Visualization of Ras-PI3K interaction in the endosome using BiFC

Tsutsumi, Kaori; Fujioka, Yoichiro; Tsuda, Masumi; Kawaguchi, Hideaki; Ohba, Yusuke

doi:10.1016/j.cellsig.2009.07.004

Cited by 28 publications

(29 citation statements)

References 43 publications

(29 reference statements)

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“…We noted that mutagenesis of the residues in a stretch of amino acids in the switch I region (Figure 1A) ( i.e. , I36 - S39), or of the interacting residues on the primary effector proteins (RAF, PI3K or RALGDS), has been reported to lower binding affinity for effector proteins to RAS proteins (Colicelli, 2004; Gysin et al, 2011; Hall et al, 2002; Huang et al, 1998; Huang et al, 1997; Karnoub and Weinberg, 2008; Malumbres and Barbacid, 2003; Pacold et al, 2000; Scheffzek et al, 1997; Shaw and Cantley, 2006; Tanaka and Rabbitts, 2010; Tsutsumi et al, 2009; Vigil et al, 2010; Walker and Olson, 2005). Analysis of the KRAS G12D (PDB: 4DSN, and see Supporting Information) structure revealed a candidate site in the switch I region (termed here the D38 site), and two additional potential binding sites near the D38 site (Figure 1B and Supporting Information): we identified a site centered on alanine 59 (termed the A59 site), located between the switch I and switch II regions; on the other side of the D38 site, we identified a potential binding site near Y32 (Figure 1B and Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Multivalent Small-Molecule Pan-RAS Inhibitors

Welsch

Kaplan

Chambers

et al. 2017

Cell

224

181

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SUMMARY Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, have potential use as chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers, and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

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Section: Resultsmentioning

confidence: 99%

Multivalent Small-Molecule Pan-RAS Inhibitors

Welsch

Kaplan

Chambers

et al. 2017

Cell

224

181

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“…One interpretation of this is that, besides Cdc42, there may be additional endomembrane effectors that need to be activated for more efficient transformation. Intriguingly, Tsutsumi et al have recently examined the binding between H-Ras and the catalytic subunit of PI3K and found that this also occurs in endosomes (48). It is possible that some fractions of PI3K may be activated by Ras in the endomembrane but not the PM, as generally assumed, during transformation.…”

Section: Discussionmentioning

confidence: 99%

Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies

Cheng

Gasman

et al. 2011

Molecular and Cellular Biology

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Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartmentspecific Ras pathways need to work cooperatively.

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“…We further demonstrated that Spred-2 also regulates the PI3K signaling pathway, as the Raf-MEK-ERK and PI3K signaling pathways functionally cross-regulate each other (43, 44). Several studies also report that PI3K signaling regulates influenza virus entry (45–47). Our findings indicated that Spred-2 deletion enhanced the phosphorylation status of Akt, a commonly used marker of PI3K activation.…”

Section: Discussionmentioning

confidence: 99%

Sprouty-Related Ena/Vasodilator-Stimulated Phosphoprotein Homology 1-Domain–Containing Protein-2 Critically Regulates Influenza A Virus–Induced Pneumonia

Ito¹,

Itakura²,

Takahashi³

et al. 2016

Critical Care Medicine

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Objective Influenza A virus causes acute respiratory infections that induce annual epidemics and occasional pandemics. While a number of studies indicated that the virus-induced intracellular signaling events are important in combating influenza virus infection, the mechanism how specific molecule plays a critical role among various intracellular signaling events remains unknown. Raf/MEK/ERK cascade is one of the key signaling pathways during influenza virus infection, and the Sprouty-related Ena/VASP homology 1-domain-containing protein (Spred) has recently been identified as a negative regulator of Raf-dependent ERK activation. Here we examined the role of Raf/MEK/ERK cascade through Spred in influenza A (H1N1) viral infection, because the expression of Spred-2 was significantly enhanced in human influenza viral-induced pneumonia autopsy samples. Design Prospective animal trial. Setting Research laboratory Subjects Wild-type (WT) and Spred-2 knockout (KO) mice inoculated with H1N1 Intervention WT or Spred-2 KO mice were infected by intranasal inoculation of H1N1(A/PR/8). An equal volume of PBS was inoculated intranasally into mock-infected mice. Measurements and Main Results H1N1 infection of Spred-2 KO mice led to higher mortality with greater viral load, excessive inflammation, and enhanced cytokine production compared with WT mice. Administration of MEK-inhibitor, U0126, improved mortality and reduced both viral load and cytokine levels. Moreover, bone marrow chimeras indicated that H1N1-induced lung pathology was most severe when Spred-2 expression was lacking in nonimmune cell populations. Furthermore, microarray analysis revealed knockdown of Spred-2 led to enhanced phosphatidylinositol 3-kinase (PI3K) signaling pathway, resulting that viral clearance was regulated by Spred-2 expression through the PI3K signaling pathway in murine lung epithelial cells. Conclusions These data support an important function of Spred-2 in controlling influenza virus-induced pneumonia and viral replication. Spred-2 may be a novel therapeutic target for controlling the immune response against influenza H1N1 virus infection.

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Visualization of Ras-PI3K interaction in the endosome using BiFC

Abstract: 3/21

Cited by 28 publications

References 43 publications

Multivalent Small-Molecule Pan-RAS Inhibitors

Multivalent Small-Molecule Pan-RAS Inhibitors

Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies

Sprouty-Related Ena/Vasodilator-Stimulated Phosphoprotein Homology 1-Domain–Containing Protein-2 Critically Regulates Influenza A Virus–Induced Pneumonia

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