MOLECULAR AND CELLULAR BIOLOGY, June 1992, p. 2653-2661

Vol. 12, No. 6

0270-7306/92/062653-09$02.00/0 Copyright © 1992, American Society for Microbiology

Anti-Cdc25 Antibodies Inhibit Guanyl Nucleotide-Dependent Adenylyl Cyclase of Saccharomyces cerevisiae and Cross-React with a 150-Kilodalton Mammalian Protein EITAN GROSS,1 IRIT MARBACH,1 DAVID

ENGELBERG,lt

MARISA SEGAL,1

GIORA SIMCHEN,2 AND ALEXANDER LEVITZKIl* Departments of Biological Chemistry' and Genetics, 2 The Alexander Silberman Institute of Life Sciences, The Hebrew University ofJerusalem, Jerusalem 91904, Israel Received 26 December 1991/Accepted 13 March 1992

mutation and can even suppress the disruption of the CDC25 (6, 28). This finding, as well as biochemical experiments (16, 20), suggests that the Cdc25 protein catalyzes the GDP-to-GTP exchange on Ras2 to convert the inactive GDP-bound Ras2 Cdc35 complex to its active GTP-bound form. Furthermore, the CDC25 gene product is essential for the glucose-induced rise of intracellular cyclic AMP (cAMP) (26), suggesting that the protein functions as a glucosemediated GDP-to-GTP exchanger. The study of the role of Cdc25, as well as that of other elements in the regulation of Ras-dependent adenylyl cyclase in S. cerevisiae, is likely to shed light on putative homologs of Cdc25 in other eukaryotes, since the Ras proteins in S. cerevisiae are highly homologous to other eukaryotic Ras proteins (4, 13). To further understand the relationship between the CDC25 gene product and Ras-dependent cyclase in S. cerevisiae and to attempt to identify the Cdc25 mammalian homolog(s), we have raised polyclonal antibodies against the C-terminal domain of this protein. Since this domain by itself is sufficient to confer GTP sensitivity of the yeast cyclase (11), we felt that such antibodies could be used to examine more directly the relationship of Cdc25 to Ras. Furthermore, this domain is highly conserved among the Cdc25 yeast homologs (5, 9, 19), and therefore antibodies raised against this domain may cross-react with mammalian Cdc25 homologs. In this study, we show for the first time that antibodies raised against the yeast Cdc25 cross-react with mammalian proteins from various tissues.

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 (30). Genetic and biochemical evidence demonstrates that the CDC25 gene product in S. cerevisiae is the upstream regulator of the Ras protein(s) (6, 15, 28). Mutations in RAS2 which lock the protein in its GTP-bound state (e.g., RAS2Val-19) suppress the cdc25's

gene

* Corresponding author. t Present address: Division of Chemistry, California Institute of Technology, Pasadena, CA 91125.

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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 RAS2Va-19 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.

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TABLE 1. S. cerevisiae and E. coli strains

TT1A-3 MS-1

T1A-4 2WT C13-ABYS86-1

C13-ABYS86-2

MATa his3 leu2 ura3 trpl ade8 canl MATa his3 leu2 ura3 trpl ade8 cdc25::URA3, with plasmid pTPKl (TPKJ 2,um LEU2) MATa leu2 ura3 trpl ade8 cdc25::URA3 his3::pHIS3-RAS2va`l9 MATot his3 leu2 ura3 trpl ade8 cdc25::URA3, with plasmid pCDC25 (CDC25 2pm LEU2) AL4Ta leu2-3,112 trpl ura3-52 ade2 canl met AM Ta pral-1 prbl-J prcl-l cpsl-3 ura3 leu2-3 his, with plasmid YEp352 (2,um URA3) MATo pral-1 prbl-l prcl-1 cpsl-3 ura3 leu2-3 his, with plasmid pRG1 (SDC25 C terminus in YEp352) A(lac, pro)F' laclqZ M1S pro', with plasmid pUR290 A(lac pro)F' lacIqZ M15 pro', with plasmid pUR290-25 C600(XcI857ABamHIAHI) lacZ XA21::TnlO, with plasmid pJM1039 C600(QcI857ABamHIAHI) lacZ XA21::TnlO, with plasmid pJM1039-25

MATERIALS AND METHODS

Preparation of the hybrid protein. The plasmids of the pUR family have been designed for inducible expression of a fusion protein between the Escherichia coli lacZ gene product (P-galactosidase (,-Gal) and the product of any desired coding region (30). The recombinant plasmid pUR290-25 was obtained by ligating a 2-kb BamHI-XhoI fragment, containing codons 1255 to 1588 coding for the catalytic portion of the Cdc25 protein, with plasmid pUR290. Cultures of B290-25 (E. coli K-12 71-18 bearing pUR290-25; Table 1) were grown to log phase, and synthesis of the fusion protein was induced with 0.2 mM isopropyl-3-D-thiogalactopyranoside (IPTG) for 120 min. Cells were centrifuged at 1,000 x g for 5 min, denatured in lysis buffer (50 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 5% 2-mercaptoethanol), and boiled for 10 min; insoluble material was removed by centrifugation at 100,000 x g for 1 h at 20°C. The supernatant was referred as the lysate. Colonies harboring the expression plasmid were found, after induction with IPTG, to express a protein of -140 kDa, the expected molecular size for the fusion protein. The expressed fusion protein exhibits p-Gal activity on SDS-gels (using 5-bromo4-chloro-3-indolyl-,-D-galactoside as the substrate for ,B-Gal) if the samples were not boiled prior to loading onto the gel (not shown). Attempts to purify the fusion protein by affinity chromatography based on the enzyme (1-Gal)-substrate interaction as described by Ullman (35) were unsuccessful. Therefore, based on the relatively large size of the fusion protein, purification was accomplished by gel filtration chromatography. One to 2 ml of the B290-25 lysate (10 mg/ml) was subjected to gel filtration chromatography as previously described by Shuman et al. (33). The initial separation of the larger from the smaller polypeptides in this mixture was accomplished by gel filtration chromatography on a Bio-Gel A-Sm column (40 cm by 1 cm2; Bio-Rad) equilibrated with 10 mM Tris (pH 7.5) containing 0.5% SDS and 5 mM 2-mercaptoethanol. The fractions containing the f3-Gal-Cdc25 fusion protein were identified by electrophoresis of a small aliquot of each fraction on SDS-6% polyacrylamide gels. These fractions were combined and concentrated to -1 ml on Centricon 30 Microconcentrators (Amicon). The concentrated sample (1 to 2 mg) was rechromatographed on the Bio-Gel A-5m column. Figure 1A shows the fractions obtained from the second run on the Bio-Gel

