Identification of a human guanine nucleotide-releasing factor (H-GRF55) specific for Ras proteins.
A critical step in the activation of cellular Ras is the release of bound GDP. Oligonucleotide primers derived from a mouse cDNA sequence homologous to the Saccharomyces cerevisiae CDC25 gene product were used to screen a human brain cDNA library. The cloning led to the isolation of a 2.8-kb cDNA predicted to encode a protein of 488 amino acids. This protein was produced in Escherichia coli as a glutathione S-transferase fusion protein and functioned in vitro as a specific guanine nucleotide-releasing factor. Polyclonal antibodies raised against the last 281 amino acids of the protein allowed a protein in the molecular weight range of 55 kDa to be identified in human cortex homogenates. Analysis by Northern blotting led to the identification of a 5.5-kb mRNA in brain poly(A)+ RNA. The functionality of the encoded protein was evaluated after expression in different cells: (i) in Saccharomyces cerevisiae the effects of the cdc25.5 and RAS2 Ala-22 mutations were reversed; (ii) in chinese hamster ovary cells, a RAS-responsive element was transactivated as demonstrated by the expression of a CAT reporter gene under the control of the polyomavirus enhancer. Finally, in situ hybridization on of human chromosomes revealed a localization on band 15q2.4.
Schweighoffer
F
F
Rhone Poulenc Rorer, Centre de Recherche de Vitry-Alfortville, Vitry sur Seine, France.
Faure
M
M
Fath
I
I
Chevallier-Multon
M C
MC
Apiou
F
F
Dutrillaux
B
B
Sturani
E
E
Jacquet
M
M
Tocque
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002540:Cerebral Cortex / Q000378:metabolism*; D002871:Chromosome Banding; D002874:Chromosome Mapping; D002884:Chromosomes, Human, Pair 15; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1
C542351:CDC25 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D011061:Poly A; D012313:RNA; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)