FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells

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.

FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells.

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, the 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 AML 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. A tyrosine kinase inhibitor midostaurin (PKC412) markedly decreases in FLT3-ITD retention and increases in the 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.

FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells

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, the 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 AML 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. A tyrosine kinase inhibitor midostaurin (PKC412) markedly decreases in FLT3-ITD retention and increases in the 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.

Arabidopsis COPPER TRANSPORTER 1 undergoes degradation in a proteasome-dependent manner

The essential nutrient copper is toxic in excess. Therefore, plants must tightly control copper uptake and distribution. Arabidopsis thaliana high-affinity copper transporters (COPTs) mediate copper uptake, partitioning, and redistribution. Here we show that COPT1 localizes to the plasma membrane and endoplasmic reticulum in stably transgenic plants expressing a COPT1–green fluorescent protein (GFP) fusion protein, and the fusion protein is rapidly degraded upon plant exposure to excess copper. MG132 treatment largely abolished copper-induced degradation of COPT1, implying a link between the proteasome and COPT1 activity in modulating copper uptake. Co-immunoprecipitation analyses revealed that COPT1 cannot be ubiquitinated in the presence of excess copper and MG132. Through site-directed mutagenesis, we identified Lys159 in the C-terminal cytoplasmic tail of COPT1 as critical for copper acquisition, but not for copper-mediated down-regulation of COPT1, in plants. Furthermore, pharmacological analysis showed that treatment with a vesicle trafficking inhibitor or a V-ATPase inhibitor does not alter the subcellular dynamics of COPT1–GFP, consistent with the absence of a connection between the endosomal recycling/vacuolar system and COPT1 degradation. Together, our data suggest that proteasomal degradation rather than vacuolar proteolysis is important for the regulation of copper transport to maintain copper homeostasis in plants.

Endocytic Pathway of Feline Coronavirus for Cell Entry: Differences in Serotype-Dependent Viral Entry Pathway

Feline coronavirus (FCoV) is a pathogen causing a lethal infectious disease in cats, feline infectious peritonitis. It has two serotypes (type I FCoV and type II FCoV). According to our previous study, type I FCoV infection is inhibited by compounds inducing intracellular cholesterol accumulation, whereas type II FCoV infection is not inhibited. Intracellular cholesterol accumulation was reported to disrupt late endosome function. Based on these findings, types I and II FCoV are considered to enter the cytosol through late and early endosomes, respectively. We investigated whether the antiviral activities of a late endosome trafficking inhibitor and cholesterol-accumulating agents are different between the FCoV serotypes. The late endosome trafficking inhibitor did not inhibit type II FCoV infection, but it inhibited type I FCoV infection. Type I FCoV infection was inhibited by cholesterol-accumulating triazoles, but not by non-cholesterol-accumulating triazoles. These phenomena were observed in both feline cell lines and feline primary macrophages. This study provides additional information on the differences in intracellular reproductive cycle between type I and type II FCoV.

A chemoenzymatic synthesis of ceramide trafficking inhibitor HPA-12

A chemoenzymatic synthesis of the title compound has been developed using an efficient and highly enantioselective lipase-catalyzed acylation in a hydrophobic ionic liquid, [bmim][PF6], followed by a diastereoselective asymmetric dihydroxylation as the key steps for incorporating the stereogenic centers. The further conversion to the appropriate intermediates and subsequent acylation with lauric acid furnished the target compound.