IRA2, an upstream negative regulator of RAS in yeast, is a RAS GTPase-activating protein.
The ras GTPase-activating protein (GAP), identified and characterized in mammalian cells, stimulates the intrinsic GTPase activity of ras proteins. We have previously proposed that the IRA genes, negative regulators of RAS genes in Saccharomyces cerevisiae, encode yeast homologs of the mammalian GAP. In this paper, we present the following evidence that a product of the IRA2 gene exhibits GAP activity similar to that of the mammalian GAP protein. (i) Extracts of yeast cells overexpressing IRA2 stimulated the GTPase activity of the yeast RAS2 protein. (ii) An epitope for a monoclonal antibody (12CA5) was added to the N terminus of the IRA2 protein. The GAP activity of extracts prepared from cells expressing this fusion protein was shown to be immunoprecipitable by 12CA5. (iii) An IRA2 protein fused to glutathione S-transferase (GST) was produced and partially purified from Escherichia coli cells. GAP activity was detected with this purified GST-IRA2 fusion protein. (iv) The GAP activity of IRA2 proteins described above did not stimulate the GTPase activity of the RAS2Val19 protein, a protein having an amino acid alteration analogous to that found in mammalian oncogenic ras proteins. This result parallels studies showing that mammalian GAP is incapable of stimulating the GTPase activity of mammalian oncogenic proteins. The remarkable conservation between the GAP activity in mammalian and yeast cells supports the idea that the function of GAP is to negatively regulate ras proteins in mammalian cells.
Tanaka
K
K
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
Lin
B K
BK
Wood
D R
DR
Tamanoi
F
F
D004926:Escherichia coli / Q000235:genetics; D020690:GTPase-Activating Proteins; D005800:Genes, Fungal; D005809:Genes, Regulator; D007700:Kinetics; D010957:Plasmids; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins