Phosphorylation of wild type FLT3 and AML-associated mutant FLT3 was recently analyzed using site-specific phosphotyrosine antibodies (15). Interestingly, the phosphorylation pattern of the different FLT3 variants showed quantitative and also qualitative differences. Although FLT3-ITD or mutations in the kinase domain resulted in ligand-independent FLT3 autophosphorylation and signaling activity, the wild type receptor is only autophosphorylated in response to stimulation with its cytokine FL.Signaling of receptor tyrosine kinases is modulated by protein-tyrosine phosphatases (PTP) (16), and aberrations in PTP function play a role in carcinogenesis (17). Some PTP, notably SHP-2, have been found to positively influence growth-stimulatory signaling pathways, and mutations leading to gain-offunction of these PTP can potentially be oncogenic. It has been demonstrated that SHP-2 directly interacts with FLT3 in a phosphorylation-dependent manner via phosphotyrosine 599. Table S1. 1 To whom correspondence should be addressed. Tel.: 49-3641-9395634; Fax:49-3641-9395602; E-mail: joerg.mueller2@med.uni-jena.de.2 The abbreviations used are: AML, acute myeloid leukemia; PTP, proteintyrosine phosphatase; FL, FLT3 ligand; PLC␥, phospholipase C␥; ITD, internal tandem duplication; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Mutations of Fms-like tyrosine kinase 3 (FLT3) are among the most frequently detected molecular abnormalities in AML patients. Internal tandem duplications (ITDs) are found in approximately 25% and point mutations within the second tyrosine kinase domain (TKD) in approximately 7% of AML patients. Patients carrying the FLT3-ITD but not the FLT3-TKD mutation have a significantly worse prognosis. Therefore, both FLT3 mutations seem to exert different biologic functions. FLT3-ITD but not FLT3-TKD has beenshown to induce robust activation of the STAT5 signaling pathway. In the present study, we investigated the mechanisms leading to differential STAT5 activation and show that FLT3-ITD but not FLT3-TKD uses SRC to activate STAT5. Coimmunoprecipitation and pull-down experiments revealed an exclusive interaction between SRC but not other Src family kinases and FLT3-ITD, which is mediated by the SRC SH2 domain. We identified tyrosines 589 and 591 of FLT3-ITD to be essential for SRC binding and subsequent STAT5 activation. Using sitespecific Abs, we found that both residues were significantly more strongly phosphorylated in FLT3-ITD compared with FLT3-TKD. SRC inhibition and knockdown blocked STAT5 activation and proliferation induced by FLT3-ITD but not by FLT3-TKD. We conclude that SRC might be a therapeutic target in FLT3-ITD ؉ AML. IntroductionThe Fms-like tyrosine kinase-3 (FLT3) receptor tyrosine kinase is expressed on blast cells in most patients with acute myeloid leukemia (AML). 1 Activating mutations of FLT3 have been detected in approximately 30% of AML patients. 2,3 Two distinct groups of FLT3 mutations have been described: (1) internal tandem duplications (FLT3-ITDs), which are most common in the juxtamembrane or the beta1-sheet of the tyrosine kinase domain-1 coding sequence in approximately 20%-27% of patients with AML; and (2) point mutations within the second tyrosine kinase domain (FLT3-TKDs) in approximately 7% of AML patients. 2,4,5 Both types of mutations activate the FLT3 receptor constitutively, leading to activation of downstream signaling proteins and resulting in factor-independent proliferation of murine lymphoid and myeloid cells. [6][7][8][9][10] Studies have identified FLT3-ITD as a prognostic factor because patients harboring this type of mutation have elevated peripheral blood and BM blast counts and an increased chance of relapse and inferior overall survival. 2,11,12 For FLT3-TKD mutations, a clear association with an inferior outcome could not be demonstrated in all studies and the relevance of FLT3-TKD for clinical prognosis is being investigated further. 13 Because FLT3 mutations can be found in approximately 30% of all AML patients and FLT3-ITD is associated with an inferior clinical prognosis, FLT3 is considered as an attractive therapeutic target. 14 Therefore, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been developed and tested in AML patients. 15,16 These studies showed that a single application of the inhibitor led to a short-term clinical response in some pat...
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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