©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase Byr2 Is a Target of Ras1 in the Fission Yeast Schizosaccharomyces pombe(*)

(Received for publication, October 31, 1994; and in revised form, November 29, 1994)

Tadayuki Masuda Ken-ichi Kariya Masayuki Shinkai Tomoyo Okada Tohru Kataoka (§)

From the Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Conservation of the structure and function of Ras proteins has been observed in a variety of eukaryotic organisms. However, the nature of their downstream effectors appears to be quite divergent; adenylyl cyclase and a protein kinase Raf-1, which do not share any structural homology with each other, are effectors of Ras in the budding yeast and in higher organisms, respectively. We show here that a protein kinase Byr2, which has been known to act downstream of Ras1 in a mating pheromone signal transduction system of Schizosaccharomyces pombe, binds directly to Ras proteins in a GTP-dependent manner. The region of Byr2 responsible for the Ras binding was mapped by a gene deletion analysis to its N-terminal segment of 206 amino acid residues, which does not possess any significant homology with the other effectors of Ras. The affinity of the Byr2 N terminus for Saccharomyces cerevisiae Ras2 was determined by measuring its activity to competitively inhibit Ras-dependent adenylyl cyclase activity and found to be comparable with those of yeast adenylyl cyclase and human Raf-1, with a dissociation constant (K) of about 1 nM. Furthermore, Byr2 inhibited a Ras GTPase-activating activity of Ira2, a S. cerevisiae homologue of neurofibromin. These results indicate that Byr2 is an immediate downstream target of Ras1 in S. pombe.


INTRODUCTION

The ras genes are widely conserved in a variety of eukaryotic organisms from yeasts to mammals. They encode small GTP-binding proteins that participate in signal transduction from plasma membrane receptors to nuclei (for reviews, see (1) and (2) ). In mammalian cells the majority of cellular Ras in quiescent cells is in the inactive GDP-bound form. Stimulation of cells with mitogens or differentiation factors activates guanine nucleotide exchange proteins and increases the abundance of the active GTP-bound form. The GTP-bound Ras interacts directly with a serine/threonine kinase Raf-1, which results in activation of a phosphorylation cascade including Raf-1, MAP (^1)kinase kinases, and MAP kinases(3, 4, 5, 6, 7) . Similar pathways involving Ras have been identified in other species including Caenorhabditis elegans and Drosophila melanogaster(1) . In the budding yeast Saccharomyces cerevisiae, a pair of Ras proteins, Ras1 and Ras2, interacts directly with adenylyl cyclase in a GTP-dependent manner and regulates its activity (for reviews, see Refs. 8 and 9). A domain comprising the leucine-rich repeats structure has been defined as a Ras protein-binding site of yeast adenylyl cyclase (10) . There is no apparent structural homology between the leucine-rich repeats of adenylyl cyclase and the Ras protein-binding region, the N-terminal regulatory domain, of Raf-1 even though they bind mutually competitively to the effector region of Ras(10) .

In the fission yeast Schizosaccharomyces pombe, its single Ras homologue, Ras1, is required for sexual responses induced by mating pheromones, namely conjugation in haploid cells and sporulation in diploid cells(11) . The function of Ras1 is mediated by control of a protein kinase cascade that ultimately regulates a member of the MAP kinase family. Spk1, which is essential for conjugation and sporulation in S. pombe, is structurally homologous to MAP kinases of vertebrates(12) , and a vertebrate MAP kinase can complement the loss of Spk1 in S. pombe(13) . Two genes BYR1 and BYR2, encoding putative protein kinases of distinctive structures, were isolated as multicopy suppressors of the ras1 defect(14, 15) , and BYR1 was shown to be epistatic to BYR2. Byr1 was found to be structurally related to vertebrate MAP kinase kinases(16) . This led to an assumption that Ras1, Byr2, Byr1, and Spk1 constitute a signal-transducing pathway analogous to the vertebrate Ras-MAP kinase cascade although direct protein-protein interactions among its constituents have not been biochemically verified. Recently from a study utilizing a yeast two-hybrid system came evidence suggesting that Byr2 interacts closely, if not directly, with Ras(7) . This led us to examine the mode of interaction of this protein with Ras by biochemical means.


