Involvement of Fps/Fes and Fer Kinases in c-Kit Signalling in Mast Cells and Mast Cell Leukemias.

The c-Kit receptor protein-tyrosine kinase (PTK) is required for mast cell differentiation and function. Signalling by c-Kit normally depends on binding its ligand, Stem Cell Factor (SCF). However, several constitutively activating mutations in c-Kit have been described in myeloid leukemias (including mast cell leukemias (MCLs)) and in solid tumors. The objective of this study is to define the involvement of two non-receptor PTKs called Fps/Fes (hereafter referred to as Fps) and Fer in c-Kit signaling in mast cells and MCLs. Recent studies using transgenic mouse strains bearing kinase-inactivating or null mutations in Fer and Fps have revealed roles in hematopoiesis, and in limiting the innate immune response. Recently, we showed that Fer and Fps are activated downstream of the high affinity IgE receptor, F_cεRI, in bone marrow-derived mast cells (BMMCs), and that Fer kinase is required for sustained p38 Mitogen-activated protein kinase (MAPK) activation and chemotaxis upon activation of either F_cεRI or c-Kit.

In this study, we focus on the roles of Fer and Fps in c-Kit signaling in BMMCs. SCF-induced c-Kit activation led to rapid association of both kinases with c-Kit, and increased tyrosine phosphorylation of Fer and Fps. Interestingly, kinase-dead Fps protein was also phosphorylated upon c-Kit activation, suggesting the involvement of an upstream kinase. Pretreatment of BMMCs with the Src family kinase inhibitor SU6656, caused reduced phosphorylation of Fer and Fps upon SCF treatment. This suggests an involvement of one or more Src family kinases (SFKs) in phosphorylating Fps and Fer upon c-Kit activation. We next examined the potential involvement of the Fyn SFK, since it is known to interact with a juxtamembrane site in activated c-Kit and has been linked to Fer activation in other cell types. SCF treatment of BMMCs from Fyn knock-out mice (fyn-null) revealed normal activation of c-Kit, Erk MAPK, but reduced phosphorylation of Fer kinase. Furthermore, we have observed reduced phosphorylation of Shp2 phosphatase, and the p38 and Jnk MAPKs in Fyn-null BMMCs compared to control. We are currently attempting to restore Fyn expression by retroviral transduction to attempt to rescue these signalling defects, and also evaluate potential requirements for Fyn in chemotaxis and cytokine production. Using MCL cell lines harbouring constitutively actived c-Kit (RBL-2H3 and HMC-1), we have observed that phosphorylation of Fer and Fps kinases are inhibited by treating cells with either a c-Kit inhibitor (SU11652) or SFK inhibitor (SU6656).

In conclusion, Fer and Fps kinases participate in c-Kit signaling in normal and leukemic mast cells, and Src family kinases likely contribute to their activation. Using mast cells from Fer/Fps-deficient mice, we will identify their downstream targets and determine if they are required for leukemogenesis by an activated c-Kit transgene.