IntroductionFms-like tyrosine kinase 3 (Flt3) functions as a growth factor receptor and is expressed primarily in multipotential hematopoietic stem cells and progenitors as well as in placenta, gonads, and brain. Together with its activating ligand Flt3 ligand (FL) it is a crucial player in assuring normal function of stem cells and the immune system. [1][2][3] Moreover, approximately 30% to 35% of patients with acute myeloid leukemia (AML) carry a mutation in Flt3, rendering Flt3 the most frequently mutated gene in AML. 4,5 Flt3 mutations are generally grouped into 2 classes: point mutations in the vicinity of codon 835 or 842 within the tyrosine kinase domain (TKD); or internal tandem duplications (ITDs) of varying lengths within the juxtamembrane domain of Flt3, which sterically represses the intrinsic kinase activity of Flt3 in the absence of ligand. 4,6 Both classes of mutations result in constitutive activation of Flt3 but distinct signaling and transforming capacities. 7-10 Although debatable as prognostic markers by themselves, ITD and TKD mutations are correlated with poor prognostic features for AML patients, suggesting Flt3 or one of its downstream effectors as potential therapeutic targets. [11][12][13][14][15] Flt3 constitutes, together with the receptor for stem cell factor (c-Kit), the receptors for platelet-derived growth factors (PDGFRs), and colony-stimulating factor-1 (CSF-1), the type III family of receptor tyrosine kinases (RTKs). 5 Type III RTKs share a common modular structure consisting of 5 extracellular Ig-like domains, a short transmembrane stretch, a juxtamembrane region followed by a bipartite kinase domain interrupted by the kinase insert, and the carboxyterminal tail. 16 Ligand binding causes receptor dimerization, kinase activation, and transphosphorylation of RTK on multiple tyrosine residues. 17 These autophosphorylated tyrosine residues together with 3 to 6 adjacent amino acids form high-affinity docking sites for relay molecules possessing either phosphotyrosine binding (PTB) or Src homology 2 (SH2) domains. 18 Upon relocation to the receptor, these signaling or adapter molecules become activated in either a phosphorylation-dependent or -independent manner and are thereby capable of transducing the signal downstream. Relay molecules reported to be recruited and/or activated upon Flt3 activation include the p85 subunit of PI3K, Ras-GAP, PLC-␥, Vav, SHIP1, SHP2, ShcA, Grb2, Cbl, and Src family kinases (SFKs) as well as Stat5. [19][20][21][22][23][24][25][26] Whereas the immediate signaling steps following ligand binding (ie, binding of signaling molecules to autophosphorylated tyrosines) are well studied in the c-Kit, PDGFRs, and CSF-1 receptor systems, 16 For personal use only. on June 19, 2019. by guest www.bloodjournal.org From Y589, Y591, Y597, or Y599, which could theoretically add to the aberrant signal relay from the autoactivated receptor. 27 Here we report that Y572, Y589, Y591, and Y599 of Flt3 are phosphorylated in vivo in Flt3-expressing cells following ligand stimu...

Objective. Fms-like tyrosine kinase-3 (FLT3), a growth factor receptor normally expressed in hematopoietic progenitor cells, has been shown to have an important role in the development of acute myeloid leukemia c due to activating mutations. FLT3 mutations are found in approximately one third of AML patients and correlate with a poor prognosis, thus making the FLT3 receptor a potential therapeutic target. The aim of the investigation is to analyze the kinetics and specificity of FLT3 autophosphorylation in wild-type FLT3 as well as in the oncogenic FLT3 mutants.Methods. We have used Ba/F3 cells stably expressing either wild-type, ITD or D835Y mutants of FLT3 in order to compare the site selectivity of tyrosine phosphorylation sites. By the use of a panel of phosphospecific antibodies directed against potential tyrosine phosphorylation sites in FLT3, we identified several novel phosphorylation sites in FLT3 and studied the kinetics and specificity of ligand-induced phosphorylation in living cells.Results. Eight phosphorylated tyrosines (pY589, pY591, pY599, pY726, pY768, pY793, pY842 and pY955) were investigated and shown to be differentially phosphorylated in the wild-type versus the mutated receptors. Furthermore, we show that tyrosines 726, 793 and 842 are novel phosphorylation sites of FLT3 in intact cells. Conclusion.In this study, we have looked at the site-specific phosphorylation in the wild-type FLT3 in comparison to the mutants found in AML. We observed not only quantitative changes but more importantly, qualitative differences in the phosphorylation patterns of the wild-type and the mutated FLT3 receptors, which might contribute to the understanding of the mechanisms by which FLT3 contributes to AML in patients with mutations in FLT3.

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