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PMID: 3088563 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Isolation of ras GTP-binding mutants using an in situ colony-binding assay.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 83 ·No. 13 ·1986-07-00 ·Pages 4607-11

Feig LA, Pan BT, Roberts TM, Cooper GM

Abstract

We have developed a strategy to isolate mutant ras genes encoding proteins defective in GTP binding. Random in vitro mutagenesis of a v-Harvey (Ha)-ras expression vector was followed by an in situ GTP-binding assay on lysed bacterial colonies. Single amino acid substitutions at ras codon 83, 119, or 144 decreased the affinity of p21 for GTP by a factor of 25-100 primarily as a consequence of increased rates of dissociation of GTP from p21. Nevertheless, these mutant genes induced transformation of NIH 3T3 cells with efficiencies comparable to wild-type v-Ha-ras. In transformed cells, mutant p21s were phosphorylated to a degree similar to that of wild-type v-Ha-ras p21, suggesting that a decrease in affinity by a factor of 100 did not prevent the mutant ras protein from binding GTP in vivo. These results are discussed with respect to the role of GTP in the regulation of p21 function.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Binding Sites Cell Transformation, Viral Chromosome Mapping Cloning, Molecular GTP-Binding Proteins/genetics Guanosine Triphosphate/metabolism Mice Mutation Oncogene Proteins, Viral/genetics,metabolism Phosphoproteins/metabolism Structure-Activity Relationship Transfection
Chemicals
Oncogene Proteins, Viral Phosphoproteins Guanosine Triphosphate GTP-Binding Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Feig L A
Pan B T
Roberts T M
Cooper G M
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38 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.83.13.4607
Published
1986-07-00
Pages
4607-11
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC323790
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.83.13.4607 PMC Full Text View journal details
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