Abstract FES protein-tyrosine kinase (PTK) is activated downstream of the KIT receptor in mast cells, and promotes mast cell migration towards Stem cell factor (SCF), the ligand for KIT. Recently, tumor-derived SCF has been implicated in promoting mast cell recruitment leading to increased tumor vascularity and poor prognosis. FES is also a potential therapeutic target in leukemias driven by oncogenic KIT receptors that are constitutively active. Thus, defining FES activation and signaling mechanisms could lead to development of FES inhibitors as potential therapeutics in mast cell-involved tumors or KIT-driven leukemias. FES was previously shown to phosphorylate Tubulin, and localize to microtubules (MTs) in an SH2 domain-dependent manner. We hypothesized that defects in MT reorganization in FES-deficient mast cells may account for the observed defects in migration towards SCF. To map potential MT binding site(s) in FES, we performed MT binding assays, and identified two modes of FES binding, one via the N-terminal F-BAR/FX domains, and also via the SH2 domain. Interestingly, mutations predicted to disrupt oligomerization of the F-BAR/FX domains enhanced MT binding in vitro, and MT localization in vivo. Using in vitro kinase assays, we show that FES preferentially phosphorylates soluble tubulin compared to MTs, suggesting that FES recruitment to MTs may depend on their phosphorylation by other PTKs (such as Src family PTKs). In mast cells treated with SCF, FES co-localizes with MTs, including some localization to the MT organizing center (MTOC). We are currently comparing MTOC orientation in polarized wild-type and FES-deficient mast cells, and examining MT subsets marked by γ-tubulin, acetylation, and detyrosination. In conclusion, we find that FES interacts directly with MTs in vitro, and co-localizes with MTs in vivo, and hypothesize that disruption of FES-MT interaction and/or activation will be relevant therapeutic targets to limit mast cell recruitment in solid tumors, and growth of KIT-driven leukemias. Funded by an operating grant from Canadian Institutes for Health Research (MOP82882) to AWBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3832. doi:10.1158/1538-7445.AM2011-3832