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Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics

Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of...

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Autores principales: Kellner, Fabienne, Keil, Andreas, Schindler, Katrin, Tschongov, Todor, Hünninger, Kerstin, Loercher, Hannah, Rhein, Peter, Böhmer, Sylvia‐Annette, Böhmer, Frank‐D., Müller, Jörg P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176853/
https://www.ncbi.nlm.nih.gov/pubmed/32155324
http://dx.doi.org/10.1111/jcmm.15132
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author Kellner, Fabienne
Keil, Andreas
Schindler, Katrin
Tschongov, Todor
Hünninger, Kerstin
Loercher, Hannah
Rhein, Peter
Böhmer, Sylvia‐Annette
Böhmer, Frank‐D.
Müller, Jörg P.
author_facet Kellner, Fabienne
Keil, Andreas
Schindler, Katrin
Tschongov, Todor
Hünninger, Kerstin
Loercher, Hannah
Rhein, Peter
Böhmer, Sylvia‐Annette
Böhmer, Frank‐D.
Müller, Jörg P.
author_sort Kellner, Fabienne
collection PubMed
description Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of the biosynthetic route and aberrantly activates STAT5, thereby promoting cellular transformation. The pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT, which is likewise essential for cell transformation. Little is known about features and mechanisms of FLT3 ligand (FL)‐dependent internalization of surface‐bound FLT3 or FLT3 ITD. We have addressed this issue by internalization experiments using human RS4‐11 and MV4‐11 cells with endogenous wild‐type FLT3 or FLT3 ITD expression, respectively, and surface biotinylation. Further, FLT3 wild‐type, or FLT3 ITD‐GFP hybrid proteins were stably expressed and characterized in 32D cells, and internalization and stability were assessed by flow cytometry, imaging flow cytometry, and immunoblotting. FL‐stimulated surface‐exposed FLT3 WT or FLT3 ITD protein showed similar endocytosis and degradation characteristics. Kinase inactivation by mutation or FLT3 inhibitor treatment strongly promoted FLT3 ITD surface localization, and attenuated but did not abrogate FL‐induced internalization. Experiments with the dynamin inhibitor dynasore suggest that active FLT3 as well as FLT3 ITD is largely endocytosed via clathrin‐dependent endocytosis. Internalization of kinase‐inactivated molecules occurred through a different yet unidentified mechanism. Our data demonstrate that FLT3 WT and constitutively active FLT3 ITD receptor follow, despite very different biogenesis kinetics, similar internalization and degradation routes.
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spelling pubmed-71768532020-04-24 Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics Kellner, Fabienne Keil, Andreas Schindler, Katrin Tschongov, Todor Hünninger, Kerstin Loercher, Hannah Rhein, Peter Böhmer, Sylvia‐Annette Böhmer, Frank‐D. Müller, Jörg P. J Cell Mol Med Original Articles Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of the biosynthetic route and aberrantly activates STAT5, thereby promoting cellular transformation. The pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT, which is likewise essential for cell transformation. Little is known about features and mechanisms of FLT3 ligand (FL)‐dependent internalization of surface‐bound FLT3 or FLT3 ITD. We have addressed this issue by internalization experiments using human RS4‐11 and MV4‐11 cells with endogenous wild‐type FLT3 or FLT3 ITD expression, respectively, and surface biotinylation. Further, FLT3 wild‐type, or FLT3 ITD‐GFP hybrid proteins were stably expressed and characterized in 32D cells, and internalization and stability were assessed by flow cytometry, imaging flow cytometry, and immunoblotting. FL‐stimulated surface‐exposed FLT3 WT or FLT3 ITD protein showed similar endocytosis and degradation characteristics. Kinase inactivation by mutation or FLT3 inhibitor treatment strongly promoted FLT3 ITD surface localization, and attenuated but did not abrogate FL‐induced internalization. Experiments with the dynamin inhibitor dynasore suggest that active FLT3 as well as FLT3 ITD is largely endocytosed via clathrin‐dependent endocytosis. Internalization of kinase‐inactivated molecules occurred through a different yet unidentified mechanism. Our data demonstrate that FLT3 WT and constitutively active FLT3 ITD receptor follow, despite very different biogenesis kinetics, similar internalization and degradation routes. John Wiley and Sons Inc. 2020-03-10 2020-04 /pmc/articles/PMC7176853/ /pubmed/32155324 http://dx.doi.org/10.1111/jcmm.15132 Text en © 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Articles
Kellner, Fabienne
Keil, Andreas
Schindler, Katrin
Tschongov, Todor
Hünninger, Kerstin
Loercher, Hannah
Rhein, Peter
Böhmer, Sylvia‐Annette
Böhmer, Frank‐D.
Müller, Jörg P.
Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics
title Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics
title_full Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics
title_fullStr Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics
title_full_unstemmed Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics
title_short Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics
title_sort wild‐type flt3 and flt3 itd exhibit similar ligand‐induced internalization characteristics
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176853/
https://www.ncbi.nlm.nih.gov/pubmed/32155324
http://dx.doi.org/10.1111/jcmm.15132
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