cdc25A (TPKJ 2p,m) cdc25A (CDC25 2,um) C13-1

C13-2 B290

B290-25 B1039 B1039-25

A-Sm column. The fractions containing the fusion protein were pooled, concentrated, and further purified by electrophoresis on a preparative SDS-5% polyacrylamide gel. Protein concentrations were determined according to Peterson (27). The recombinant plasmid pJM1039-25 was obtained by ligating a 2-kb BglII-PvuII fragment, containing codons 877 to 1588 of the CDC25 open reading frame, with plasmid pJM1039 (see below). E. coli cultures bearing pJM1039-25, designated B1039-25 (Table 1), were grown and prepared as described above for B290-25 except that for induction, cultures were incubated at 42°C. Rabbit immunization and antibody purification. Gel slices from the preparative gel containing 200 ,ug of the fusion protein were homogenized with sterile phosphate-buffered saline (PBS), mixed with RIBI adjuvant R-730, and injected into rabbits at multiple intradermal sites. After two subsequent injections at 2-week intervals, each with about 100 ,ug of protein, the antisera of one rabbit showed a positive reaction with the antigen in enzyme-linked immunosorbent assays (ELISA) and on Western immunoblots. The antibodies were purified in two consecutive steps. First, the antiserum was passed through a Sepharose 4B column to which the lysate of E. coli harboring the parent plasmid pUR290 was coupled. Then, the eluate was purified on a Sepharose 4B column to which the lysate of E. coli harboring plasmid pUR290-25 was coupled, and it was eluted at low pH. Five to 10 mg of B290-25 lysate or 50 to 100 mg of B290 lysate was dialyzed against coupling buffer (0.1 M NaHCO3, 0.5 M NaCl [pH 8.3]) for 16 h at room temperature and incubated with 1 ml of packed CNBr-activated Sepharose 4B beads (Pharmacia) overnight at 4°C. Derivatized beads were then thoroughly washed with coupling buffer and transferred to the same buffer containing 0.2 M glycine (pH 8) for 2 h at room temperature. The beads were then washed three times alternately with coupling buffer and with acetate buffer (0.1 M acetic acid, 0.1 M sodium acetate, 0.5 M NaCl [pH 4]), once with coupling buffer, and once with PBS (30 mM NaH2PO4-NaHPO4, 0.85 M NaCl [pH 7.4]). The beads were stored in PBS containing 0.02% NaN3 at 4°C. Antiserum (5 to 10 ml) was incubated with end-over-end mixing with B290-derivatized beads overnight at 4°C. The beads were centrifuged (3 min, 250 x g), washed with 20 ml of PBS, and recentrifuged. The supernatants (containing spe-

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E. coli K-12 71-18-1 K-12 71-18-2 2097-1 2097-2

Abbreviation

Genotype

Strain

S. cerevisiae SP-1

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FIG. 1. Purification of the ,3-Gal-Cdc25 fusion protein. (A) Fractions obtained from the second run on the Bio-Gel A-Sm column. Fractions 10 to 24 (-1 ml each) were obtained from the column, and a small aliquot of each fraction was loaded on an SDS-6% polyacrylamide gel. (B) Scanner plot of the gel. Integration of the plot indicates that the fusion protein composes -70% of the total protein obtained from the column.

cific antibodies that did not bind to the column) were pooled and incubated with the B290-25-derivatized beads. After overnight end-over-end mixing, the beads were loaded onto a column and washed with -40 column volumes of PBS at 0°C. Bound antibodies were eluted with 0.1 M glycine-HCI (pH 2.3) at 0°C and immediately neutralized with 1 M Tris (pH 8) to approximately pH 7. Protein concentration was monitored at 280 nm. Fractions were then assayed for their ability to recognize the P-Gal-Cdc25 fusion protein in ELISA, and those containing activity were pooled and stored at -20°C with 1 mg of bovine serum albumin per ml and 0.02% NaN3. Preparation of protein extracts and cell fractionation. Yeast strains used are listed in Table 1. Medium commonly used was YEPD (1% yeast extract, 2% peptone, 2% glucose). Yeast strains C13-ABYS86-1 and -2 were grown on SD (0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulfate, 2% glucose) plus all of the amino acids but without uracil. Cells were harvested in log phase. Total protein extracts and membranes were prepared as described by Garreau et al. (17). Yeast cells were broken by vigorous vortexing with glass beads (0.4- to 0.6-mm diameter) in ice-cold buffer containing 50 mM 4-morpholinethanesulfonic acid (MES; pH 6), 0.1 mM MgCl2, 0.1 mM EGTA, 1 mM 2-mercaptoethanol, and the following protease inhibitors: benzamidine (313 ,ug/ml), pepstatin A (1.36 ,ug/ml), leupeptin