EXPERIMENTAL PROCEDURES

Materials and Cell Strains

S. cerevisiae Ras2, Ras2, Ras2, and human H-Ras proteins were overproduced in Sf9 cells by using recombinant baculoviruses expressing the respective cDNAs(17) . The posttranslationally modified forms of these proteins were extracted and purified as described before(17) . Plasmid pGEX-5X-BYR2 was constructed by inserting the whole coding sequence of the BYR2 into pGEX-5X-1 (Pharmacia Biotech Inc.) so that Byr2 was expressed as a fusion protein with GST. Various deletions were introduced into the Byr2 sequence by digestion from the C terminus with Escherichia coli exonuclease III and mung bean nuclease or by cleavage with suitable restriction endonucleases. The GST-Byr2 fusion proteins were purified from E. coli harboring the corresponding expression plasmids by using glutathione-Sepharose 4B columns. They were designated as GST-Byr2(x-x), in which x-x represents the range of expressed polypeptides in amino acid positions. The genotypes and growth conditions of yeast strains used in this study were described before (18) .

Adenylyl Cyclase Assay

Yeast adenylyl cyclase, overproduced in yeast TK35-1 harboring a plasmid YEP24-ADC1-CYR1, was solubilized from the crude membrane fraction with buffer C (50 mM MES/NaOH, pH 6.2, 0.1 mM MgCl(2), 0.1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM 2-mercaptoethanol) containing 1.5 M NaCl as described previously(18) . The supernatant (10 µg of protein) after centrifugation at 100,000 times g for 1 h was used for the adenylyl cyclase assay. Measurements of adenylyl cyclase activity dependent on the GTPS-bound form of Ras and of its inhibition by purified GST-Byr2 polypeptides were carried out similarly as described before(10) .

Assay for GTPase Activity

Ras2 protein was first loaded with [-P]GTP by incubation in a mixture containing 20 mM Tris/HCl, pH 7.5, 5 mM MgCl(2), 10 mM EDTA, 1 mM DTT, and 1 mM [-P]GTP (6,500 cpm/pmol), followed by addition of MgCl(2) to 15 mM. The GTPase reactions were initiated by addition of a fixed amount of the [-P]GTP-bound Ras2 into a mixture (40 µl) containing 25 mM Tris/HCl, pH 7.5, 10 mM MgCl(2), 5 mM EDTA, 1 mM DTT, and 1 mM GTP, with or without 48 pmol of GST-Byr2(1-236) and with or without Ira2. After 5 min of incubation at 30 °C, the reaction was stopped by rapid filtration through a nitrocellulose filter, and the GTPase activity was estimated by measuring the residual amount of [-P]GTP bound to Ras2 retained on the filter. An aliquot (11.4 µg of protein) of a Triton X-100 extract of yeast SP1 cells harboring pKT10-IRA2 (19) was used as a source of Ira2 for each assay. An aliquot (11.4 µg of protein) of the extract of SP1 cells harboring pKT10 vector instead of pKT10-IRA2 was used for the reaction without Ira2.

Assay for Binding of Ras to Byr2

GST-Byr2(1-236) was attached to glutathione-Sepharose 4B resin in buffer B (25 mM Tris/HCl, pH 7.5, 5 mM MgCl(2), 1 mM EDTA, and 1 mM DTT) by continuous mixing for 10 min, and the resin was washed once with buffer B to remove unbound GST-Byr2(1-236). Aliquots of the resin with 2 µg each of GST-Byr2(1-236) attached were incubated with 10 pmol (quantitated by GTPS binding) of various Ras proteins, which had been loaded with GDP or GTPS, for 2 h at 4 °C, and, after washing three times with buffer B containing 0.1% Lubrol PX, the bound Ras was separated by SDS-polyacrylamide gel electrophoresis (12% gel) and quantitated by Western immunoblotting with an anti-Ras2 polyclonal antibody (17) for Ras2 and its mutants and with a monoclonal antibody Y13-259 for Ha-Ras.


RESULTS

Inhibition of Ras-dependent Activation of Adenylyl Cyclase by the N-terminal Segment of Byr2

We examined whether the N-terminal region of Byr2 could bind to Ras protein and thereby compete for it with yeast adenylyl cyclase in vitro as observed for the human Raf-1 N-terminal polypeptide(10) . A suggestion for using this region of Byr2 came from our observation that overexpression of the N-terminal 392 amino acid residues suppressed heat shock sensitivity of S. cerevisiae cells bearing the activated RAS2 gene (data not shown). The purified GST-Byr2(1-236) and GST-Byr2(1-206) inhibited the Ras2-dependent adenylyl cyclase activity in a dose-dependent manner, whereas GST-Byr2(1-151) and GST-Byr2(88-236) failed to inhibit the activity (Fig. 1A). In contrast, GST-Byr2(1-236) had no effect on the Mn-dependent adenylyl cyclase activity, suggesting that GST-Byr2(1-236) exerted its inhibitory effect by interacting with Ras but not with adenylyl cyclase itself.


