N822K- or V560G-mutated KIT activation preferentially occurs in lipid rafts of the Golgi apparatus in leukemia cells

Abstract

Background KIT tyrosine kinase is expressed in mast cells, interstitial cells of Cajal, and hematopoietic cells. Permanently active KIT mutations lead these host cells to tumorigenesis, and to such diseases as mast cell leukemia (MCL), gastrointestinal stromal tumor (GIST), and acute myeloid leukemia (AML). Recently, we reported that in MCL, KIT with mutations ( D816V , human; D814Y , mouse) traffics to endolysosomes (EL), where it can then initiate oncogenic signaling. On the other hand, KIT mutants including KIT D814Y in GIST accumulate on the Golgi, and from there, activate downstream. KIT mutations, such as N822K , have been found in 30% of core binding factor-AML (CBF-AML) patients. However, how the mutants are tyrosine-phosphorylated and where they activate downstream molecules remain unknown. Moreover, it is unclear whether a KIT mutant other than KIT D816V in MCL is able to signal on EL. Methods We used leukemia cell lines, such as Kasumi-1 ( KIT N822K , AML), SKNO-1 ( KIT N822K , AML), and HMC-1.1 ( KIT V560G , MCL), to explore how KIT transduces signals in these cells and to examine the signal platform for the mutants using immunofluorescence microscopy and inhibition of intracellular trafficking. Results In AML cell lines, KIT N822K aberrantly localizes to EL. After biosynthesis, KIT traffics to the cell surface via the Golgi and immediately migrates to EL through endocytosis in a manner dependent on its kinase activity. However, results of phosphorylation imaging show that KIT is preferentially activated on the Golgi. Indeed, blockade of KIT N822K migration to the Golgi with BFA/M-COPA inhibits the activation of KIT downstream molecules, such as AKT, ERK, and STAT5, indicating that KIT signaling occurs on the Golgi. Moreover, lipid rafts in the Golgi play a role in KIT signaling. Interestingly, KIT V560G in HMC-1.1 migrates and activates downstream in a similar manner to KIT N822K in Kasumi-1. Conclusions In AML, KIT N822K mislocalizes to EL. Our findings, however, suggest that the mutant transduces phosphorylation signals on lipid rafts of the Golgi in leukemia cells. Unexpectedly, the KIT V560G signal platform in MCL is similar to that of KIT N822K in AML. These observations provide new insights into the pathogenic role of KIT mutants as well as that of other mutant molecules. Electronic supplementary material The online version of this article (10.1186/s12964-01...

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

“…Recently, we reported that in MCL, KIT D816V (human) or KIT D814Y (mouse) activates STAT5 and AKT on the ER and endolysosomes, respectively 21,25 , whereas KIT V560G in MCL activates downstream at the Golgi apparatus 24 . Furthermore, KIT mutants including KIT D816V in cells other than MCL, such as GIST and leukemia cells, cause oncogenic signals on the Golgi apparatus 22,24,33 . As previously described 4,18,19 , we confirmed that after biosynthesis in the ER, FLT3-ITD causes STAT5 tyrosine phosphorylation in a manner similar to KIT D816V in MCL.…”

“…Mutant KIT in leukemia localizes to endosome-lysosome compartments through endocytosis, whereas that in GIST stops in the Golgi region during early secretory trafficking. We further showed that blockade of KIT trafficking to the signal platform inhibits oncogenic signals [24][25][26] , suggesting that trafficking suppression is a novel strategy for suppression of tyrosine phosphorylation signals.…”

“…Recently, we reported that constitutively active KIT mutants in MCL, GIST, or AML accumulate in organelles in a manner dependent on their tyrosine kinase activity 21,22,24 . Thus, we asked whether FLT3-ITD tyrosine kinase activity was required for retention of the mutant in the Golgi region.…”

“…Previous studies showed that BFA, an inhibitor of ER export 39 , suppresses the activation of AKT and ERK but not STAT5 in MV4-11 cells 4,40 . Recently, we reported that in addition to BFA, M-COPA blocks trafficking of KIT mutants from the ER [24][25][26] . Thus, we treated AML cells with M-COPA as well as BFA to confirm the effect of blockade of ER export on FLT3 signaling.…”

“…Recently, we reported that KIT, a type III RTK, accumulates in intracellular compartments, such as endosomal/lysosomal membrane and the Golgi apparatus, in mast cell leukemia (MCL), gastrointestinal stromal tumor (GIST), and AML [21][22][23][24] . Mutant KIT in leukemia localizes to endosome-lysosome compartments through endocytosis, whereas that in GIST stops in the Golgi region during early secretory trafficking.…”