(5 ,ig/ml), antipain (2 ,ug/ml), chymostatin (2 ,ug/ml), aprotinin (10 ,ug/ml), soybean trypsin inhibitor (10 ,ug/ml), 2 mM phenylmethylsulfonyl fluoride, and 4 mM o-phenanthroline (all from Sigma). The mixture was then centrifuged at 800 x g for 5 min at 4°C to remove unbroken cells and glass beads. The supernatant is referred as the lysate. Crude membranes were prepared by centrifuging the lysate at 100,000 x g for 30 min at 4°C. For adenylyl cyclase assays and for Cdc25 localization assays, lysates were prepared by the mild Glusulase procedure as described by Engelberg et al. (16). Lysates of mammalian tissues were prepared by Dounce homogenization as described by Wolfman and Macara (38). Protein concentrations were determined according to Lowry et al. (23) or Peterson (27). Immunoprecipitation, electrophoresis, and immunoblot-

ting. For immunoprecipitations, membranes were solubilized at a detergent-to-protein ratio of 3:1 by using HLN buffer (50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.4], 0.6% lubrol PX, 0.1 M NaCI) containing protease inhibitors as described above. For immunoprecipitation experiments, detergent extracts containing 150 ,ug of protein were incubated with antibody (2 to 3 ,ug of affinity-purified anti-Cdc25 antibodies) overnight at 0°C. The immune complexes were recovered by incubation with end-over-end mixing with 300 ,ul of a 10% suspension of protein A sepharose for 1 h at 4°C. The immunoprecipitates were then washed five times with HLN buffer containing 0.06% lubrol PX, boiled in Laemmli sample buffer (22), and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose by using a semidry blotter (LKB) at 0.8 mA/cm2 for 2.5 h. Blots were blocked for 1 h at room temperature with Tris-buffered saline containing 5% powdered skim milk (Cadbury) and 0.3% Tween 20 and allowed to react overnight at 4°C with the primary antibody in the same solution. The immunopurified antiCdc25 antibody was used at a 1:40 dilution (10 ,ug/ml) and incubated with 1 mg of B290 lysate (E. coli harboring plasmid pUR290) per ml for 1 h at room temperature prior to reaction with the blot. Alternatively, the antibody solution was incubated with 1 mg of B290-25 lysate (E. coli harboring plasmid pUR290-25) per ml to block the anti-Cdc25 antibodies. Blots were washed seven times for 5 to 10 min each time with Tris-buffered saline containing 0.2% Tween 20 (without 0.02% NaN3) and incubated with the second antibody for 2 h at room temperature in the same solution used for the first antibody except without 0.02% NaN3. Horseradish peroxidase-conjugated protein A was used at a 1:1,000 dilution. The blots were visualized by using the ECL detection system (Amersham). Plasmids. Plasmids pTPKJ and pCDC25, which are present in strains TT1A-3 and TT1A-4, respectively (Table 1), have been described by Broek et al. (6). Both plasmids are derived from plasmid YEp13, which is a 2,um-based plasmid (32). pTPKI contains the gene TPKJ, and pCDC25 contains the gene CDC25. Plasmid pHIS3-RAS2v ,9, which was transformed into yeast strain TT1A-3 (strain MS-1; Table 1), has

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been described by Kataoka et al. (21). Plasmids YEp352 and pRG1, which were transformed into the protease-deficient yeast strain C13-ABYS86 (Table 1), have been described by Damak et al. (10). Plasmid pJM1039-25 is derived from plasmid pJM1039, which has been described by Mahajna et al. (24). Adenylyl cyclase assay. Adenylyl cyclase activity was measured as described by Casperson et al. (8). The reaction mixture contained 50 to 100 ,g of protein, 50 mM MES (pH 6), 0.1 mM EDTA, 20 mM creatine phosphate, 1 mg of creatine kinase per ml, 2 mM 2-mercaptoethanol, 1 mM (a-32P]ATP (10 to 20 cpm/pmol), and 1 mM [3H]cAMP (10,000 cpm) in a final volume of 100 ,ul. The assay was carried out at 30°C for 60 min. Antibodies, divalent cations, and nucleotides were added as described in the figure legends. The reaction was stopped by adding 100 ,u of stopping solution (2% SDS, 1 mM cAMP, 12 mM ATP), and the [,2P]cAMP was determined as described by Salomon et al. (31).

RESULTS Interaction of the anti-Cdc25 antibodies with yeast Cdc25 and with the C-terminal domain of Sdc25. Antiserum raised against the fusion protein was immunoaffinity purified (see Materials and Methods) and was shown to interact specifically with the Cdc25 portion of the -140-kDa P-Gal-Cdc25 fusion protein (Fig. 2, lane 1) as well as with a bacterially expressed -70-kDa C-terminal domain of the CDC25 gene product (Fig. 2, lane 3). The affinity-purified anti-Cdc25 antibodies recognize a -180-kDa protein on immunoblots in membranes of wildtype yeast cells (strain SP-1; Fig. 3C, lane 1) and in membranes of isogenic cells which harbor a multicopy (2,m) plasmid harboring the complete CDC25 gene (strain TT1A-4; Fig. 3A, lane 2, and Fig. 3C, lane 2). Membranes of yeast cells in which the CDC25 gene was disrupted did not interact with anti-Cdc25 antibodies (strain Tl1A-3; Fig. 3A, lane 4). Recognition of the -180-kDa protein was inhibited on immunoblots by addition of B290-25 lysate containing the

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1 2 3 4 FIG. 2. Evidence that immunopurified anti-Cdc25 antibodies specifically recognize the Cdc25 portion of the 1-Gal-Cdc25 fusion protein and a truncated Cdc25 protein. Lanes: 1 and 2, B290-25 lysate (expressing the ,-Gal-Cdc25 fusion protein) and B290 lysate (expressing the n-Gal protein), respectively; 3 and 4, B1039-25 lysate (expressing the truncated Cdc25 protein) and B1039 lysate (expressing the P-Gal protein), respectively. The samples were loaded on an SDS-6% polyacrylamide gel. The purified antibodies were used at a 1:40 dilution (10 xg/ml). Sizes are indicated in kilodaltons.

