Analysis of Ras Activation in Living Cells with GFP‐RBD

Bivona, Trever G.; Quatela, Steven; Philips, Mark R.

doi:10.1016/s0076-6879(05)07012-6

Cited by 30 publications

(32 citation statements)

References 16 publications

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“…A probe for the activation of Ras, a monomeric small G protein, consisted of Ras-binding domain (RBD)-CFP and CD8-YFP (63). Although the direct interaction between RBD and CD8 has not been elucidated, the close proximity, which was dependent on extracellular stimuli, was detected by FRET (bystander FRET) (64). CD8-YFP was localized to the entire secretory apparatus of cells, and the FRET measurement detected Ras activation on the endomembrane, which occurred relatively slowly compared with that on the plasma membrane.…”

Section: Bystander Fretmentioning

confidence: 99%

Development of Probes for Cellular Functions Using Fluorescent Proteins and Fluorescence Resonance Energy Transfer

Miyawaki

2011

Annu. Rev. Biochem.

208

193

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Many genetically encoded probes that employ fluorescent proteins and fluorescence resonance energy transfer (FRET) have been developed to better understand the spatiotemporal regulation of various cellular processes. The different types of FRET and measurement techniques necessitate characterization of their specific features. Here I provide theoretical and practical comparisons of bimolecular and unimolecular FRET constructs, intensity-based and lifetime-based FRET measurements, FRET imaging using live- and fixed-cell samples, green fluorescent protein-based and chemical fluorophore-based FRET, and FRET efficiency and indices. The potential benefits and limitations of a variety of features in the technologies using fluorescent proteins and FRET are discussed.

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Section: Bystander Fretmentioning

confidence: 99%

Development of Probes for Cellular Functions Using Fluorescent Proteins and Fluorescence Resonance Energy Transfer

Miyawaki

2011

Annu. Rev. Biochem.

208

193

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“…Recruitment assays [42][43][44][45][46] using GFP-tagged RBDs for Rhotekin, POSH, and PAK were used to monitor changes in the subcellular distribution of active endogenous Rho GTPases. Fixed cells expressing HA-Ect2 in each specific location (vector: diffuse; WT and DH-mutant: nucleus; NLSmutant: cytoplasm) that co-expressed GFP-RBD were scored for GFP-RBD localization based on the following categories: nuclear-excluded, plasma membrane-highlighted, diffuse nuclear and cytoplasmic localization, nuclear-highlighted.…”

Section: Ovarian Tumor Tissue Microarray (Tma) Immunohistochemistrymentioning

confidence: 99%

The Role of Ect2 Nuclear RhoGEF Activity in Ovarian Cancer Cell Transformation

et al. 2013

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Ect2, a Rho guanine nucleotide exchange factor (RhoGEF), is atypical among RhoGEFs in its predominantly nuclear localization in interphase cells. One current model suggests that Ect2 mislocalization drives cellular transformation by promoting aberrant activation of cytoplasmic Rho family GTPase substrates. However, in ovarian cancers, where Ect2 is both amplified and overexpressed at the mRNA level, we observed that the protein is highly expressed and predominantly nuclear and that nuclear but not cytoplasmic Ect2 increases with advanced disease. Knockdown of Ect2 in ovarian cancer cell lines impaired their anchorage-independent growth without affecting their growth on plastic. Restoration of Ect2 expression rescued the anchorage-independent growth defect, but not if either the DH catalytic domain or the nuclear localization sequences of Ect2 were mutated. These results suggested a novel mechanism whereby Ect2 could drive transformation in ovarian cancer cells by acting as a RhoGEF specifically within the nucleus. Interestingly, Ect2 had an intrinsically distinct GTPase specificity profile in the nucleus versus the cytoplasm. Nuclear Ect2 bound preferentially to Rac1, while cytoplasmic Ect2 bound to RhoA but not Rac. Consistent with nuclear activation of endogenous Rac, Ect2 overexpression was sufficient to recruit Rac effectors to the nucleus, a process that required a functional Ect2 catalytic domain. Furthermore, expression of active nuclearly targeted Rac1 rescued the defect in transformed growth caused by Ect2 knockdown. Our work suggests a novel mechanism of Ect2-driven transformation, identifies subcellular localization as a regulator of GEF specificity, and implicates activation of nuclear Rac1 in cellular transformation.

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“…H-Ras and H-RasG12V are in pDCR, pEGFP, or monomeric red fluorescent protein (mRFP) plasmid (Rotblat et al, 2004). The RBD-GFP was a gift from Mark Philips (New York University, New York, NY) (Bivona et al, 2005). Pleckstrin homology (PH)-phospholipase (PL) C␦ was in pEGFP or pECFP and PH-protein kinase B (Akt) in mRFP were from Tamas Balla (National Institutes of Health, Bethesda, MD).…”

Section: Cells Plasmids and Transfectionmentioning

confidence: 99%

“…Next, we examined the effect of EGF treatment on Ras activation in live COS-7 cells cotransfected with a RBD-GFP chimera, which binds Ras-GTP specifically (Bivona et al, 2005). Within 30 s of EGF addition, RBD-GFP was recruited from the cytosol onto the PM colocalizing with H-Ras in PM ruffles ( Figure 1D and Supplemental Video 1).…”

Section: H-ras Associates With the Cie Pathwaymentioning

confidence: 99%