regulatory kinase Csk causes intramolecular engagement of the SH2 domain to suppress kinase activity. Substitution of the Fgr tail tyrosine with phenylalanine did not further enhance the transforming or kinase activity of Fgr in Rat-2 cells, suggesting that its kinase domain is uncoupled from regulation by its non-catalytic SH3-SH2 region. To explore the regulatory mechanism further, we expressed near-full-length Fgr in insect cells, purified it to homogeneity, and confirmed that it was singly phosphorylated on its negative regulatory tail (and not the activation loop). Hydrogen-deuterium exchange mass spectrometry demonstrated that the SH3 and SH2 domains are protected from deuterium uptake as observed previously for recombinant, downregulated Hck, suggesting that they are packed against the back of the kinase domain. We then tested recombinant Fgr for sensitivity to activation in vitro using peptide ligands for the SH3 and SH2 domains, as well as a peptide that engages both domains simultaneously. Fgr kinase activity was unaffected by these peptides, providing further evidence that the kinase domain is not allosterically coupled to SH3 and SH2 control. In contrast, Hck and c-Src were both stimulated by these peptides, consistent with a domain displacement mechanism of activation described previously. Control experiments using surface plasmon resonance spectroscopy confirmed that the SH3 and SH2 domains of both Hck and Fgr bind to each of the peptide ligands with similar kinetics and affinity. Taken together, our data show that Fgr is a unique Src family member in that its kinase domain is not subject to regulatory domain control despite evidence for intramolecular interactions with the SH3 and SH2 domains. As a consequence, simple over-expression of Fgr is sufficient to induce transformation of rodent fibroblasts, unlike Hck or other Src family members. By extension, over-expression of Fgr may contribute to AML development and selective targeting of its kinase activity may be of therapeutic benefit. Citation Format: Kexin Shen, Heather R. Dorman, Haibin Shi, Ravi K. Patel, Jamie A. Moroco, John R. Engen, Thomas E. Smithgall. The AML linked Src family kinase Fgr is uncoupled from SH2 and SH3 domain regulation and drives oncogenic transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2377. doi:10.1158/1538-7445.AM2017-2377