P-Gal-Cdc25 fusion protein (Fig. 3B, lane 4) or by addition of B1039-25 lysate containing the truncated Cdc25 protein (not shown). Recognition of the -180-kDa protein was not inhibited when the antibodies were preincubated with B290 lysate or B1039 lysate containing the P-Gal protein. The anti-Cdc25 antibody solution prepared for Western blots always contained one of these two lysates in order to block nonspecific interactions. The antibodies immunoprecipitated a protein of -180 kDa from membranes of TT1A-4 cells (Fig. 3A, lane 5, and Fig. 3B, lane 2) but not from membranes of TT1A-3 cells (Fig. 3B, lane 1). The weak bands that appear in addition to the intense Cdc25 band in the immunoprecipitate (Fig. 3A, lane 5) do not appear in immunoprecipitates of TT1A-3 cells (not shown). The protein was not immunoprecipitated with preimmune serum (not shown). The antibodies cross-reacted on immunoblots with the C-terminal domain of the product of the SDC25 gene, a homolog of the CDC25 gene, when expressed on a multicopy (2,um) plasmid in a wild-type yeast strain (strain C13-2; Fig. 3D, lane 2). In cells from the same yeast strain harboring the parent plasmid, the C-terminal domain of Sdc25 was not detected with the antibodies (strain C13-1; not shown). Localization of the Cdc25 protein in wild-type yeast strains. To determine the localization of the Cdc25 protein, lysates prepared by the mild Glusulase procedure (see Materials and Methods) from wild-type yeast strains (strain SP-1) were fractionated for 30 min at 100,000 x g (4°C). The resulting pellet (total membrane fraction) and supernatant (soluble fraction) were analyzed on immunoblots. All of the Cdc25 protein was found in the total membrane fraction (Fig. 4A, lanes 1 and 2). To further determine whether Cdc25 is a peripheral membrane protein, membranes were incubated for 30 min at 0°C in the presence of either 1% lubrol PX, 0.5 M NaCl, 0.1 M Na2CO3 (pH 11.5), or 2.5 M urea as described by Hicke and Schekman (18) and centrifuged at 100,000 x g for 30 min at 4°C in a TLA-100 microcentrifuge to obtain the soluble and membrane fractions. Figure 4A shows that these treatments failed to release Cdc25 from the membrane fraction. Furthermore, treatment with combinations of detergent (1% lubrol PX or 1% deoxycholate) plus either 2.5 M urea or 0.5 M NaCl also failed to release Cdc25 from the membrane fraction. Even 8 M urea alone or the combination of 8 M urea plus 1% deoxycholate only partially released Cdc25 (Fig. 4A, lanes 15 to 18). We also examined the possibility that Cdc25 is bound to the membrane by a fatty acid that is attached to the polypeptide through a thiol ester bond. Treatment of the membranes with hydroxylamine as described by Bolanowski et al. (3), which would have cleaved the thiol ester bond, also failed to release Cdc25 (Fig. 4A, lanes 31 and 32). Another possibility was that the Cdc25 protein interacts with the cytoskeletal matrix. However, treatment of the membranes with 0.6 M KI or 1 M sodium thiocyanate (NaSCN), which are known to release loosely bound cytoskeletal proteins, failed to release Cdc25 (Fig. 4A, lanes 19 to 22). Combination of KI or NaSCN together with 1% Triton also failed to release the protein. Finally, treatment of the membranes with 2 mM EDTA (pH 12) as described by Casey et al. (7) completely released Cdc25 from the membrane fraction (Fig. 4A, lanes 35 and 36). Also, treatment with 1% SDS completely solubilized the Cdc25 protein (lanes 33 and 34). Lysates prepared by vigorous glass bead homogenization (see Materials and Methods) and treated with the same reagents have shown identical results (data not shown). We also investigated the association of the Cdc25 protein

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FIG. 3. Evidence that anti-Cdc25 antibodies recognize the CDC25 gene product in wild-type yeast cells and cross-react with the C-terminal domain of the SDC25 gene product. (A) Lanes: 1 and 2, lysate and membrane preparations, respectively, of yeast cells which harbor a multicopy (2,um) plasmid harboring the complete CDC25 gene (TT1A-4) (-1 ,Lg of protein was loaded onto each lane); 3 and 4, lysate and membrane preparations, respectively, of yeast cells in which the CDC25 gene was disrupted (TT1A-3); 5, immunoprecipitate from TT1A-4 membranes. (B) Lanes: 1 and 2, immunoprecipitates of TT1A-3 and TT1A-4 membranes, respectively; 3 and 4, identical to lanes 1 and 2 but treated with the addition of the ,B-Gal-Cdc25 fusion protein to the primary antibody solution. (C) Membranes of the wild-type strain (SP-1) (lane 1) and of strain TT1A-4 (lane 2) (the samples were loaded on an SDS-4% polyacrylamide gel). (D) Lanes: 1, identical to lane 2 in panel A; 2, lysate of yeast cells which harbor a multicopy (2,um) plasmid harboring the C-terminal portion of the SDC25 gene (strain C13-2). The anti-Cdc25 antibodies were used at a 1:40 dilution (10 pLg/ml) and incubated with B290 lysate prior to reaction with the blot. Horseradish peroxidase-conjugated protein A was used at a 1:1,000 dilution, and blots were visualized by using the ECL detection system (Amersham). Sizes are indicated in kilodaltons.

