Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac

Dupuy, Aurélien G.; L’Hoste, Sébastien; Cherfils, Jacqueline; Camonis, Jacques; Gaudriault, Georges; Gunzburg, Jean de

doi:10.1038/sj.onc.1208647

Cited by 26 publications

(27 citation statements)

References 61 publications

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“…In Fig. 4B, the effects of expressing dominant-negative Rap1, which blocks the activation of endogenous Rap1 by titrating GEFs (Dupuy et al, 2005;Feig, 1999), takes place within 2 hours. Comparatively, Rap1 is inactivated within 10 minutes of the onset of mitosis (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Because integrin activation is a prerequisite for integrin outside-in signalling, Rap1 activity ultimately influences integrin outside-in signalling and thereby regulates cell morphology (Arthur et al, 2004;Tsukamoto et al, 1999). To determine whether Rap1 activity controls HeLa-cell morphology, we expressed a constitutively active mutant (Rap1[Q63E]) or a powerful dominant-negative mutant (Rap1[S17A]) (Dupuy et al, 2005) of Rap1 in cells seeded on fibronectin-coated plates. Transfected cells were easily identifiable by their increased immunofluorescence when stained with an anti-Rap1 antibody (data not shown).…”

Section: Modulation Of Rap1 Activity Affects the Morphology Of Hela Cmentioning

confidence: 99%

“…Because a previous study had shown that Rap1 mutants are not fully active when fused to a N-terminal tag (Dupuy et al, 2005), we co-transfected cells with untagged mutant alleles of Rap1 and a vector encoding red fluorescent protein (RFP) in the ratio 3:1. Under these conditions, more than 95% of RFP-expressing cells showed markedly increased Rap1 levels compared with control untransfected cells, as assessed by immunofluorescence using anti-Rap1 antibodies (not shown).…”

Section: Misregulation Of Rap1 Affects the Regulation Of Cell Morpholmentioning

confidence: 99%

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Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis

Dao

Dupuy

Gavet

et al. 2009

Journal of Cell Science

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114

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At the onset of mitosis, most adherent cells undergo cell retraction characterised by the disassembly of focal adhesions and actin stress fibres. Mitosis takes place in rounded cells, and the two daughter cells spread again after cytokinesis. Because of the well-documented ability of the small GTPase Rap1 to stimulate integrin-dependent adhesion and spreading, we assessed its role during mitosis. We show that Rap1 activity is regulated during this process. Changes in Rap1 activity play an essential role in regulating cell retraction and spreading, respectively, before and after mitosis of HeLa cells. Indeed, endogenous Rap1 is inhibited at the onset of mitosis; conversely, constitutive activation of Rap1 inhibits the disassembly of premitotic focal adhesions and of the actin cytoskeleton, leading to delayed mitosis and to cytokinesis defects. Rap1 activity slowly increases after mitosis ends; inhibition of Rap1 activation by the ectopic expression of the dominant-negative Rap1[S17A] mutant prevents the rounded cells from spreading after mitosis. For the first time, we provide evidence for the direct regulation of adhesion processes during mitosis via the activity of the Rap1 GTPase.

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Section: Discussionmentioning

confidence: 99%

Section: Modulation Of Rap1 Activity Affects the Morphology Of Hela Cmentioning

confidence: 99%

Section: Misregulation Of Rap1 Affects the Regulation Of Cell Morpholmentioning

confidence: 99%

See 1 more Smart Citation

Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis

Dao

Dupuy

Gavet

et al. 2009

Journal of Cell Science

Self Cite

114

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“…Rap1 was also shown to interact directly with Vav2 and Tiam1 (Arthur et al, 2004), two Rac GEFs, as well as with IQGAP (Jeong et al, 2007). In addition to these examples, Rap1 also interacts with DOCK4 (Yajnik et al, 2003), as well as with C3G (Dupuy et al, 2005) and PDZ-GEF1/PDZ-GEF2 (de Rooij et al, 1999;, Rap GEFs that associate directly with cell adhesion proteins (Hogan et al, 2004;Sakurai et al, 2006). It is noteworthy that RNA silencing of DOCK4 was shown to perturb cell adhesion (Yajnik et al, 2003).…”

Section: Cytokines and Growth Factorsmentioning

confidence: 99%

Anchoring Junctions As Drug Targets: Role in Contraceptive Development

2008

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“…One explanation for the discrepancy could be compensatory changes mediated by other small GTPases, which may alleviate spatial memory deficits in the KOs but not in the N17Rap1 transgenic mice. The interfering N17Rap1 mutant is believed to act by titrating out Rap1 GDP/GTP-exchange factors (Dupuy et al, 2005) including EPAC (exchange nucleotide protein directly activated by cyclic adenosine-3Ј,5Ј-monophosphate), which activates not only Rap1 but several other small GTPases as well (Bos, 2006). Such broader inhibition of small GTPase signaling may block compensatory changes in the N17Rap1 transgenics.…”

Section: Contribution Of Cortical Input To La In Fear Learningmentioning

confidence: 99%

Enhanced Cortico-Amygdala Efficacy and Suppressed Fear in Absence of Rap1

Pan¹,

Vautier²,

Ito³

et al. 2008

J. Neurosci.

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Auditory fear conditioning, a model for fear learning, is thought to be mediated by synaptic changes in the cortical and thalamic inputs to the lateral amygdala (LA); however, the specific roles of both pathways are still debated. Here, we report that a CaMKII-␣-Cre-mediated knock-out (KO) of the rap1a and rap1b genes impaired synaptic plasticity and increased basal synaptic transmission in the cortical but not thalamic input to the LA via presynaptic changes: increases in glutamate release probability and the number of glutamate quanta released by a single action potential. Moreover, KO mice with alterations in the cortico-LA pathway had impaired fear learning, which could be rescued by training with a more aversive unconditional stimulus. These results suggest that Rap1-mediated suppression of synaptic transmission enables plasticity in the cortico-amygdala pathway, which is required for fear learning with a moderately aversive unconditional stimulus.

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Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac

Cited by 26 publications

References 61 publications

Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis

Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis

Anchoring Junctions As Drug Targets: Role in Contraceptive Development

Enhanced Cortico-Amygdala Efficacy and Suppressed Fear in Absence of Rap1

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