Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

Lavoie, Hugo; Thevakumaran, Neroshan; Gavory, Gwenaëlle; Li, John J.; Padeganeh, Abbas; Guiral, Sébastien; Duchaine, Jean; Mao, Daniel Y.L.; Bouvier, Michel; Sicheri, Frank; Therrien, Marc

doi:10.1038/nchembio.1257

Cited by 146 publications

(170 citation statements)

References 51 publications

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“…Sorafenib-induced B-/C-Raf heterodimers have been linked to specific increases in the nanoscale clustering of oncogenic K-ras4B (here, K-ras) (60,61), which resides in acidic lipid nanodomains (62). While evidence for the localization of SPRED1 to acidic lipid domains exists (55), details on the composition are not known.…”

Section: Resultsmentioning

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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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: Resultsmentioning

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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“…1C). Using the biosensor assay to screen a drug library, Lavoie et al 26 further found that certain ATP-competitive inhibitors for BCR-ABL, p38, and VEGFR also promote Raf dimerization. In addition, these inhibitors induced the paradoxical activation of ERK, indicating the clinical importance of screening kinase-targeted drugs for their potential to promote Raf dimerization.…”

Section: Revisiting Raf Dimerization In Human Disease Statesmentioning

confidence: 99%

“…Therrien and Sicheri has recently provided key insight regarding how the ATP-competitive Raf inhibitors promote Raf dimerization. 26 Protein kinase domains consist of N-and C-lobes, and due to their dynamic nature, there is a significant degree of flexibility between the N-and C-lobes as well as within the N-lobe itself. The side-to-side dimer interface spans both the N-and C-lobes, and by binding the ATP pocket, Lavoie and coworkers 26 found that the inhibitors stabilize the closed conformation of the kinase domain, thus generating a static dimer interface surface that is conducive for dimerization.…”

Section: Revisiting Raf Dimerization In Human Disease Statesmentioning

confidence: 99%

“…26 Protein kinase domains consist of N-and C-lobes, and due to their dynamic nature, there is a significant degree of flexibility between the N-and C-lobes as well as within the N-lobe itself. The side-to-side dimer interface spans both the N-and C-lobes, and by binding the ATP pocket, Lavoie and coworkers 26 found that the inhibitors stabilize the closed conformation of the kinase domain, thus generating a static dimer interface surface that is conducive for dimerization. These investigators went on to engineer Raf dimerization biosensors to examine dimerization induced by a panel of ATP-competitive Raf inhibitors.…”

Section: Revisiting Raf Dimerization In Human Disease Statesmentioning

confidence: 99%

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The importance of Raf dimerization in cell signaling

2013

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“…These considerations gain further relevance in the light of recent insights into the Ras-dependent activation mechanism of Raf. A wealth of experimental data has recently established that Raf kinases function as homo-and heterodimers [57][58][59][60]. Although many details of Raf regulation remain obscure it is evident that only the dimeric form is responsive and sensitive to activation by Ras-GTP [57].…”

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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Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

Cited by 146 publications

References 51 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

The importance of Raf dimerization in cell signaling

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

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