with the membrane in isogenic yeast cells which overexpress Cdc25 (strain TT1A-4; Table 1). In lysates of these cells, Cdc25 fractionates in both the pellet and supernatant of a 100,000 x g centrifugation (Fig. 4B, lanes 1 and 2). Further treatments of the pellet produced results identical to the results obtained for wild-type cells (Fig. 4B). Anti-Cdc25 antibodies inhibit adenylyl cyclase activity. When anti-Cdc25 antibodies were added to yeast lysates, adenylyl cyclase was strongly inhibited. Anti-Cdc25 antibodies inhibited both Mg2+/Guanylylimidodiphosphate (GppNHp)-dependent cyclase activity and, with much less potency, the Mn2+-dependent activity. These activities were not inhibited by preimmune serum (Fig. 5). The 50% inhibitory concentration (IC50) value for the inhibition of GppNHp-supported activity is -3-fold lower than the IC50 value for the inhibition of Mn2+-supported activity. Anti-p21Haras monoclonal antibodies (Y13-259) also inhibited the Mg2+/ GppNHp-dependent activity 3.5-fold more potently than the Mn2-dependent activity (Fig. 6A). Inhibition of Mg2+/ GppNHp- and Mn2+-dependent activities by anti-Cdc25 antibodies was identical whether the enzyme system was preloaded with GppNHp prior to antibody addition or exposed simultaneously to GppNHp and antibody. Similar results were obtained with anti-p21Haras monoclonal antibodies (Fig. 6A). However, addition of the anti-Cdc25 antibodies to membranes of yeast strain MS-1 (a strain disrupted in the CDC25 gene and carrying the RAS2Val-19 mutation) had no effect on the cyclase activity whereas significant inhibition was observed with the isogenic wild-type strain (Fig. 6B).

Anti-Cdc25 antibodies cross-react with mammalian proteins. To identify putative mammalian homologs of Cdc25, we performed Western blot analysis utilizing the purified anti-Cdc25 antibodies on lysates and membranes from various tissues of several species of animals. Figure 7A shows that the anti-Cdc25 antibody cross-reacted with a number of proteins: a -140-kDa membrane protein from brain tissue of 3-days-postnatal mice and from brain tissue of adult rats and adult guinea pigs and a -160-kDa membrane protein from ovary tissue of adult rats. Lysates of the same tissues showed no response with the antibodies (data not shown). These signals were completely abolished when the antibody solution was preincubated with B290-25 lysate (containing the ,-Gal-Cdc25 fusion protein) prior to reaction with the blot (shown only for mouse brain and rat ovary). The signals were not abolished when the antibodies were preincubated with B290 lysate (containing the ,B-Gal protein; the antibody solution was routinely preincubated with this lysate). Lysates and membranes prepared from PC12 and NIH-3T3 cell lines showed no response with the antibodies (shown only for the membranes). We further examined membranes from brain tissue of postnatal (3, 7, 10, and 14 days old) and adult mice. Figure 7B shows that the cross-reacting -140-kDa signal faded as the mice grew older (days 3 to day 14) and completely disappeared in adult mice. DISCUSSION In this study, we have shown for the first time that antibodies raised against the C-terminal domain of the

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FIG. 4. Evidence that Cdc25 is tightly bound to the membrane. Aliquots of wild-type yeast lysates and membranes (strain SP-1) (A) or lysates and membranes of isogenic yeast cells overexpressing Cdc25 (strain TT1A-4) (B) were diluted into fractionation buffer containing various reagents, incubated on ice for 30 min, and centrifuged (30 min, 100,000 x g, 4°C) in a TLA-100 microcentrifuge. The pelleted material (lanes P) and supernatant (lanes S) were separated and loaded on an SDS-6% polyacrylamide gel. An immunoblot of these samples was incubated with the purified anti-Cdc25 antibody. (A) Wild-type yeast strain. Lanes 1 and 2 contain untreated lysate. Membranes treated with various reagents are shown in lanes 3 and 4 (fractionation buffer); 5 and 6 (1% lubrol PX), 7 and 8 (0.1 M Na2CO3, pH 11.5), 9 and 10 (0.5 M NaCl), 11 and 12 (1% deoxycholate [Doc]), 13 and 14 (1% deoxycholate plus 0.5 M NaCI), 15 and 16 (8 M urea), 17 and 18 (1% deoxycholate plus 8 M urea), 19 and 20 (1 M NaSCN), 21 and 22 (0.6 M KI), 23 and 24 (1% Triton), 25 and 26 (1 M NaSCN plus 1% Triton), 27 and 28 (0.6 M KI plus 1% Triton), 29 and 30 (5% taurocholate), 31 and 32 (1 M NH20H * HCI, pH 9.8), 33 and 34 (1% SDS), and 35 and 36 (2 mM EDTA, pH 12). (B) Yeast strain overexpressing the Cdc25 protein. Lanes: 1 to 12, same as lanes 1 to 4 and 11 to 18 in panel A; 13 and 14, same as lanes 35 and 36 in panel A.

