Three-dimensional structures of H-ras p21 mutants: Molecular basis for their inability to function as signal switch molecules

Krengel, Ute; Schlichting, Ilme; Scherer, Anna; Schumann, Renate; Frech, Matthias; John, Jacob; Kabsch, Wolfgang; Pai, E.F.; Wittinghofer, Alfred

doi:10.1016/0092-8674(90)90018-a

Cited by 375 publications

(334 citation statements)

References 42 publications

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“…Structural studies, mutational experiments and the use of TS analogs have proven unable to elucidate the origin of the catalytic role of Gln 61 . In fact, all early works (e.g., Krengel et al 1990 ;Pai et al 1990) concluded that glutamine must be crucial as a general base that extracts a proton from the nucleophilic water molecule. This view was drastically changed by our study (Langen et al 1992), in which we demonstrated that Gln 61 cannot be the general base in Ras (since the energy of the proposed PT step in Ras is higher than the actual observed barrier for the GTPase reaction in solution).…”

Section: The Ras/gap System and The Mechanism Of The Corresponding Phmentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

333

448

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Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

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Section: The Ras/gap System and The Mechanism Of The Corresponding Phmentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

333

448

View full text Add to dashboard Cite

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“…This region comprises part of the switch I domain, a region of Ras (residues 30 ± 37) whose conformation di ers between GTP-and GDP-bound forms (Tong et al, 1989;Milburn et al, 1990;Krengel et al, 1990). In addition to the three Raf's, several proteins have been identi®ed with these characteristics (Figure 5), but a central goal in the ®eld, is to decide which of these are actually downstream targets of Ras.…”

Section: Ras Mediates Its Actions Through Interaction With Multiple Ementioning

confidence: 99%

“…The conformation of Ras-GTP di ers from Ras-GDP in two distinct regions, designated switch I (Ras residues 30 ± 37) and switch II (Ras residues 59 ± 76) (Tong et al, 1989;Milburn et al, 1990;Krengel et al, 1990). Since switch I overlaps with the Ras e ector domain sequences, a common feature of candidate Ras e ectors is the loss of binding to Ras-GTP due to mutations in the core Ras e ector domain (32 ± 40).…”

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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“…[13][14][15][16][17][18][19][20][21] Other, older reports have analyzed the interaction of the protooncogenic effector domain mutants with various guanine exchange factors and GTPase activating proteins. [22][23][24][25] In addition, mutating the highly conserved cysteine at position 186 of the CAAX box domain to serine results in a nonfarnesylated protein that fails to associate with membranes and becomes cytosolic. 26,27 Localization to the membranes and transforming activity of oncogenic Ras/S186 are rescued when Ras is targeted to the membranes by a myristoylation signal in its N-terminal domain.…”

mentioning

confidence: 99%

Inhibition of Ras oncogenic activity by Ras protooncogenes

Díaz

Lue

Mathews

et al. 2004

Intl Journal of Cancer

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Point mutations in ras genes have been found in a large number and wide variety of human tumors. These oncogenic Ras mutants are locked in an active GTP-bound state that leads to a constitutive and deregulated activation of Ras function. The dogma that ras oncogenes are dominant, whereby the mutation of a single allele in a cell will predispose the host cell to transformation regardless of the presence of the normal allele, is being challenged. We have seen that increasing amounts of Ras protooncogenes are able to inhibit the activity of the N-Ras oncogene in the activation of Elk in NIH 3T3 cells and in the formation of foci. We have been able to determine that the inhibitory effect is by competition between Ras protooncogenes and the N-Ras oncogene that occurs first at the effector level at the membranes, then at the processing level and lastly at the effector level in the cytosol. In addition, coexpression of the N-Ras protooncogene in thymic lymphomas induced by the N-Ras oncogene is associated with increased levels of p107, p130 and cyclin A and decreased levels of Rb. In the present report, we have shown that the N-Ras oncogene is not truly dominant over Ras protooncogenes and their competing activities might be depending on cellular context. Key words: oncogene inhibition; N-Ras; protooncogene; Ras processing; Ras effectors ras genes play an important role in a variety of differentiation processes and signal transduction, including the regulation of cell proliferation, vesicle movement, cell survival, T-cell activation, apoptosis and cytoskeleton. 1,2 The 3 ras genes encode 4 highly related proteins of 21 kd in size that are ubiquitously expressed: N-Ras, H-Ras, K-RasA and K-RasB. 2,3 Ras is synthesized on free ribosomes as a soluble inactive protein that requires several posttranslational modifications in order to reach the cell membranes where it exhibits its biologic activity. These modifications include prenylation, proteolysis, carboxymethylation and palmitoylation. 4 -7 Once activated by ligand-mediated extracellular stimuli, Ras induces the activation of effector molecules to propagate downstream signals in the cytoplasm and nucleus of the cell. Ras effectors preferentially bind to Ras-GTP through the effector domain loop found in amino acids 32-40. There are several different mammalian proteins that have been described as Ras effectors: Raf, phosphatidylinositol 3-kinase (PI3K), Ral GDP dissociation stimulator (RalGDS), members of the Rho family, mitogen-activating protein kinase 1 (MEKK1), Nore1, among others. 2,3,8 Specific point mutations in ras genes at codons 12, 13 and 61 have been found in a large number and wide variety of human tumors. 9 These mutations render Ras as an oncogene since it is constitutively active (in the GTP-bound form), leading to a deregulated activation of Ras function. 2,3,5,9 -11 In order to dissect the effects of the different downstream pathways of Ras, oncogenic mutants in the effector domain of Ras have been characterized. Thus, Ras/V12/E38 binds Raf and not PI3...

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Three-dimensional structures of H-ras p21 mutants: Molecular basis for their inability to function as signal switch molecules

Cited by 375 publications

References 42 publications

Why nature really chose phosphate

Why nature really chose phosphate

Increasing complexity of Ras signaling

Inhibition of Ras oncogenic activity by Ras protooncogenes

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