The Aberrant Localization of Oncogenic Kit Tyrosine Kinase Receptor Mutants Is Reversed on Specific Inhibitory Treatment

Abstract

Kit is a cell surface type III tyrosine kinase (TK) receptor implicated in cell transformation through overexpression or oncogenic mutation. Two categories of Kit mutants displaying mutations either in the juxtamembrane intracellular domain (regulatory mutants) or in the catalytic domain (catalytic mutants) have been described. To explore the effect of Kit oncogenic mutations on its subcellular localization, we constructed enhanced green fluorescent protein (EGFP)-tagged human Kit chimeras harboring mutations either in the regulatory (V560G) or in the catalytic (D816V) domain. When expressed in Chinese hamster ovary cells, EGFP-tagged wild-type Kit was activated on stem cell factor stimulation, whereas both EGFP-tagged Kit mutants displayed a constitutive TK activity. Constitutively activated mutants exhibited a high-mannose-type N-glycosylation pattern and an intracellular localization, suggesting that these mutants induce downstream oncogenic signaling without the need to reach the cell surface. Inhibition of constitutive Kit TK activity with dasatinib induced a complex, mature N-glycosylation pattern identical to unstimulated wild-type Kit and resulted in the redistribution of the mutants to the plasma membrane. This relocalization was clearly correlated to the inhibition of TK activity because imatinib, a specific inhibitor of the V560G mutant, inactive on the catalytic D816V mutant, induced only the relocalization of the V560G mutant. These data show that on TK inhibition, the aberrant localization of Kit mutants can be fully reversed. Kit mutants are then exported and/or stabilized at the cell surface as inactive and fully N-glycosylated isoforms. (Mol Cancer Res 2009;7(9):1525-33)

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

“…2 A and B, the presence of WT KIT and the N505I mutant on the cell surface is higher than that of T417IΔ418-419 and Dup A502Y503 mutants. The presence of the cytoplasmic domain mutants V560D, D816V, and V560DY823D on the cell surface was very low, in accordance with previous reports (18,19). We also used fluorescence microscopy to visualize the cellular distribution of a green fluorescent protein (GFP) fused to WT or each oncogenic KIT mutant expressed in COS7 cells.…”

“…We and others (18,19) have shown that glycosylated WT KIT migrates on SDS/PAGE as two distinct bands with apparent molecular masses of 145 kDa and 125 kDa ( Fig. 2 C and D).…”

“…Analysis of cellular localization using fluorescence microscopy showed that, unlike WT KIT, which is mostly confined to the cell membrane, oncogenic KIT with mutations in the TKD or JM domains is primarily localized inside the cell (18,19). Because anti-RTK antibodies that have been successfully applied in targeted therapy of cancers (20) exert their inhibitory activity by binding receptor molecules that are located on the cell surface, we compared the cellular localization and dynamic properties of the most common recurrent oncogenic KIT mutations in human cancers.…”

Sustained Mutant KIT Activation in the Golgi Complex Is Mediated by PKC-θ in Gastrointestinal Stromal Tumors

“…Furthermore, the cellular localization of MT-KIT differs from that of WT-KIT and is not fully understood. Most WT-KITs are localized to the plasma membrane before ligand binding, but some MT-KITs are abnormally localized to the intracellular compartment (9,10).…”

Les tumeurs stromales gastro-intestinales (GIST)

“…De plus, les formes immatures de KIT peuvent être phosphorylées et transmettre un signal d'activation cellulaire ( Figure 5). Des résultats similaires ont été obtenus par d'autres équipes [31]. Les formes mutées et sauvages de KIT ayant des devenirs biologiques très différents, et les GIST coexprimant les deux formes, il nous a semblé important d'étudier la biologie des formes mutées de KIT en comparant leur expression isolée et leur coexpression avec les formes sauvages.…”