CDC25 gene product recognize the Cdc25 protein in wildtype yeast strains. We have also shown that in yeast strains overexpressing Cdc25, the antibodies were effective in both Western blotting and immunoprecipitation (Fig. 3). The additional bands that appear in the immunoprecipitate are most probably degradation products of the Cdc25 protein. Moreover, these antibodies recognized the C-terminal domain of the SDC25 gene product extremely well (Fig. 3). By using these antibodies, we have also been successful in detecting, on Western blots, a number of putative mammalian homologs of Cdc25 from brain membranes of postnatal mice, adult rats, and adult guinea pigs and from ovary membranes of adult rats (Fig. 7A). The cross-reacting signal from postnatal mouse brain faded within 14 days and completely disappeared in adult mouse brain (Fig. 7B). This finding suggests that the concentration of the cross-reacting protein is significantly higher in the early (and more accelerated) stages of developing mouse brain. These crossreacting proteins probably represent putative Cdc25 homologs. Further characterization of the proteins is necessary to determine whether they are true Cdc25 homologs or

represent more remote members of the family which regulate small G proteins other than Ras. The antibodies potently inhibited the Mg2+/GppNHpdependent cyclase activity and with a much lower efficacy the Mn2'-dependent cyclase activity (Fig. 5). This result suggests that the C-terminal domain, against which the antibody was raised, interacts with the Ras protein which, in turn, activates the adenylyl cyclase moiety. These findings support our previous observations that the C-terminal portion of the CDC25 gene is sufficient to suppress cdc25ts and, correspondingly, to confer guanyl nucleotide sensitivity to adenylyl cyclase (11). Direct inhibition of Ras function by anti-p21Haras monoclonal antibodies also resulted in strong inhibition of the guanyl nucleotide-sensitive cyclase activity, with a much weaker effect on the Mn2'-sensitive activity (Fig. 6). There was no difference in the inhibition efficiency of the antibody toward Mg2+/GppNHp- and Mn2+-dependent cyclase activity when Ras was preloaded with GppNHp prior to addition of the antibody compared with the results obtained without preloading (Fig. 6). In this respect also, the results with anti-p21Haras monoclonal antibodies were sim-

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19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

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ANTI-Cdc25 ANTIBODIES

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A preloaded >

80-

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-

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----

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anti Cdc25 Ab (ig) FIG. 5. Dose-dependent inhibition of adenylyl cyclase by antiCdc25 antibodies. The adenylyl cyclase activity is expressed as percentage of maximal activity, which was 26.3 pmol of cAMP min-' mg of protein-' in the presence of 10 mM Mg2' and 100 ,uM GppNHp and 18.9 pmol of cAMP min-' mg of protein-' in the presence of 2.5 mM Mn2". Adenylyl cyclase was analyzed in the presence of increasing amounts of anti-Cdc25 antibodies (Ab) (0 to 5 of antibodies per assay). The IC50s for Mg2+/GppNHp- and Mn '-dependent activity are 1.5 and 4.2 p,g of anti-Cdc25 antibodies per assay, respectively. Symbols: A, Mg2+/GppNHp; El, Mn2+; E, preimmune serum.

B

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wild type

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100

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ilar to those observed for the anti-Cdc25 antibodies (Fig. 6). On the other hand, addition of anti-Cdc25 antibodies to a yeast strain carrying a mutationally active RAS gene (RAS2vai-l9) in a cdc25-negative background had no effect on the adenylyl cyclase activity (Fig. 6B). The findings from the preloading assay suggest that the anti-Cdc25 antibodies do not effect the GDP-to-GTP exchange reaction on Ras, but rather that their main mode of action is by steric interference with the complex Cdc25-Ras2-Cdc35 through their binding to the Cdc25 protein. The findings obtained with the mutationally activated RAS gene may be considered as supporting evidence. The finding that anti-Cdc25 antibodies affect the Mg2+/GppNHp-dependent activity more potently than the Mn2+-dependent activity of adenylyl cyclase suggests that they interfere mainly with Ras-dependent activity and have a lesser effect on the cyclase catalyst. It is likely that the effect of the anti-Cdc25 antibodies on the Mn2+-dependent activity reflects the fact that Cdc25, Ras, and Cdc35/ Cyrl proteins are all in one macromolecular complex. Previous results from our laboratory demonstrated that the cyclase protein (Cdc35) is found in the cytoplasm in a rasl ras2 bcyl strain. When the CDC25 gene is introduced into these cells on a multicopy plasmid, cyclase relocalizes to the membrane, suggesting that Cdc25 and Cdc35 interact even in the absence of Ras proteins (16). Our findings that the anti-Cdc25 antibodies also inhibit the Mn2+-dependent activity, although less potently, support this view. We also used the antibodies to localize the Cdc25 protein in wild-type yeast strains. We showed that all of the Cdc25 protein is associated with the membrane fraction (Fig. 4A). Further investigation of the nature of this association by treating membranes prepared by the mild Glusulase procedure with several reagents showed that Cdc25 was not loosely bound to the membrane. Classical treatments that

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FIG. 6. Effect of anti-Cdc25 antibodies on adenylyl cyclase activity when the enzyme system was preloaded with GppNHp prior to antibody (Ab) addition (A) or in a yeast strain carrying a mutationally activated RAS gene (B). (A) Adenylyl cyclase inhibition by anti-Cdc25 antibodies compared with inhibition by antip2lHa,as monoclonal antibodies was measured after simultaneous exposure to GppNHp and anti-Cdc25 antibodies or after preincubation with GppNHp followed by exposure to the antibodies. The lysates were preloaded by incubation with 500 pLM GppNHp for 10 min at 4°C and then 15 min at 30°C. Control lysates were incubated under the same conditions; 4.2 ,ug of anti-Cdc25 antibodies or 24 p.g of anti-p21Haras monoclonal antibodies was added to the reaction mixture at the onset of the assay. The cyclase activity is expressed as percentage of maximal activity. The maximal specific activity of GppNHp-preloaded lysates was 15 pmol of cAMP min-' mg of protein-1, and that of nonpreloaded lysates was 10.2 pmol of cAMP min-' mg of protein-'. The maximal specific activity of Mn2+ was 19 pmol of cAMP min-' mg of protein-'. Other details are given in Materials and Methods. (B) The effect of anti-Cdc25 antibodies on adenylyl cyclase activity in strain MS-1 (carrying the RAS2VaI-l9 mutation in a cdc25-negative background) was compared with its effect on the isogenic wild-type strain (SP-1); 6.4 ,ug of anti-Cdc25 antibodies was added to the reaction mixture at the onset of the assay. Adenylyl cyclase activity was measured in a crude membrane preparation. The maximal specific activity of GppNHp-activated membranes of strain MS-1 was 17.4 pmol of cAMP min-' mg of protein-l, and that of strain SP-1 was 23.4 pmol of cAMP min'- mg of protein-'. The maximal specific activity of Mn2+-activated membranes of MS-1 was 22.3 pmol of cAMP min-' mg of protein-', and that of SP-1 was 19.6 pmol of cAMP min-' mg of protein-1. The experiment is a representation of a number of independent experiments with the same results within a variation of less than 10%.

