An activating mutant of Cdc42 that fails to interact with Rho GDP-dissociation inhibitor localizes to the plasma membrane and mediates actin reorganization

Gibson, Richard; Gandhi, Payal N.; Tong, Xiaofeng; Miyoshi, Jun; Takai, Yoshimi; Konieczkowski, Martha; Sedor, John R.; Wilson-Delfosse, Amy L.

doi:10.1016/j.yexcr.2004.07.033

Cited by 19 publications

(20 citation statements)

References 30 publications

(53 reference statements)

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“…BiFC data indicated that mutant Cdc42(R66Ep) could interact with Rdi1p, in contrast to previous studies of mammalian cells (18,36), but the membrane extraction and growth inhibition assays indicated that Rdi1p could not extract Cdc42(R66Ep) from the plasma membrane (Fig. 5), which agrees with analysis of the mammalian Cdc42(R66E) mutant protein.…”

Section: Discussionsupporting

confidence: 72%

Use of Bimolecular Fluorescence Complementation To Study In Vivo Interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae

2007

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Saccharomyces cerevisiae Cdc42p functions as a GTPase molecular switch, activating multiple signaling pathways required to regulate cell cycle progression and the actin cytoskeleton. Regulatory proteins control its GTP binding and hydrolysis and its subcellular localization, ensuring that Cdc42p is appropriately activated and localized at sites of polarized growth during the cell cycle. One of these, the Rdi1p guanine nucleotide dissociation inhibitor, negatively regulates Cdc42p by extracting it from cellular membranes. In this study, the technique of bimolecular fluorescence complementation (BiFC) was used to study the dynamic in vivo interactions between Cdc42p and Rdi1p. The BiFC data indicated that Cdc42p and Rdi1p interacted in the cytoplasm and around the periphery of the cell at the plasma membrane and that this interaction was enhanced at sites of polarized cell growth during the cell cycle, i.e., incipient bud sites, tips and sides of smalland medium-sized buds, and the mother-bud neck region. In addition, a ring-like structure containing the Cdc42p-Rdi1p complex transiently appeared following release from G 1 -phase cell cycle arrest. A homology model of the Cdc42p-Rdi1p complex was used to introduce mutations that were predicted to affect complex formation. These mutations resulted in altered BiFC interactions, restricting the complex exclusively to either the plasma membrane or the cytoplasm. Data from these studies have facilitated the temporal and spatial modeling of Rdi1p-dependent extraction of Cdc42p from the plasma membrane during the cell cycle.

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Section: Discussionsupporting

confidence: 72%

Use of Bimolecular Fluorescence Complementation To Study In Vivo Interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae

2007

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“…By expressing constitutively active RHO GTPases, they found no significant difference in the signalling between the forms that were able or unable to bind to RHOGDIs. Similarly, RHOGDI-null cells responded the same way as their wild-type counterparts when constitutively active mutants of RAC1 and CDC42 were overexpressed 19, 20 . This led them to conclude that RHOGDIs are not required for the translocation of RHO GTPases to their membrane destinations 19-21 .…”

Section: Rhogdi Functionsmentioning

confidence: 85%

“…The existence of two pathways by which RHO GTPases reach their membrane destinations provides an explanation for how constitutively active RHO proteins that cannot interact with RHOGDI can still exert their effects 19, 20 . Without binding to RHOGDI, a fraction of RHO GTPases continues to be transported to the plasma membrane through vesicle trafficking; this occurs at a reduced rate, but one sufficient to account for their phenotype.…”

Section: Rhogdi Functionsmentioning

confidence: 99%

“…Arg66, which is located in the switch II region and forms hydrogen bonds with Asp185, Pro30 and Ala31, is another key residue in the RHOGDI -RHO GTPase interaction; substitution of the RHO GTPase Arg66 for Glu (or of Arg68 in RHOA) abolishes the interaction with RHOGDIs 19-21 . Interestingly, the RHOGDI- RHO GTPase interaction can be rescued with a charge-reversing substitution in Asp185 in RHOGDI (Asp185Arg) 20 .…”

