Abstract
FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30–40% of acute myelogenous leukemia (AML) patients. Thus, drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from leukemia patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.
Highlights
FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), transduces growth signals
In human leukemia cells, wild‐type FLT3 localizes to the PM, whereas FLT3‐internal tandem duplication (ITD) accumulates in the perinuclear region
Fig. 1), supporting the results of FLT3ITD mislocalization. Since these three cell lines have different ITD sequences[27,28,29], the accumulation of FLT3 in the perinuclear region was independent of the inserted amino acid sequences but dependent on ITD insertion. These results suggest that ITD causes FLT3 retention in the perinuclear compartment in acute myelogenous leukemia (AML) cells
Summary
FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), transduces growth signals. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. Upon stimulation with FLT3 ligand, the receptor undergoes dimerization and autophosphorylates its tyrosine residues, such as Tyr[591] and Tyr8423–5 It activates downstream molecules, such as AKT, extracellular signal-regulated kinase (ERK), and transcription factors[3,6]. FLT3-ITD is suggested to activate signal transducers and activators of transcription 5 (STAT5) soon after synthesis[4,18,19,20], the precise subcellular localization of the mutant and the relationship between the mislocalization and growth signals remain unclear. We further showed that blockade of KIT trafficking to the signal platform inhibits oncogenic s ignals[24,25,26], suggesting that trafficking suppression is a novel strategy for suppression of tyrosine phosphorylation signals
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