c-Raf-1 RBD Associates with a Subset of Active v-H-Ras

Fridman, Masha; Walker, Francesca; Catimel, Bruno; Domagała, Teresa; Nice, Edouard C.; Burgess, Antony W.

doi:10.1021/bi001224x

Cited by 11 publications

(9 citation statements)

References 41 publications

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“…As discussed previously, R89 is the single residue of the Raf RBD, which is critical for binding to h-ras. Interestingly, Q66A and T68A decrease the affinity for h-ras (22), whereas A85K increase the affinity putatively by allowing for binding to a wider range of GTPase conformers (23,24). Consistent with the latter observation, our data indicates a strong selection for lysine at position 85.…”

Section: Hierarchy In the Hydrophobicsupporting

confidence: 87%

Massive sequence perturbation of a small protein

Campbell-Valois

Tarassov²,

Michnick³

2005

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

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Most protein topologies rarely occur in nature, thus limiting our ability to extract sequence information that could be used to predict structure, function, and evolutionary constraints on protein folds. In principle, the sequence diversity explored by a given protein topology could be expanded by introducing sequence perturbations and selecting variant proteins that fold correctly. However, our capacity to explore sequence space is intrinsically limited by the enormous number of sequences generated from the 20 amino acids and the limited number of variants likely to fold. Here we sought to test whether the sequence space for naturally existing proteins can be explored by simple, sequential degeneration of a complete set of short sequence segments of a model protein, without long-range covariation. Using the Raf ras binding domain as a model of a small protein capable of autonomous folding, we degenerated 72 of 76 positions of the primary structure for the 20 amino acids in segments of four to seven residues defined by secondary structure and selected the folded species for interaction with h-ras by using an in vivo survival-selection assay. The methodology presented allowed for rigorous statistical analysis and comparison of sequence diversity. The ensemble of sequence variants of Raf ras binding domain obtained have recaptured the diversity observed for the ubiquitin-roll topology. A signature sequence for this fold and the implication of this strategy to protein design and structure prediction are discussed.massive mutagenesis ͉ protein-fragment complementation assay ͉ protein structure topology ͉ ubiquitin superfold ͉ Raf ras binding domain

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Section: Hierarchy In the Hydrophobicsupporting

confidence: 87%

Massive sequence perturbation of a small protein

Campbell-Valois

Tarassov²,

Michnick³

2005

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

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“…The Ras proteins were loaded with nucleotides as described (30). The samples were stored at Ϫ20°C in the presence of nucleotides and MgCl 2 , since removal of these reagents led to inactivation of Ras-GTP upon prolonged storage, an observation that is in accordance with findings already reported (35). Plasmids for H-RasG12V and H-RasG12V-C186S have been described elsewhere (36).…”

Section: Journal Of Biological Chemistry 26505mentioning

confidence: 54%

B- and C-RAF Display Essential Differences in Their Binding to Ras

Fischer

Hekman

Kuhlmann

et al. 2007

Journal of Biological Chemistry

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Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD).Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B-and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

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“…Importin-␣, importin-␤, IBB domain, and anti-GST IgG were immobilized onto a carboxymethyldextran sensor chip using NHS/EDC coupling as described previously (25). GST-importin-␤ and GST-IBB were captured onto immobilized anti-GST IgG, and biotinylated NLS peptides were immobilized onto streptavidin-coated surfaces according to protocols described previously (26,27). The samples for analyses were prepared at various concentrations in HBS buffer (10 mM Hepes, pH 7.4, containing 3.4 mM EDTA, 0.15 mM NaCl, and 0.005% (v/v) Tween 20) and were injected (30 l) over the sensor surface at a flow rate of 10 l min Ϫ1 .…”

Section: Methodsmentioning

confidence: 99%

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Catimel¹,

Teh²,

Fontes³

et al. 2001

Journal of Biological Chemistry

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144

128

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Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-␣/␤ heterodimer. Importin-␣ contains the NLS binding site, whereas importin-␤ mediates the translocation through the nuclear pore. We characterized the interactions involving importin-␣ during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-␣ is shown to exist in a monomeric autoinhibited state (as- Nucleocytoplasmic transport occurs through nuclear pore complexes, large supramolecular structures that penetrate the double lipid layer of the nuclear envelope. Most macromolecules require an active, signal-mediated transport process that enables the passage of particles up to 25 nm in diameter (ϳ25 MDa). The first and best characterized nuclear targeting signals are the classical nuclear localization sequences (NLSs) 1 that contain one or more clusters of basic amino acids (1). NLSs do not conform to a specific consensus sequence and fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters.Despite the sequence variability, the classical NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of ␣ and ␤ subunits (for recent reviews see Refs. 2-6). Importin-␣ contains the NLS-binding site, and importin-␤ is responsible for the translocation of the importincargo complex through the pore. Transfer through the pore is facilitated by the proteins Ran (Ras-related nuclear protein) and nuclear transport factor-2. Once inside the nucleus, importin-␤ binds to Ran-GTP to effect the dissociation of the import complex; the importin subunits return to the cytoplasm separately and without the cargo. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP-and GDP-bound forms of Ran between the cytoplasm and the nucleus. This distribution is in turn controlled by various Ran-binding regulatory proteins.Importin-␣ consists of two structural and functional domains, a short basic N-terminal importin-␤-binding (IBB) domain (7-9), and a large NLS-binding domain comprising armadillo (Arm) repeats (10). The monopartite NLSs bind at a major site located between the first and fourth Arm repeats and additionally at a minor site spanning repeats 4 -8 (11-13). The bipartite NLSs span the two binding sites, with each site recognizing one of the basic clusters (12, 13). The linker sequence between the two basic clusters makes few contacts with the receptor, consistent with its tolerance to mutations. The affinity of the importin-targeting sequence interaction is a critical parameter in determining transport efficiency (3).The structure of mouse importin-␣ showed that in the absence of importin-␤ or NLS-containing proteins, a part of the IBB domain occupies the major NLS-binding site (14). Based on this observation, it was sugges...

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c-Raf-1 RBD Associates with a Subset of Active v-H-Ras

Cited by 11 publications

References 41 publications

Massive sequence perturbation of a small protein

Massive sequence perturbation of a small protein

B- and C-RAF Display Essential Differences in Their Binding to Ras

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

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