Section: Structural Analysis Of Rhogdi–rho Gtpasementioning

confidence: 99%

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The 'invisible hand': regulation of RHO GTPases by RHOGDIs

Garcı́a-Mata

Boulter

Burridge

2011

Nat Rev Mol Cell Biol

477

561

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Preface The 'invisible hand' is a term originally coined by Adam Smith in the Theory of Moral Sentiments to describe the forces of self-interest, competition, and supply and demand that regulate the resources in society. This metaphor continues to be used by economists to describe the self-regulating nature of a market economy. The same metaphor can be used to describe the RHO-specific guanine nucleotide dissociation inhibitor (RHOGDI) family, which operates in the background, as an invisible hand, using similar forces to regulate the RHO GTPase cycle.

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“…49 Specific mutations in Cdc42 that block its interaction with RhoGDI alter the membrane distribution of Cdc42, but do not prevent its activation of filopodia. 50,51 The importance of the RhoGDI interaction in recycling Cdc42 is probably in the redistribution and/or delivery of Cdc42 to an appropriate site. Given that ERM proteins can form discrete complexes with RhoGDI and DG, it could be that the ERM-RhoGDI complex serves to deliver inactive Cdc42 to the GEF, in this case Dbl, and does so by docking with DG where it releases Cdc42 for activation and RhoGDI for recharging.…”

Section: Discussionmentioning

confidence: 99%

Recruitment of Dbl by Ezrin and Dystroglycan Drives Membrane Proximal Cdc42 Activation and Filopodia Formation

Batchelor¹,

Higginson²,

Chen³

et al. 2007

Cell Cycle

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ACKnoWLEdgEmEntSWe are grateful to the following for supplies of antisera or plasmids, Art Alberts, P. Aspenstrom, Louise Anderson, Francis Barr and Sam Crouch, and to Kathryn Ayscough for comments on the manuscript. The work was funded by an MRC Career Establishment Grant to S.J.W., J.R.H. was in receipt of a BBSRC studentship and A.E. was supported by grants from the Italian Association for Cancer Research, Compagnia S. Paolo, and Ministero della Salute. ABStRACtDystroglycan is an essential laminin binding cell adhesion molecule, which is also an adaptor for several SH2 domain-containing signaling molecules and as a scaffold for the ERK-MAP kinase cascade. Loss of dystroglycan function is implicated in muscular dystrophies and the aetiology of epithelial cancers. We have previously demonstrated a role for dystroglycan and ezrin in the formation of filopodia structures. Here we demonstrate the existence of a dystroglycan:ezrin:Dbl complex that is targeted to the membrane by dystroglycan where it drives local Cdc42 activation and the formation of filopodia. Deletion of an ezrin binding site in dystroglycan prevented the association with ezrin and Dbl and the formation of filopodia. Furthermore, expression of the dystroglycan cytoplasmic domain alone had a dominant-negative effect on filopodia formation and Cdc42 activation by sequestering ezrin and Dbl away from the membrane. Depletion of dystroglycan inhibited Cdc42-induced filopodia formation. For the first time we also demonstrate co-localization of Cdc42 and dystroglycan at the tips of dynamic filopodia.

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An activating mutant of Cdc42 that fails to interact with Rho GDP-dissociation inhibitor localizes to the plasma membrane and mediates actin reorganization

Cited by 19 publications

References 30 publications

Use of Bimolecular Fluorescence Complementation To Study In Vivo Interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae

Use of Bimolecular Fluorescence Complementation To Study In Vivo Interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae

The 'invisible hand': regulation of RHO GTPases by RHOGDIs

Recruitment of Dbl by Ezrin and Dystroglycan Drives Membrane Proximal Cdc42 Activation and Filopodia Formation

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