TCR-Induced Activation of Ras Proceeds at the Plasma Membrane and Requires Palmitoylation of N-Ras

Rubio, Ignacio; Grund, Stefan; Song, Shu-Ping; Biskup, Christoph; Bandemer, Sabine; Fricke, Melanie; Förster, Martin; Graziani, Andrea; Wittig, Ute; Kliche, Stefanie

doi:10.4049/jimmunol.1000334

Cited by 50 publications

(53 citation statements)

References 37 publications

(64 reference statements)

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“…In accordance with the notion that de-palmitoylated versions of H-Ras and N-Ras transit through Golgi and ER (collectively known as endomembranes) on their way to the PM (Choy et al, 1999), several studies reported the presence of active GTP-loaded Ras at endomembranes of growth factor challenged cells (reviewed by Fehrenbacher et al, 2009). However, although there is agreement that activation of Ras downstream of multiple types of receptors occurs initially at the PM, there is an ongoing controversy regarding the possible existence of meaningful amounts of Ras-GTP at intracellular sites, a debate fueled by the fact that endomembrane Ras activation has been detectable in cells that overexpress Ras (Chiu et al, 2002) but could not be observed in the case of native, endogenous Ras (Augsten et al, 2006;Fehrenbacher et al, 2009;Rubio et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

“…To assess the localization of FLT3-induced K-Ras and N-Ras activation, we monitored the subcellular distribution of E3-R3(A/ D), a trivalent fluorescent affinity probe for Ras-GTP (see the Materials and Methods for more details; shown in the figures in green; RBD) that has previously been employed to image Ras activation in a number of settings (Augsten et al, 2006;Leadsham et al, 2009;Rubio et al, 2010;Wu et al, 2010). The specificity of this probe for detection of activated Ras proteins in the employed cell system was confirmed by cotransfection with different N-Ras versions (supplementary material (Fig.…”

Section: K-ras and N-ras Are Activated Downstream Of Wild-type Flt3mentioning

confidence: 99%

“…PmCherry-K-Ras and pmCherry-N-Ras were generated as previously described (Rubio et al, 2010). A previously characterized multivalent fluorescent reporter of Ras-GTP, based on the trimerization of the Ras binding domain (RBD) of c-Raf, was used to visualize Ras-GTP in live cells (Augsten et al, 2006;Rubio et al, 2010).…”

Section: Plasmidsmentioning

confidence: 99%

“…Activation of all three isoforms of Ras proteins can be assessed biochemically by employing the Ras-binding domain (RBD) of the Ras effector Raf as an affinity probe for Ras-GTP isolation. This strategy can also be exploited for visualizing the subcellular sites of Ras-GTP accumulation in intact cells by using fluorescently labeled RBD reporter probes (Augsten et al, 2006;Chiu et al, 2002;Rubio et al, 2010). In accordance with the notion that de-palmitoylated versions of H-Ras and N-Ras transit through Golgi and ER (collectively known as endomembranes) on their way to the PM (Choy et al, 1999), several studies reported the presence of active GTP-loaded Ras at endomembranes of growth factor challenged cells (reviewed by Fehrenbacher et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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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

Köthe

Müller

Böhmer

et al. 2013

Journal of Cell Science

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Summary FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3 ITD) is an important oncoprotein in acute myeloid leukemia (AML).Owing to its constitutive kinase activity FLT3 ITD partially accumulates at endomembranes, a feature shared with other diseaseassociated, mutated receptor tyrosine kinases. Because Ras proteins also transit through endomembranes we have investigated the possible existence of an intracellular FLT3-ITD/Ras signaling pathway by comparing Ras signaling of FLT3 ITD with that of wild-type FLT3. Ligand stimulation activated both K-and N-Ras in cells expressing wild-type FLT3. Live-cell Ras-GTP imaging revealed ligandinduced Ras activation at the plasma membrane (PM). FLT3-ITD-dependent constitutive activation of K-Ras and N-Ras was also observed primarily at the PM, supporting the view that the PM-resident pool of FLT3 ITD engaged the Ras/Erk pathway in AML cells. Accordingly, specific interference with FLT3-ITD/Ras signaling at the PM using PM-restricted dominant negative K-RasS17N potently inhibited cell proliferation and promoted apoptosis. In conclusion, Ras signaling is crucial for FLT3-ITD-dependent cell transformation and FLT3 ITD addresses PM-bound Ras despite its pronounced mislocalization to endomembranes.

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Section: Introductionmentioning

confidence: 99%

Section: K-ras and N-ras Are Activated Downstream Of Wild-type Flt3mentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

Köthe

Müller

Böhmer

et al. 2013

Journal of Cell Science

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“…Fused to EGFP, these oligomers are instrumental for the visualization of growth factor-stimulated activation of endogenous Ras in live cells [38][39][40][41]. In the course of those studies we noticed that oligomeric RBD-variants sequestered Ras-GTP in vitro in an oligomerization grade-dependent fashion and interfered with Ras-dependent signaling in COS-7 cells [38].…”

Section: Oligovalent Ras-binding Domains Block Oncogenic Ras-induced mentioning

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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Tumor microenvironmental influences on dendritic cell and T cell function: A focus on clinically relevant immunologic and metabolic checkpoints

Hargadon

2020

Clinical & Translational Med

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Cancer immunotherapy is fast becoming one of the most promising means of treating malignant disease. Cancer vaccines, adoptive cell transfer therapies, and immune checkpoint blockade have all shown varying levels of success in the clinical management of several cancer types in recent years. However, despite the clinical benefits often achieved by these regimens, an ongoing problem for many patients is the inherent or acquired resistance of their cancer to immunotherapy. It is now appreciated that dendritic cells and T lymphocytes both play key roles in antitumor immune responses and that the tumor microenvironment presents a number of barriers to the function of these cells that can ultimately limit the success of immunotherapy. In particular, the engagement of several immunologic and metabolic checkpoints within the hostile tumor microenvironment can severely compromise the antitumor functions of these important immune populations. This review highlights work from both preclinical and clinical studies that has shaped our understanding of the tumor microenvironment and its influence on dendritic cell and T cell function. It focuses on clinically relevant targeted and immunotherapeutic strategies that have emerged from these studies in an effort to prevent or overcome immune subversion within the tumor microenvironment. Emphasis is also placed on the potential of next‐generation combinatorial regimens that target metabolic and immunologic impediments to dendritic cell and T lymphocyte function as strategies to improve antitumor immune reactivity and the clinical outcome of cancer immunotherapy going forward.

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TCR-Induced Activation of Ras Proceeds at the Plasma Membrane and Requires Palmitoylation of N-Ras

Abstract: Material Supplementary 4.DC1http://www.jimmunol.org/content/suppl/2010/08/16/jimmunol.100033

Cited by 50 publications

References 37 publications

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

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

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

Tumor microenvironmental influences on dendritic cell and T cell function: A focus on clinically relevant immunologic and metabolic checkpoints

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