Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

Abstract

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a 13-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, -3-fold less potently, the Mn2'-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyrl proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of -150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the 1B-galactosidase-Cdc25 fusion protein.ras is a family of proto-oncogenes which are highly conserved in evolution from yeasts to humans. ras genes are expressed in most mammalian tissues, in which it is believed that they play a major role in mediating the transduction of proliferative signals. Nevertheless, the pathway that leads to the activation of ras and its effector, as well as their exact function in mammalian cells, remains unknown (2). The products of mammalian ras genes are membrane-associated GTP-binding proteins which exert their effect on the putative effector(s) in their GTP-bound form (4) and require facilitated hydrolysis of the GTP by GTPase-activating protein to inactivate the protein (25). GDP has a low rate of dissociation from these proteins, and reactivation requires an exchange factor. Although exchange activity of guanyl nucleotides bound to mammalian Ras has been detected in mammalian cells (14,37,38), the factors which catalyze guanyl nucleotide exchange have not yet been identified.In Saccharomyces cerevisiae, which contains two RAS genes, the Ras effector is adenylyl cyclase, the product of the CDC35ICYRI gene (...

Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins.

“…2A). These findings are in agreement with a previous report that analyzed endogenous CDC25 protein in wild-type cells (18). Of potential biological significance, but irrelevant to the issue at hand, we found that the level of expression of full-length CDC25 was considerably lower than that of any of the deletion mutants that lacked residues 1 to 877 (Fig.…”

Biochemical and Genetic Analysis of the Mitochondrial Response of Yeast to BAX and BCL-XL

“…Total-protein extracts were prepared following cell breakage using glass beads (21) or the Y-Per reagent (Pierce). Cells were broken with glass beads (0.4 to 0.6 mm in diameter) by vigorous vortexing in ice-cold buffer containing 100 mM HEPES (pH 8.0), 1% sodium dodecyl sulfate (SDS), 1% 2-mercaptoethanol, 5 mM EDTA, 10% glycerol, 2% Triton X-100, and a protease inhibitor mixture (Sigma).…”

A Candida albicans homolog of CDC25 is functional in Saccharomyces cerevisiae

“…Proteins were transferred to nitrocellulose using a semi-dry blotter (LKl3). The blot was blocked for 1 h at room temperature with Tris-buffered saline containing 5% powdered skimmed milk (Cadbury), 0.02% NaN, and 0.3% Tween 20 and reacted overnight in the same solution with immunopurified anti-Cdc25 antibody diluted 1 : 40 [26]. Specific blocking of the antibody was carried out by a l-h incubation at room temperature with a cell lysate prepared from E. coli expressing the C-terminus of Cdc25 against which the antibody was raised.…”

“…We have checked the expression of the gene CSC25 in strain cdc25'"[CSC25, 2 p] at three different temperatures by using polyclonal anti-Cdc25 antibodies [26] as described in Materials and Methods. As shown in Fig.…”

“…Ras activity in yeast cells is regulated by the Ira proteins, Ira1 and Ira2, which are homologous to the mammalian proteins GAP and NF1 [18,191, and by Cdc25. Genetic and biochemical experiments suggest that Cdc25 is the upstream regulator of Ras, serving to catalyze the GDP to GTP exchange reaction [20-251. Cdc25 is a large protein with a molecular mass of 200-180kDa and is associated with the plasma membrane [26,271. TWO point mutations in the CDC25 gene render the cells temperature-sensitive.…”