title: 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.
authors.affiliation: Laboratoire de Biotechnologie et Pharmacologie Génétique Appliquée, Centre National de la Recherche Scientifique, Ecole Normale Supérieure de Cachan, 94235 Cachan, France.
authors.forename: Houcine
authors.initials: H
authors.lastname: Bougherara
authors.forename: Frédéric
authors.initials: F
authors.lastname: Subra
authors.forename: Ronan
authors.initials: R
authors.lastname: Crépin
authors.forename: Patrick
authors.initials: P
authors.lastname: Tauc
authors.forename: Christian
authors.initials: C
authors.lastname: Auclair
authors.forename: Marie-Alix
authors.initials: MA
authors.lastname: Poul
mesh_terms: D000818:Animals; D001549:Benzamides; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D000069439:Dasatinib; D005434:Flow Cytometry; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000068877:Imatinib Mesylate; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000378:metabolism; D009154:Mutation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D019009:Proto-Oncogene Proteins c-kit / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects*; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit; D000069439:Dasatinib