The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

Abstract

Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

Expression of myeloid Src-family kinases is associated with poor prognosis in AML and influences Flt3-ITD kinase inhibitor acquired resistance

“…However, the T338F and T338M mutants of Fgr both transformed TF-1 cells to cytokine independence. The difference between Hck and Fgr in this regard may relate to the intrinsic transforming activity Fgr relative to Hck, which has been linked to differences in its activation loop relative to all other Src-family members [24]. TF-1 cells expressing Flt3-ITD (but not wild type Flt3) grew equally well in the presence or absence of GM-CSF as expected, providing a positive control for this experiment.…”

“…Thus, the partial rescue of growth inhibition in cells co-expressing the F691L mutant with Fgr may be related to transforming signals generated by Fgr itself. Recently, we reported that Fgr is unique among Src-family kinases in that it can induce fibroblast transformation by simple over-expression without mutation, and that it also reduces the requirement of TF-1 cells for GM-CSF when expressed alone [24]. More generally, these studies with TF-1 cells suggest that Flt3-ITD is the primary inhibitor target for A-419259 in Flt3-ITD + AML, although the presence of Hck and Fgr may modulate Flt3-ITD inhibitor sensitivity especially in the presence of Flt3-ITD kinase domain mutations.…”

“…In addition to Hck, recent evidence has identified Fgr as an oncogene and a therapeutic target in AML. In contrast to Hck, expression of wild-type Fgr without mutation induces oncogenic transformation of rodent fibroblasts in vitro, and reduces the cytokine dependence of a human myeloid leukemia cell line in colony-forming assays [24]. In addition, an N -phenylbenzamide kinase inhibitor, TL02-59, potently inhibits Fgr kinase activity in vitro and in vivo.…”

Dynamic regulatory features of the protein tyrosine kinases

“…Underscoring differences between closely related kinases, the catalytic activity of one SRC family member, FGR, seems to have evolved unfettered by SH3 and SH2 mediated regulation (77). Despite having all the hallmarks of a regulated kinase, the activation loop of FGR is phosphorylated regardless of the phosphorylation status of the regulatory tail and FGR activity is not increased by binding of peptides to the SH3 or SH2 domains.…”

“…Constitutive activity is attributed to observations that overexpression of wild type FGR occurs in acute myeloid leukemia cells and FGR knockdown reduces cell growth (78). Characterization of FGR by HDX-MS suggests that, despite the unconstrained catalytic function, FGR adopts the compact autoinhibited structure of other SRC family membersand yet the SH3 and SH2 regulatory features are effectively bypassed (77). The authors of this work show that sequence differences between FGR and other Src family members in the activation loop are responsible, at least in part, for the high level of FGR kinase activity.…”