The structure of Ras protein: a model for a universal molecular switch

Wittinghofer, Alfred; Pai, Emil F.

doi:10.1016/0968-0004(91)90156-p

Cited by 299 publications

(229 citation statements)

References 27 publications

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“…The latter observation suggests that the placement of a hydrophobic amino acid at this relative position is critical, possibly because of its influence on local secondary and tertiary structures. That the substitution of a glycine at this position could have a detrimental effect is supported by the finding that the transforming potential of Ras can be activated as a result of the converse change, that is, the substitution of a valine for a glycine residue at position 12 of the Ras protein that is associated with a local, though critical, conformational change (Wittinghofer and Pai 1991). Additionally, the activity of chicken EGFR is stimulated by the deliberate replacement of an isoleucine for an invariant valine residue (position 702 in human EGFR) within the ATP-binding pocket (Shu et al 1990).…”

Section: Discussionmentioning

confidence: 68%

The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.

Luetteke¹,

Phillips²,

Qiu³

et al. 1994

Genes Dev.

406

286

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Mice harboring the waved-1 {wa-1) and waved-2 {wa-2) mutations exhibit skin and eye abnormalities that are strikingly similar to those of TGF-a-deficient mice, and wa-1 and TGF-a were recently shown to be allelic. Because the wa-2 mutation was mapped previously to the vicinity of the EGF/TGF-a receptor (EGFR) gene on mouse chromosome 11, we hypothesized that the wa-2 phenotype might result from a defect in either the expression or activity of EGFR, or both. In the present report, we show that EGFR mRNA and protein of normal size are expressed in wa-2 liver and skin at levels that are comparable to those in the corresponding normal tissues, and that the ability of wa-2 EGFR to bind ligand is unaltered. However, ligand-dependent autophosphorylation of wa-2 EGFR is diminished 5-to 10-fold in vitro, and the ability of wa-2 EGFR to phosphorylate an exogenous substrate is reduced by >90% compared with that of the control receptor. EGF-induced tyrosine phosphorylation, including that of EGFR itself, is also diminished in skin, particularly at lower doses of exogenous EGF. To establish the nature of the wa-2 mutation, we determined the nucleotide sequence of the coding region of normal and wa-2 murine EGFR cDNAs. A comparison of these sequences revealed a single-nucleotide transversion resulting in the substitution of a glycine for a conserved valine residue near the amino terminus of the tyrosine kinase domain. The importance of this mutation was confirmed by showing that its introduction into an otherwise normal EGFR markedly reduced the receptor's tyrosine kinase activity in transfected Chinese hamster ovary cells. Finally, in situ hybridization analysis demonstrated expression of EGFR predominantly in the outer root sheath of active hair follicles in neonatal mice. As we previously localized TGF-a mRNA to the inner root sheath, this pattern of EGFR expression is consistent with the effect of the wa-2 mutation on hair structure, and together with our previous characterization of TGF-a-deficient mice, reveals a critical role for signaling by this ligand/receptor system in skin.[Key Words: EGF receptor; mutation; tyrosine kinase; in sitU; hair follicle] Received October 12, 1993; revised version accepted January 6, 1994. A range of cellular activities, including proliferation, migration, and differentiation, are controlled by signal transduction systems that are activated by the binding of polypeptide growth factors to cell-surface receptors. The majority of these receptors consist of an extracellular ligand-binding domain linked to a cytoplasmic sequence with intrinsic protein kinase activity that is specific for tyrosine residues (for review, see Ullrich and Schlessinger 1990; also see Carpenter and Wahl 1990). One of the better characterized tyrosine kinase receptors is the epidermal growth factor receptor (EGFR), a 170-kD integral membrane glycoprotein. The extracellular domain of EGFR includes two cysteine-rich regions. The intraConesponding author. cellular domain comprises a juxtamembrane region containing si...

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Section: Discussionmentioning

confidence: 68%

The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.

Luetteke¹,

Phillips²,

Qiu³

et al. 1994

Genes Dev.

406

286

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“…S4 B and C). Interestingly, the side chain of Asp80 in the G3 motif of GIMAP2 points toward the β-phosphate, whereas a glycine at the equivalent position acts as a sensor for the γ-phosphate in most other TRAFAC class GTPases, including the closely related Tocs and the more distantly related Ras (26). A glutamine or histidine residue positions the catalytic water molecule in Ras-like small GTPases, EF1/EF-TU, EF2/EF-G, eIF2, IF2, and SelB (27).…”

Section: Resultsmentioning

confidence: 99%

Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)

Schwefel

Fröhlich²,

Eichhorst³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix α7 packing against switch II. In the absence of α7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of α7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin-and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes. G protein | protein structure

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“…The behaviour of truncated c-Haras (amino acid residues 1 -166) blotted to nitrocellulose was strongly different from that in solution where biochemical properties like binding of guanine nucleotides were essentially unaltered compared to the full-length protein [8]. However, it should be noted that thermal stability and binding to nitrocellulose filters were different [ 81.…”

Section: Discussionmentioning

confidence: 99%

GTP‐blot Analysis of Small GTP‐binding Proteins

Klinz

1994

European Journal of Biochemistry

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Recombinant c-Ha-ras, ralA and rap2, but not raplA or raplB proteins retained their ability to bind [a-32P]GTP after SDSPAGE and transfer to nitrocellulose. Recombinant c-Ha-ras missing the C-terminal 23 amino acid residues failed to bind [a-32P]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [a-32P]GTP was decreased manyfold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process.Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of [a-32P]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [a-32P]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [CX-~~P]GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras.Our results demonstrate that detection and quantification of small GTP-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.Guanine nucleotide binding regulatory proteins such as ras proteins and a-subunits of heterotrimeric GTP-binding proteins exert their function by cycling between an inactive, GDP-bound, and an active, Members of the mammalian ras protooncogene group, c-Ha-ras, c-Ki-ras and N-ras each encode small guanine nucleotide binding proteins of 21 kDa which are associated with the inner side of the plasma membrane. Mammalian ras genes acquire transformation-inducing properties by single point mutations within their coding sequences [l, 31. Cloning and sequencing of cDNAs has revealed that a large and diverse family of ras-related small GTP-binding proteins exist with calculated molecular masses between 21 -25 kDa. Groups belonging to this family include the rap, ral, rho, rac and rab proteins [4,5]. Members of the family of small GTPbinding proteins show an extraordinary conservation of sequences dedicated to guanine nucleotide binding.To crystallize the human c-Ha-ras protooncogene product, John et al. [6] expressed the protein with a truncated Cterminus of 23 amino acids. The resulting c-Ha-ras (amino acid residues 1-166) was crystallized as a complex with the slowly hydrolyzing GTP analogue guanosine 5'- [P,yimido]triphosphate (GppNHp) and analyzed by X-ray crys-

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The structure of Ras protein: a model for a universal molecular switch

Cited by 299 publications

References 27 publications

The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.

The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.

Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)

GTP‐blot Analysis of Small GTP‐binding Proteins

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