Determinants of Ras proteins specifying the sensitivity to yeast Ira2p and human p120-GAP.

Parrini, Maria Carla; Bernardi, Alberto De; Parmeggiani, Andrea

doi:10.1002/j.1460-2075.1996.tb00448.x

Cited by 19 publications

(22 citation statements)

References 22 publications

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“…In addition, mutations in this domain lead to diminished responses to Cdc24p-mediated nucleotide exchange activity (58), suggesting that this domain of Cdc42p plays an important role in its function. The analogous domain in S. cerevisiae Ras2p is involved in the interaction between Ras2p and its GTPaseactivating protein, Ira2p (70,71). This domain in the Ras crystal structure corresponds to the turn between loop 6 and the ␣3 helix (72), a region of the protein that is predicted to be in close proximity to bound nucleotide.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the Mechanisms of Action of the Saccharomyces cerevisiae Dominant Lethal cdc42 G12V and Dominant Negative cdc42 D118A Mutations

Davis

Richman

Deliduka

et al. 1998

Journal of Biological Chemistry

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The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42 D118A mutant and the lethal, gain of function cdc42 G12V mutant were examined. Cdc42 D118A,C188S p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the twohybrid system, which correlated with the temperature dependence of the cdc42 D118A phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42 D118A action. Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42 D118A,C188S p-Cdc24p interactions and could suppress the cdc42 D118A mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. The cdc42 G12V mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1⌬ and cla4⌬ cells but not ste20⌬ cells, suggesting that the mechanism of cdc42 G12V action is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors of cdc42 G12V were also identified that displayed a dominant negative phenotype at 16°C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.The establishment of cell polarity is crucial for the control of many cellular and developmental processes, such as the generation of cell shape, the intracellular movement of organelles, and the secretion and deposition of new cell surface constituents (1). Polarized growth in the yeast Saccharomyces cerevisiae occurs in response to both internal and external signals, resulting in different morphological structures (2-5). The mechanics of cell polarity initiation during the mitotic cell cycle can be divided into three sequential phases: (i) nonrandom bud site selection; (ii) organization of proteins at the bud site; and (iii) bud emergence and polarized growth. Genetic and biochemical studies have identified over 25 proteins, including several GTPases and components of the actin cytoskeleton, that are involved in the regulation of the cell polarity pathway in S. cerevisiae (1, 6, 7).At least six members of the Ras superfamily of GTPases (Rsr1p/Bud1p, Cdc42p, Rho1p, Rho2p, Rho3p, and Rho4p) are involved in controlling cell polarity in S. cerevisiae. These proteins are active when in the GTP-bound state and inactive in the GDP-bound state (8, 9). The activity of these GTPases is controlled by regulatory proteins, such as guanine-nucleotide exchange factors, GTPase-activating proteins, and guanine-nucleotide dissociation inhibitors, as well as by the intracellular localization of the GTPase. Rsr1p/Bud1p is a member of the Ras subfamily and is responsible for bud site selection at one of the two cell poles, but it is not required for bud emergence or polari...

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Section: Discussionmentioning

confidence: 99%

Analysis of the Mechanisms of Action of the Saccharomyces cerevisiae Dominant Lethal cdc42 G12V and Dominant Negative cdc42 D118A Mutations

Davis

Richman

Deliduka

et al. 1998

Journal of Biological Chemistry

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“…Whether switch II is important for interaction with all Ras e ectors is presently not known. Mutation of yet other residues (e.g., residues 92 or 106) suggests a third region of contact between Ras and Ras GAPs and possibly other e ectors (Morcos et al, 1996;Parrini et al, 1996;Yoder-Hill et al, 1995). The three dimensional structure of Ras GDP bound to GAP-CAT has recently been solved (Sche zek et al, 1997) and consistent with mutational studies, residues of the phosphate binding loop (residues 10 to 16), switch regions I (30 to 37) and II (59 to 76) and possibly helix a3 (87 to 98) on Ras participate in binding interactions.…”

Section: Ras Mediates Its Actions Through Interaction With Multiple Ementioning

confidence: 99%

Increasing complexity of Ras signaling

et al. 1998

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The initial discovery that ras genes endowed retroviruses with potent carcinogenic properties and the subsequent determination that mutated ras genes were present in a wide variety of human cancers, prompted a strong suspicion that the growth-promoting actions of mutated Ras proteins contribute to their aberrant regulation of growth stimulatory signaling pathways. In 1993, a remarkable convergence of experimental observations from genetic analyses of Drosophila, S. cerevisiae and C. elegans as well as biochemical and biological studies in mammalian cells came together to de®ne a clear role for Ras in signal transduction. What emerged was an elegant linear signaling pathway where Ras functions as a relay switch that is positioned downstream of cell surface receptor tyrosine kinases and upstream of a cytoplasmic cascade of kinases that included the mitogen-activated protein kinases (MAPKs). Activated MAPKs in turn regulated the activities of nuclear transcription factors. Thus, a signaling cascade where every component between the cell surface and the nucleus was de®ned and conserved in worms,¯ies and man. This was a remarkable achievement in our e orts to appreciate how the aberrant function of Ras proteins may contribute to the malignant growth properties of the cancer cell. However, the identi®cation of this pathway has proven to be just the beginning, rather than the culmination, of our understanding of Ras in signal transduction. Instead, we now appreciate that this simple linear pathway represents but a minor component of a very complex signaling circuitry. Ras signaling has emerged to involve a complex array of signaling pathways, where cross-talk, feedback loops, branch points and multi-component signaling complexes are recurring themes. The simplest concept of a signaling cascade, where each component simply relays the same message to the next, is clearly not the case. In this review, we summarize our current understanding of Ras signal transduction with an emphasis on new complexities associated with the recognition and/or activation of cellular e ectors, and the diverse array of signaling pathways mediated by interaction between Ras and Ras-subfamily proteins with multiple e ectors.