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are used to solubilize integral membrane proteins only partially released the Cdc25. Also, treatments that are known to release loosely bound cytoskeletal proteins failed to release the protein. By using a method that removes all but the very intrinsic components of the membrane (EDTA, pH 12) (7), we have been successful in completely solubilizing the Cdc25 protein (Fig. 4). These results suggest that Cdc25 is tightly associated with the cytoskeleton. It was indeed recently suggested that the N-terminal domain of Cdc25 is homologous to the SH-3 domain (29), which is believed to mediate interactions with the cytoskeleton (12). These results are in agreement with observations made by Vanoni et al. (36), who also suggested a strong interaction between the Cdc25 protein and the cytoskeletal matrix, and with the finding that Cdc25 lacks a consensus N-terminal signal sequence or a sequence suggesting a transmembrane domain as determined by the PROSITE computer program (1). Interestingly, in isogenic cells which overexpress the Cdc25 protein, Cdc25 fractionates in both the pellet and supernatant of a 100,000 x g centrifugation (Fig. 4B). This finding suggests that a fraction of the overexpressed Cdc25 protein possesses biochemical properties somewhat different from those of the endogenous Cdc25. Our observations are in agreement with observations made by Jones et al. (20), who reported that in cells overexpressing the Cdc25 protein, Cdc25 fractionates in both the pellet and the supernatant of a 100,000 x g centrifugation. On the other hand, further treatments of the pellet showed that in overexpressing Cdc25 cells, as in wild-type cells, Cdc25 was tightly bound to the pellet fraction, and only alkaline conditions in the presence of EDTA completely solubilized the protein (Fig. 4B). The anti-Cdc25 antibodies are being used to further analyze the biochemical properties of the Cdc25 protein in S. cerevisiae and to further characterize the cross-reacting mammalian proteins. ACKNOWLEDGMENTS We thank D. R. Lowy (NCI, NIH) for monoclonal antibody Y13-259. We thank Amos Oppenheim and Hilla Giladi (Center of Molecular Biology, Faculty of Medicine, Hebrew University of Jerusalem) for valuable assistance in preparing the bacterial Cdc25 expression vectors. We thank K. Matsumoto and M. Wigler (Cold

Spring Harbor Laboratory) for providing yeast strains and plasmids and D. H. Wolf (Institute for Biochemistry, University of Stuttgart) for the protease-deficient strain C13-ABYS86. We also thank Motti Anafi for valuable advice on the immunological assays. We also acknowledge I. Cabanchik from our department, who pointed out to us the EDTA (pH 12) solubilization protocol. This study was supported by a grant from the Israel Academy of Sciences. REFERENCES 1. Bairoch, A. 1990. PROSITE: a dictionary of protein sites and patterns, fifth release. University of Geneva, Geneva, Switzerland. 2. Barbacid, M. 1987. ras genes. Annu. Rev. Biochem. 56:779827. 3. Bolanowski, M. A., B. J. Earles, and W. J. Lennarz. 1984. Fatty acylation of proteins during development of sea urchin embryos. J. Biol. Chem. 259:4934-4940. 4. Bourne, H. R., D. A. Sanders, and F. McCormicl. 1991. The GTPase superfamily: conserved structure and molecular mechanism. Nature (London) 349:117-127. 5. Boy-Marcotte, E., F. Damak, J. Camonis, H. Garreau, and M. Jacquet. 1989. The C-terminal part of a gene partially homologous to CDC25 gene suppresses the cdc25-5 mutation in Saccharomyces cerevisiae. Gene 77:21-30. 6. Broek, D., T. Toda, T. Michaeli, L. Levin, C. Birchmeier, M. Zoller, S. Powers, and M. Wigler. 1987. The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell 48:789-799. 7. Casey, J. R., D. M. Lieberman, and R. A. F. Reithmeier. 1989. Purification and characterization of band 3 protein. Methods Enzymol. 173:494-512. 8. Casperson, G. F., N. Walker, A. R. Brasier, and H. R. Bourne. 1983. A guanine nucleotide-sensitive adenylate cyclase in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 258:7911-7914. 9. Chant, J., K. Corrado, J. R. Pringle, and I. Herskowitz. 1991. Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for the site selection and interacts with bud formation gene BEM1. Cell 65:1213-1224. 10. Damak, F., E. Boy-Marcotte, D. Le-Roscouet, R. Guilband, and M. Jacquet. 1991. SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae. Mol. Cell. Biol. 11:202-212. 11. Daniel, J., M. J. Becker, E. Enari, and A. Levitzki. 1987. The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the cdc25 start gene product. Mol. Cell. Biol. 7:3857-3861.

