Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals via K-Ras at the plasma membrane of Acute Myeloid Leukemia cells

Köthe, Susanne; Müller, Jörg P.; Böhmer, Sylvia‐Annette; Tschongov, Todor; Fricke, Melanie; Koch, Siegfried; Thiede, Christian; Requardt, Robert P.; Rubio, Ignacio; Böhmer, Frank D.

doi:10.1242/jcs.131789

Cited by 26 publications

(25 citation statements)

References 44 publications

(68 reference statements)

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“…RS4‐11 cells and MV4‐11 cells were obtained from German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany) and were maintained as previously described . The FLT3‐GFP hybrid genes were lentivirally packed and subsequently transduced in 32D cells.…”

Section: Methodsmentioning

confidence: 99%

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

Kellner

Keil

Schindler

et al. 2020

J Cellular Molecular Medi

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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. K E Y W O R D S degradation, Fms-like tyrosine kinase 3 internal tandem duplications, GFP hybrid genes, oncogene, plasma membrane, receptor endocytosis, receptor tyrosine kinase | 4669 KELLNER Et aL.

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Section: Methodsmentioning

confidence: 99%

“…The intracellular pool of FLT3 ITD effectively activates STAT5 and upregulates its targets, Pim‐1/2, but ineffectively activates PI3K/AKT and RAS/MAPK pathways. In contrast, the pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT …”

Section: Introductionmentioning

confidence: 99%

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

Kellner

Keil

Schindler

et al. 2020

J Cellular Molecular Medi

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“…Alternatively, the partial only blockade of a Ras effector pathway like the Raf/MEK/Erk cascade may not suffice to compromise all-or-nothing, switch-like type of threshold-controlled processes [52,53]. Furthermore the final outcome to Ras/Erk pathway activation is subject to regulation by intrincate, as yet not fully understood positive and negative feedback loops [54-56] that may add further levels of complexity in settings of incomplete Ras-GTP blockade by MSOR. Taken together, these considerations indicate that the degree of MSOR-mediated inhibition of a proximal downstream effector of Ras such as Erk, will not necessarily translate into the same degree of inhibition of a given Ras-dependent tumorigenic hallmark.…”

Section: Discussionmentioning

confidence: 99%

Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

et al. 2014

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BackgroundRas is a membrane-associated small G-protein that funnels growth and differentiation signals into downstream signal transduction pathways by cycling between an inactive, GDP-bound and an active, GTP-bound state. Aberrant Ras activity as a result of oncogenic mutations causes de novo cell transformation and promotes tumor growth and progression.ResultsHere, we describe a novel strategy to block deregulated Ras activity by means of oligomerized cognate protein modules derived from the Ras-binding domain of c-Raf (RBD), which we named MSOR for multivalent scavengers of oncogenic Ras. The introduction of well-characterized mutations into RBD was used to adjust the affinity and hence the blocking potency of MSOR towards activated Ras. MSOR inhibited several oncogenic Ras-stimulated processes including downstream activation of Erk1/2, induction of matrix-degrading enzymes, cell motility and invasiveness in a graded fashion depending on the oligomerization grade and the nature of the individual RBD-modules. The amenability to accurate experimental regulation was further improved by engineering an inducible MSOR-expression system to render the reversal of oncogenic Ras effects controllable.ConclusionMSOR represent a new tool for the experimental and possibly therapeutic selective blockade of oncogenic Ras signals.

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“…Furthermore the final outcome to Ras/Erk pathway activation is subject to regulation by intrincate, as yet not fully understood positive and negative feedback loops [54][55][56] that may add further levels of complexity in settings of incomplete Ras-GTP blockade by MSOR. Taken together, these considerations indicate that the degree of MSOR-mediated inhibition of a proximal downstream effector of Ras such as Erk, will not necessarily translate into the same degree of inhibition of a given Rasdependent tumorigenic hallmark.…”

Section: Discussionmentioning

confidence: 99%

Graded Inhibition of Oncogenic Ras-Signaling by Multivalent Ras-Binding Domains

Augsten¹,

Böttcher²,

Spanbroek³

et al. 2014

Cancer Cell Signaling

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Background: Ras is a membrane-associated small G-protein that funnels growth and differentiation signals into downstream signal transduction pathways by cycling between an inactive, GDP-bound and an active, GTP-bound state. Aberrant Ras activity as a result of oncogenic mutations causes de novo cell transformation and promotes tumor growth and progression. Results: Here, we describe a novel strategy to block deregulated Ras activity by means of oligomerized cognate protein modules derived from the Ras-binding domain of c-Raf (RBD), which we named MSOR for multivalent scavengers of oncogenic Ras. The introduction of well-characterized mutations into RBD was used to adjust the affinity and hence the blocking potency of MSOR towards activated Ras. MSOR inhibited several oncogenic Ras-stimulated processes including downstream activation of Erk1/2, induction of matrixdegrading enzymes, cell motility and invasiveness in a graded fashion depending on the oligomerization grade and the nature of the individual RBD-modules. The amenability to accurate experimental regulation was further improved by engineering an inducible MSOR-expression system to render the reversal of oncogenic Ras effects controllable. Conclusion: MSOR represent a new tool for the experimental and possibly therapeutic selective blockade of oncogenic Ras signals.

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Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals via K-Ras at the plasma membrane of Acute Myeloid Leukemia cells

Cited by 26 publications

References 44 publications

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

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

Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

Graded Inhibition of Oncogenic Ras-Signaling by Multivalent Ras-Binding Domains

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