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“…32 P]GTP (227 Bq⅐pmol Ϫ1 ) for 10 min at 30°C, as described above for the p21⅐[␥- 35 S]GTP␥S complex. Specific Activity of p120-GAP-The p120-GAP activity was measured at a concentration of 10 nM Ha-Ras p21-bound GTP and the obtained values extrapolated to 1 M Ha-Ras p21-bound GTP for comparison with the data of the literature.…”

Section: Methodsmentioning

confidence: 99%

“…To determine the effect of GAP on the activity of the exchange factor on both active and inactive states of Ras, the complex formed by p21 with the nonhydrolyzable GTP analogue [␥- 35 S]GTP␥S or with [ 3 H]GDP was used. It is known that p120-GAP and C-GAP have an affinity for GTP-bound p21 much higher (Ͼ100 times) than for GDPbound p21 (9,24).…”

Section: Stimulation By C-sdc25p Of the Ha-ras Gtp␥s/gtp␥s Exchange Imentioning

confidence: 99%

“…Since the apparent dissociation rate constant of p21⅐[␥- 35 S]GTP␥S is a measure of the GEF activity, this corresponds to a 46% inhibition of the GEF activity in the GTP␥S/GTP␥S exchange. Most important, this effect was specific for the active form of p21, since no GAP inhibition was detectable if p21⅐[␥- 35 S]GTP␥S was replaced by p21⅐ [ 3 H]GDP (Fig. 2B).…”

Section: Stimulation By C-sdc25p Of the Ha-ras Gtp␥s/gtp␥s Exchange Imentioning

confidence: 99%

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A New Function of p120-GTPase-activating Protein

Giglione

Parrini

Baouz

et al. 1997

Journal of Biological Chemistry

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This work studies the coordination of the action of GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF) on activated human c-HaRas p21. Purified human p120-GAP was obtained with a new efficient procedure. To distinguish the GTPase-activating effect of p120-GAP from other effects dependent on the interaction with activated Ha-Ras, the nonhydrolyzable GTP analogue guanosine 5-O-(thiotriphosphate) (GTP␥S) was used. The results showed that the GTP␥S/GTP␥S exchange enhanced by the C-terminal catalytic domain of the yeast GEF Sdc25p (C-Sdc25p) is prevented by p120-GAP. This effect is strictly specific for the activated form of Ha-Ras, the target of GAP; no effect on Ha-Ras⅐GDP was detectable. The GAP catalytic domain also inhibited C-Sdc25p but to a lower extent. The interfering effect by p120-GAP was also evident in a homologous mammalian system, using full-length mouse RasGEF, its C-terminal half-molecule, or C-terminal catalytic domain. As a consequence of this inhibition, presence of p120-GAP enhanced the regeneration of HaRas⅐GTP␥S by GEF at a GDP:GTP␥S ratio mimicking the in vivo GDP:GTP ratio. Our work describes a novel function of p120-GAP and suggests a mechanism by which GAP protects Ha-Ras⅐GTP in vivo against unproductive exchanges. This constrain is likely involved in the regulation of the physiological GDP/GTP cycle of Ras and in the action of p120-GAP as downstream effector of Ras. Helix ␣3 is proposed as a Ras element playing a key-role in the interference between GAP and GEF on Ras.Ras proteins are molecular switches of a pathway regulating cell growth and differentiation by cycling between two conformations: the active GTP-bound state and the inactive GDPbound state (1). As for most GTPases, the GDP/GTP cycle of Ha-Ras is controlled by two kinds of regulators, the GTPase activating protein (GAP) 1 and the GDP/GTP exchange factor (GEF). Thus, the level of Ha-Ras⅐GTP depends on the ratio of the activities of these two regulators and consequently, conditions influencing their activity affect the function of Ha-Ras. The mechanisms involved in the regulation of these two ligands have only been in part clarified. For instance, why only in particular conditions is the decrease in GAP activity associated with the increase in the concentration of p21⅐GTP (2, 3)? Why can overexpression of Ras be associated with a very low percentage of activated GTP-bound state (Ͻ0.3%) (4)? Upon activation of receptor or nonreceptor tyrosine kinase, p120-GAP is phosphorylated in vivo, but it is as yet unclear whether this modification is involved in the regulation of its negative effect on Ha-Ras⅐GTP (5). Concerning GEF, the activation of Ha-Ras by tyrosine kinase-linked receptors has been reported to depend on the transient translocation of the ubiquitous Ras⅐GEF SOS to the cell membrane (5), a process whose regulation still presents several unclear aspects. Recent work in vivo has suggested that Ca 2ϩ , calmodulin (6), and phosphorylation (7) control the activity of the neuronal CDC25-like RasGEF, b...

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Determinants of Ras proteins specifying the sensitivity to yeast Ira2p and human p120-GAP.

Cited by 19 publications

References 22 publications

Analysis of the Mechanisms of Action of the Saccharomyces cerevisiae Dominant Lethal cdc42 G12V and Dominant Negative cdc42 D118A Mutations

Analysis of the Mechanisms of Action of the Saccharomyces cerevisiae Dominant Lethal cdc42 G12V and Dominant Negative cdc42 D118A Mutations

Increasing complexity of Ras signaling

A New Function of p120-GTPase-activating Protein

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