p21-ras effector domain mutants constructed by "cassette" mutagenesis.

Stone, James C.; Vass, William C.; Willumsen, Berthe M.; Lowy, Douglas R.

doi:10.1128/mcb.8.8.3565

Cited by 40 publications

(35 citation statements)

References 25 publications

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“…The increased responsiveness of rv2 and rv68 to weak and leaky mutations throughout the effector domain suggests that they may affect elements that function downstream of p21-target interaction. This interpretation seems quite likely in the case of rv2, since Met-36 has detectable transforming activity in NIH 3T3 cells (34). On the other hand, its dramatic responsiveness to several null v-H-ras mutations suggests that rv68 may represent a cell mutation that influences the way in which p21 combines with its target, in which case rv68 may genetically define the actual target of ras action.…”

Section: Resultsmentioning

confidence: 97%

“…Virus stocks were prepared by rescuing these temperature-sensitive alleles with Moloney murine leukemia virus. The pBW1406 linker substitution mutation (34) encodes a p21 species with a substantially remodelled effector domain (see Table 2) and was used as a negative control virus. The pBR1631 (c-H-ras) retroviral vector has been described elsewhere (34).…”

Section: Methodsmentioning

confidence: 99%

“…The pBW1406 linker substitution mutation (34) encodes a p21 species with a substantially remodelled effector domain (see Table 2) and was used as a negative control virus. The pBR1631 (c-H-ras) retroviral vector has been described elsewhere (34). The pBW1225 vector expresses the Cys-186-to-Ser mutation, encoding the same nonisoprenylated, non-membrane-localized form of p21 as pBW858 (42).…”

Section: Methodsmentioning

confidence: 99%

“…Effector domain mutations in v-Hras were constructed by the cassette method as described previously (34), and each mutation was transferred to a GV16-based retroviral vector (22) (12). Virus stocks were prepared by rescuing these temperature-sensitive alleles with Moloney murine leukemia virus.…”

Section: Methodsmentioning

confidence: 99%

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Genetic definition of ras effector elements.

Stone¹,

Blanchard²

1991

Mol. Cell. Biol.

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The products of ras genes may function as GTP-binding signal transducers, but the nature of their targets is largely unknown. To define genetically the cellular effector(s) of ras in rat fibroblast transformation, somatic variants that suppress the nontransforming phenotype of v-H-ras effector domain mutations were sought. Variant cell lines perturbed in the ras effector pathway were recovered, and the properties of one suggest that the primary target of ras action may be altered. In this cell variant, no single residue in the ras protein effector domain must be wild type to bring about transformation. In parental rat cells, conservative substitutions are tolerated in six of nine residues. Functional interaction with the target may not require a high degree of structural specificity in the ras protein effector domain.

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Section: Resultsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Genetic definition of ras effector elements.

Stone¹,

Blanchard²

1991

Mol. Cell. Biol.

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“…Included in this study were the v-H-ras effector domain substitutions described previously (68,70). Also studied were new v-H-ras effector domain mutations, including a complete set of codon 32 mutations constructed by the cassette mutagenesis technique (70).…”

Section: Ras Mutationsmentioning

confidence: 99%

Interaction of Activated Ras with Raf-1 Alone May Be Sufficient for Transformation of rat2 Cells

Stang

Bottorff

Stone

1997

Molecular and Cellular Biology

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v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.A number of cellular proteins bind Ras and are candidates for downstream effectors that function in cell transformation by the Ras oncogene. The Raf protein kinases (38,54,72,75,76,82), phosphatidylinositol-3-OH kinase (63), P120 GAP (71, 74), neurofibromin (4, 6), ralGDS (35), zeta PKC (18), and a number of other less well-defined species isolated in two hybrid screens (28, 73) all have the property of binding Ras. In each case, the interaction depends on Ras being in the GTPdependent "on" state, and in many cases the interaction has been documented with purified components in vitro. Each interaction also depends on the integrity of the effector region; severely impaired effector mutants generally block the interaction. In contrast, the interaction of Ras with proteins related to the CDC25 class of guanyl nucleotide exchange protein bind Ras in a complex with either GTP or GDP, and this interaction is not generally sensitive to mutations that remodel the effector loop.There is copious evidence that Ras regulation of the Raf family of protein kinases is important for growth and cell transformation. Raf members phosphorylate and activate MEK, a dual-specificity kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK), a serine kinase that has multiple targets (14,15,30,40). Growth factors that activate MAPK do so largely through activation of Ras (64). Both Raf-1 and MEK1 can be transforming (8,13,46). Ras signaling can largely be blocked by dominant negative Raf-1 and MEK1 mutants (13, 39) and by the drug PD098059, a specific inhibitor of MEK (20).Also, there is growing evidence that Ras can function by Raf-independent mechanisms to bring about oncogenic transformation in some situations. Transformation-impaired mutant Ras species with different binding specificity toward putative effectors show strong synergy in transformation assays, suggesting that multiple Ras-regulated pathways contribute to cell transformation (34,78). Different Ras effector mutants were also reported to cooperate to bring about stimulation of DNA synthesis in f...

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Ein photochemischer Schalter zur Kontrolle von Protein-Protein-Wechselwirkungen

Pollitt

Schultz

1998

Angewandte Chemie

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Durch Einbau nichtnatürlicher Aminosäuren, deren Seitenkette eine photolabile Gruppe trägt, läßt sich die Wechselwirkung zweier Proteine gezielt verhindern. Ein mutiertes Ras‐Protein, bei dem Asp 38 gegen den β‐o‐Nitrobenzylester (Nb) dieser Aminosäure ersetzt ist, wechselwirkt nicht mehr mit seinem Effektorprotein p120‐GAP (siehe unten) und weist daher nur seine intrinsische GTP‐Hydrolyseaktivität auf. Durch photoinduzierte Abspaltung der Nb‐Gruppe werden 50 % der p120‐GAP‐abhängigen GTPase‐Aktivität des Wildtyp‐Proteins wiederhergestellt.

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p21-ras effector domain mutants constructed by "cassette" mutagenesis.

Cited by 40 publications

References 25 publications

Genetic definition of ras effector elements.

Genetic definition of ras effector elements.

Interaction of Activated Ras with Raf-1 Alone May Be Sufficient for Transformation of rat2 Cells

Ein photochemischer Schalter zur Kontrolle von Protein-Protein-Wechselwirkungen

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