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FIG. 7. Cross-reactions of anti-Cdc25 antibodies with mammalian proteins. (A) Western blot analysis of membranes prepared from various tissues of several species of animals. From left to right: membranes from adult guinea pig brain, membranes from adult rat brain, membranes from NIH 3T3 cell lines, membranes from PC12 cell lines, membranes from 3-days-postnatal mouse brain, membranes from adult rat ovary, membranes of yeast cells which harbor a multicopy (2,um) plasmid harboring the complete CDC25 gene, and duplicates of the last three lanes. The duplicate lanes (blocked) were incubated at a 1:40 dilution (10 ±g/ml) with anti-Cdc25 antibodies that had been preincubated with B290-25 lysate (1 mg/ml) containing the P-Gal-Cdc25 fusion protein. The other lanes were incubated with antibodies at the same dilution but were preincubated with B290 lysate containing the I-Gal protein; 30 pg of each tissue preparation was loaded onto the gel. (B) Membrane preparations of postnatal (3, 7, 10, and 14 days old) and adult mouse brain. The blot was treated as described for panel A for the nonblocked lanes. Further details are given in Materials and Methods. Sizes are indicated in kilodaltons.

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25. McCormick, F. 1989. ras GTPase activating protein: signal transmitter and signal terminator. Cell 56:5-8. 26. Munder, T., and H. Kuntzel. 1989. Glucose-induced cAMP signalling in Saccharomyces cerevisiae is mediated by the CDC25 protein. FEBS Lett. 242:341-345. 27. Peterson, G. L. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83:346-356. 28. Robinson, L. C., J. B. Gibbs, M. S. Marshall, I. S. Sigal, and K. Tatchell. 1987. CDC25: a component of the Ras-adenylate cyclase pathway in Saccharomyces cerevisiae. Science 235: 1218-1221. 29. Rodway, A. R. F., M. J. E. Sternberg, and D. L. Bentley. 1989. Similarity in membrane proteins. Nature (London) 342:624. 30. Ruther, U., and B. Muller-Hill. 1983. Easy identification of cDNA clones. EMBO J. 2:1791-1794. 31. Salomon, Y., C. Londos, and M. Rodbell. 1974. A highly sensitive adenylate cyclase assay. Anal. Biochem. 58:541-548. 32. Sherman, F., G. R. Fink, and J. B. Hicks. 1982. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 33. Shuman, H. A., T. J. Silhavy, and J. R. Beckwith. 1980. Labeling of proteins with 3-galactosidase by gene fusion. J. Biol. Chem. 255:168-174. 34. Toda, T., I. Uno, T. Ishikawa, S. Powers, T. Kataoka, D. Broek, S. Cameron, J. Broach, K. Matsumoto, and M. Wigler. 1985. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell 40:27-36. 35. Ullman, A. 1984. One step purification of hybrid proteins which have 3-galactosidase activity. Gene 29:27-31. 36. Vanoni, M., M. Vavassori, G. Frascotti, E. Martegani, and L. Alberghina. 1990. Overexpression of the CDC25 gene, an upstream element of the RAS/adenylyl cyclase pathway in Saccharomyces cerevisiae, allows immunological identification and characterization of its gene product. Biochem. Biophys. Res. Commun. 172:61-69. 37. West, M., H. Kung, and T. Kamata. 1990. A novel membrane factor stimulates guanine nucleotide exchange reactions of ras proteins. FEBS Lett. 259:245-248. 38. Wolfman, A., and I. G. Macara. 1990. A cytosolic protein catalyzes the release of GDP from p2lras. Science 248:67-69.

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12. Drobin, D. J., J. Mulholland, Z. Zhu, and D. Botestein. 1990. Homology of a yeast actin-binding protein to signal transduction proteins and myosin-1. Nature (London) 343:288-290. 13. Downward, J. 1990. The ras superfamily of small GTP-binding proteins. Trends Biochem. Sci. 15:469-472. 14. Downward, J., R. Rebecca, L. Wu, and R. A. Weinberg. 1990. Identification of a nucleotide exchange-promoting activity for p2lras. Proc. Natl. Acad. Sci. USA 87:5998-6002. 15. Engelberg, D., R. Perlman, and A. Levitzid. 1989. Transmembrane signalling in Saccharomyces cerevisiae. Cell. Signalling 1:1-7. 16. Engelberg, D., G. Simchen, and A. Levitzki. 1990. In vitro reconstitution of CDC25 regulated S. cerevisiae adenylyl cyclase and its kinetic properties. EMBO J. 9:641-651. 17. Garreau, H., J. H. Camonis, C. Guitton, and M. Jacquet. 1990. The Saccharomyces cerevisiae CDC25 gene product is a 180 kDa polypeptide and is associated with a membrane fraction. FEBS Lett. 269:53-59. 18. Hicke, L., and R. Schekman. 1989. Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro. EMBO J. 8:1677-1684. 19. Hughes, D. A., Y. Fukui, and M. Yamamoto. 1990. Homologous activators of ras in fission and budding yeast. Nature (London) 344:355. 20. Jones, S., M. L. Vignais, and J. R. Broach. 1991. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to Ras. Mol. Cell. Biol. 11:26412646. 21. Kataoka, T., S. Powers, C. McGill, 0. Fasano, J. Strathern, J. Broach, and M. Wigler. 1984. Genetic analysis of yeast RASI and RAS2 genes. Cell 37:437-445. 22. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 23. Lowry, 0. H., N. J. Rosebrough, A. L. Faff, and R. J. Randall. 1951. Protein measurements with Folin phenol reagent. J. Biol. Chem. 193:265-275. 24. Mahajna, J., A. Oppenheim, A. Rattray, and M. Gottesman. 1986. Translation initiation of bacteriophage lambda gene cII requires integration host factor. J. Bacteriol. 165:167-174.

ANTI-Cdc25 ANTIBODIES