The Saccharomyces cerevisiae SDC25 C-domain gene product overcomes the dominant inhibitory activity of Ha-Ras Asn-17.

Schweighoffer, Fabien; Cai, Hong; Chevallier-Multon, M C; Fath, I; Cooper, Geoffrey M.; Tocqué, Bruno

doi:10.1128/mcb.13.1.39

Cited by 52 publications

(58 citation statements)

References 43 publications

(51 reference statements)

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“…This argument is supported by the finding that overexpressing a dominant active form of Ras (e.g. RasG12V) or an activator domain usually abolishes the inhibitory effect of dominant negative Ras (3,7). However, other explanations have been also proposed (8 -9) including low affinity of RasS17N for GTP and the inability of GTP to induce the RasS17N conformation necessary for binding and activating downstream effectors (4,10).…”

mentioning

confidence: 55%

Structure of the G60A Mutant of Ras

Ford¹,

Skowronek²,

Boykevisch

et al. 2005

Journal of Biological Chemistry

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Substituting alanine for glycine at position 60 in v-HRas generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 cells and to induce germinal vesicle breakdown in Xenopus oocytes. The crystal structure of the GppNpbound form of RasG60A unexpectedly shows that the switch regions adopt an open conformation reminiscent of the structure of the nucleotide-free form of Ras in complex with Sos. Critical residues that normally stabilize the guanine nucleotide and the Mg 2؉ ion have moved considerably. Sos binds to RasG60A but is unable to catalyze nucleotide exchange. Our data suggest that the dominant negative effect observed for RasG60A⅐GTP could result from the sequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchange factor ternary complex.Ras is an essential component of signal transduction pathways that regulate growth, proliferation, differentiation, and apoptosis in response to the activation of membrane-bound receptors (1, 2). Dominant negative Ras mutants have been widely used to elucidate the role of Ras in a variety of signaling pathways. The asparagine for serine mutant at position 17, RasS17N, is probably the most frequently used dominant negative form of Ras (3) and the success of this mutant popularized the use of dominant negative mutants to study the signaling of other small GTPases. Despite this success, the exact molecular details by which dominant negative GTPases exert their inhibitory function are a matter of debate in the literature. It is widely accepted that RasS17N blocks the ability of endogenous Ras to function by sequestering and depleting the intracellular pool of available guanine nucleotide exchange factor (GEF), 1 thereby blocking the activation of endogenous Ras (4 -6). This argument is supported by the finding that overexpressing a dominant active form of Ras (e.g. RasG12V) or an activator domain usually abolishes the inhibitory effect of dominant negative Ras (3, 7). However, other explanations have been also proposed (8 -9) including low affinity of RasS17N for GTP and the inability of GTP to induce the RasS17N conformation necessary for binding and activating downstream effectors (4, 10). To complicate matters, the S17N mutant of Rap1A, which a priori should behave like RasS17N, is unable to inhibit the activation of Rap1A by its exchange factor, C3G, in vitro (11). Understanding at the molecular level how a dominant negative Ras functions should shed light on its cellular role and help in designing new tools to dissect the signaling of Ras and other small G-proteins. Because it inhibits the activation of endogenous Ras, dissecting the action of a dominant negative Ras should also better our understanding of the reaction of nucleotide exchange. So far, the structure of a dominant negative Ras complex is lacking in the literature.The substitution of alanine for glycine at position 60 in v-H-Ras, v-H-RasG60A, generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 ...

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mentioning

confidence: 55%

Structure of the G60A Mutant of Ras

Ford¹,

Skowronek²,

Boykevisch

et al. 2005

Journal of Biological Chemistry

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“…These mutant proteins specifically block growth factor-induced morphological changes acting through endogenous Rac1 and Cdc42Hs, respectively (27,46). They are thought to function by titrating out specific activating factors, possibly GDP/GTP exchange factors (15,16,36,49). Rac1 T17N blocked membrane ruffling ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The GTPase-Activating Protein n-Chimaerin Cooperates with Rac1 and Cdc42Hs To Induce the Formation of Lamellipodia and Filopodia

Kozma

Ahmed

Best³

et al. 1996

Molecular and Cellular Biology

144

121

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n-Chimaerin is a GTPase-activating protein (GAP) mainly for Rac1 and less so for Cdc42Hs in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1 T17N and Cdc42Hs T17N , respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding was ineffective in inducing morphological changes, while a mutant lacking GAP activity alone was effective. Microinjected n-chimaerin colocalized in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases.

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“…These "dead-end complexes" competitively inhibit the formation of the GTP-bound, activated form of the endogenous GTPase. This mechanism has been demonstrated to underlie the dominant-negative effects observed by expression of GTPbinding mutant forms of Sec4p WalchSolimena et al, 1997), Ras (Feig and Cooper, 1988;Hwang et al, 1993;Schweighoffer et al, 1993), Rab3a (Burstein et al, 1992), Rab3d (Chen et al, 2003), and Ypt1 (Jones et al, 1995). Thus the dominant effects of Rab10-T23N may reflect competitive binding of the mutant protein to a Rab10 GEF.…”

Section: Mechanism Of the Inhibitory Effects Of Mutant Forms Of Gfp-rmentioning

confidence: 99%

Rab10 Regulates Membrane Transport through Early Endosomes of Polarized Madin-Darby Canine Kidney Cells

Babbey

Ahktar

Wang

et al. 2006

MBoC

155

129

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The Saccharomyces cerevisiae SDC25 C-domain gene product overcomes the dominant inhibitory activity of Ha-Ras Asn-17.

Cited by 52 publications

References 43 publications

Structure of the G60A Mutant of Ras

Structure of the G60A Mutant of Ras

The GTPase-Activating Protein n-Chimaerin Cooperates with Rac1 and Cdc42Hs To Induce the Formation of Lamellipodia and Filopodia

Rab10 Regulates Membrane Transport through Early Endosomes of Polarized Madin-Darby Canine Kidney Cells

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