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PMID: 3313005 Published · ppublish English Journal Article

Heterologous expression and characterization of the human R-ras gene product.

Molecular and cellular biology ·Vol. 7 ·No. 8 ·1987-08-00 ·Pages 2845-56

Lowe DG, Goeddel DV

Abstract

We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.

MeSH Terms
Alleles Antibodies Antigen-Antibody Complex Cell Line Cloning, Molecular Escherichia coli/genetics Fibrosarcoma Humans Molecular Weight Oncogenes Plasmids Proto-Oncogene Proteins/genetics Proto-Oncogene Proteins p21(ras) Transcription, Genetic
Chemicals
Antibodies Antigen-Antibody Complex Proto-Oncogene Proteins HRAS protein, human Proto-Oncogene Proteins p21(ras)
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Lowe D G
Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Goeddel D V
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.7.8.2845-2856.1987
Published
1987-08-00
Pages
2845-56
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC367902
Subset
IM
External Links
PubMed source DOI: 10.1128/mcb.7.8.2845-2856.1987 PMC Full Text View journal details
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