Tyrosine kinase inhibitors induce down-regulation of c-Kit by targeting the ATP pocket

• Published in: PloS one 2013-04, Vol.8 (4), p.e60961
• Main Authors: D'allard, Diane, Gay, Julie, Descarpentries, Clotilde, Frisan, Emilie, Adam, Kevin, Verdier, Frederique, Floquet, Célia, Dubreuil, Patrice, Lacombe, Catherine, Fontenay, Michaela, Mayeux, Patrick, Kosmider, Olivier
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Analysis; ATP; Benzamides - pharmacology; Binding; Binding Sites - drug effects; Biology; c-Kit protein; Cancer; Cell Line; Cell proliferation; Cell surface; Cell survival; Down-regulation; Down-Regulation - drug effects; Enzyme inhibitors; Epidermal growth factor; Erythroblasts; Erythroblasts - cytology; Erythroblasts - drug effects; Erythroblasts - metabolism; Erythropoietin; Glycoproteins; Humans; Imatinib; Imatinib Mesylate; Internalization; Kinases; Ligands; Lysosomes - drug effects; Lysosomes - metabolism; Maturation; Medicine; Methyl-β-Cyclodextrin; Mutation; Peptides; Phenols (Class of compounds); Phosphorylation; Phosphotyrosine; Piperazines - pharmacology; Proteasomes; Protein Kinase Inhibitors - pharmacology; Protein Transport - drug effects; Protein-tyrosine kinase; Protein-Tyrosine Kinases - antagonists & inhibitors; Proteolysis - drug effects; Proteomics; Proto-Oncogene Proteins c-kit - metabolism; Pyrimidines - pharmacology; Stem cell factor; Stem cells; Thiazoles - pharmacology; Tyrosine; Tyrosine kinase inhibitors; Ubiquitin; Ubiquitin-protein ligase
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0060961

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.