The Efficacy of Raf Kinase Recruitment to the GTPase H-ras Depends on H-ras Membrane Conformer-specific Nanoclustering

Guzmán, Camilo; Šolman, Maja; Ligabue, Alessio; Blaževitš, Olga; Andrade, Débora M.; Reymond, Luc; Eggeling, Christian; Abankwa, Daniel

doi:10.1074/jbc.m113.537001

Cited by 49 publications

(89 citation statements)

References 63 publications

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“…In addition, it served to calibrate a fixed setting that allows acquisition of data from cells with comparable expression levels. Two or three biological repeats were performed, and the apparent fluorescence resonance energy transfer (FRET) efficiency was calculated from obtained fluorescence lifetimes (51). Our well-established membrane FRET assay has been described elsewhere (45,(52)(53)(54).…”

Section: Methodsmentioning

confidence: 99%

“…Cells were then fixed with 4% PFA and mounted with Mowiol 4-88 on microscope slides. Fluorescence lifetimes of the mGFP-or EGFP-tagged donor constructs were measured using a fluorescence lifetime imaging attachment (Lambert Instruments, Leutingwolde, The Netherlands) on an inverted microscope (Zeiss Axio Observer.D1) as previously described (51). Fluorescein (0.01 mM, pH 9) was used as a lifetime reference standard.…”

Section: Methodsmentioning

confidence: 99%

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SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

Siljamäki

Abankwa

2016

Molecular and Cellular Biology

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The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B-and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras-and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner.S PRED (Sprouty-related proteins with an EVH1 domain) proteins are Sprouty-related negative modulators of Ras/mitogen-activated protein kinase (MAPK) signaling, and they have been shown to attenuate extracellular signal-regulated kinase (ERK) activity following various extracellular mitogenic stimuli (e.g., growth factors, cytokines, and chemokines) (1-5). The mammalian SPRED family contains four members, SPRED1, SPRED2, SPRED3, and Eve-3, the last of which is a splice variant of SPRED3 (1, 2, 6). SPRED1 and SPRED2 proteins contain an N-terminal enabled/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain and a cysteine-rich C-terminal Sprouty-related (SPR) domain, separated by a small c-Kit-binding domain (KBD) (2, 7). The SPR domain is necessary for membrane anchoring following growth factor stimulation (5), and both EVH1 and SPR domains have been implicated in ERK suppression (5,8,9). SPRED3 lacks a functional KBD and shows lower inhibitory activity than SPRED1 and -2, suggesting that the KBD also participates in inhibiting ERK activity (1-5).The precise mechanism of action of how SPRED proteins block MAPK signaling remains unclear. Overexpression of SPRED1 was suggested to increase the recruitment of the Ras effector Raf to the plasma membrane, where its association with Ras does not, however, lead to Raf activation (1, 2, 6). Instead, SPRED1 was reported to prolong Ras-Raf complexation, thus withdrawing Raf from activation by phosphorylation (2, 7). Another study showed that overexpression of SPRED1 inhibits Ras activation, as evidenced by decreased levels of GTP-bound ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

Siljamäki

Abankwa

2016

Molecular and Cellular Biology

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“…Recent work has suggested a role for nanoclusters in both EGF receptor signaling (Wang et al, 2014b) and neural circuits (Broadhead et al, 2016). The nanoclustering of H-Ras has a direct effect on specific Raf effector recruitment (Guzman et al, 2014). We have now documented changes in the molecular organization of signaling complexes following TCR stimulation.…”

Section: Discussionmentioning

confidence: 81%

Development of nanoscale structure in LAT-based signaling complexes

Barr¹,

Sherman

Yi³

et al. 2016

Journal of Cell Science

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The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we have demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 (also known as LCP2) localizes to the periphery of LAT clusters. In this study, we show that initially LAT and SLP-76 are randomly dispersed throughout the clusters that form upon TCR engagement. The segregation of LAT and SLP-76 develops near the end of the spreading process. The local concentration of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that the nanoscale organization of LATbased signaling complexes is dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation.

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“…Residues R169 and K170 interact with the higher population of cholesterol and sphingomyelin, most likely via hydrogen bonding (54). Although R169A/K170A mutations promote constitutively active Ras, the R128A/R135A mutant shows decreased signaling output, indicating that these residues in the allosteric lobe are critical to maintaining a signaling conducive Rasmembrane orientation (54,56).…”

Section: Nucleotide-induced Changes In Ras-membrane Orientationmentioning

confidence: 99%

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

Parker

Mattos

2015

Molecular Cancer Research

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The small GTPase Ras is mutated in about 20% of human cancers, primarily at active site amino acid residues G12, G13, and Q61. Thus, structural biology research has focused on the active site, impairment of GTP hydrolysis by oncogenic mutants, and characterization of protein-protein interactions in the effector lobe half of the protein. The C-terminal hypervariable region has increasingly gained attention due to its importance in H-Ras, N-Ras, and K-Ras differences in membrane association. A highresolution molecular view of the Ras-membrane interaction involving the allosteric lobe of the catalytic domain has lagged behind, although evidence suggests that it contributes to isoform specificity. The allosteric lobe has recently gained interest for harboring potential sites for more selective targeting of this elusive "undruggable" protein. The present review reveals critical insight that isoform-specific differences appear prominently at these potentially targetable sites and integrates these differences with knowledge of Ras plasma membrane localization, with the intent to better understand the structure-function relationships needed to design isoform-specific Ras inhibitors. Mol Cancer Res; 13(4); 595-603. Ó2015 AACR.

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The Efficacy of Raf Kinase Recruitment to the GTPase H-ras Depends on H-ras Membrane Conformer-specific Nanoclustering

Cited by 49 publications

References 63 publications

SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

Development of nanoscale structure in LAT-based signaling complexes

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

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