Figure 1: Measurement of Ras2 binding to GST-Byr2 by adenylyl cyclase inhibition assay. A, adenylyl cyclase activity was measured in the presence of 0.5 pmol of Ras2 with the addition of various amounts of GST-Byr2(1-236) (bullet), GST-Byr2(1-206) (), GST-Byr2(1-151) (), and GST-Byr2(88-236) (). An essentially similar assay was carried out in the presence of 2.5 mM Mn instead of Mg and Ras2 with the addition of various amounts of GST-Byr2(1-236) (circle). B, adenylyl cyclase activities dependent on various concentrations of Ras2 were measured in the presence of various amounts of GST-Byr2(1-236) as follows: 0 (bullet), 5 (), 10 (), and 20 pmol (). One unit of activity is defined as 1 pmol of cAMP formed in 1 min of incubation with 1 mg of protein at 30 °C under standard assay conditions. C, double-reciprocal plot analysis of the binding reaction between GST-Byr2(1-236) and Ras2. The amounts of free and Byr2-bound Ras were calculated as described in the text.



To prove the competitive nature of the inhibition, we extended the analysis by examining the patterns of inhibition in the presence of various concentrations of GST-Byr2(1-236) or Ras2 (Fig. 1B). We assumed that GST-Byr2 and adenylyl cyclase bound mutually exclusively to Ras2. At each point of Ras2 concentration in the curves obtained for various amounts of GST-Byr2, we obtained free Ras2 concentration available for adenylyl cyclase activation as that required for giving the same adenylyl cyclase activity in the absence of the competitor. A difference between the original and the free concentrations of Ras2 was regarded as that bound to GST-Byr2(1-236), and a reciprocal of this value was plotted against a reciprocal of the free Ras2 concentration (Fig. 1C). This gave a series of straight lines for each value of GST-Byr2(1-236), which converged on the horizontal axis. The data confirmed our assumption that GST-Byr2(1-236) polypeptide bound directly to Ras2 protein and competitively sequestered it from adenylyl cyclase. The K(d) value of GST-Byr2(1-236) for Ras2 was calculated from the point of intersection with the horizontal axis and determined to be about 1 nM, which is comparable with the value of human Raf-1 for H-Ras, 3.5 nM, or of yeast adenylyl cyclase for Ras2, 7 nM (see (10) for the data and a detailed description on the kinetic analysis).

The Effect of Byr2 N Terminus on Kinetic Properties of Ras Protein

A number of regulatory factors have been identified that interact directly with a specific set of small GTP-binding proteins and affect their intrinsic kinetic properties: various GAPs and proteins regulating guanine nucleotide exchanges (for reviews see (20) ). We examined whether the GST-Byr2 polypeptide had any effect on the intrinsic GDP dissociation and GTPase activities of Ras and found no effect detectable (data not shown and Fig. 2, respectively).


Figure 2: Inhibition of Ira2-stimulated GAP activity of Ras2 by GST-Byr2(1-236). An openbar stands for the amount of [-P]GTP bound to Ras2 (column 1) or Ras2 (column 7) put into each reaction at the start of incubation. Blackbars stand for the residual amounts of [-P]GTP bound to Ras2 (columns 2-6) or Ras2 (columns 8 and 9) after 5 min of incubation. The reactions contained: none (column 2); 48 pmol of GST-Byr2(1-236) (column 3); the lysate from yeast cells harboring pKT10 vector (columns 4 and 8); the lysate from yeast cells harboring pKT10-IRA2 (columns 5 and 9); 48 pmol of GST-Byr2(1-236) and the lysate from yeast cells harboring pKT10-IRA2 (column 6). The experiment was carried out as described under ``Experimental Procedures.''



In S. cerevisiae two proteins, Ira1 and Ira2, were identified as GAPs for Ras1 and Ras2(19) . Next we examined the effect of GST-Byr2 on the stimulatory activity of Ira2 protein on Ras2 GTPase. As shown in Fig. 2, the addition of GST-Byr2(1-236) efficiently inhibited the GAP activity of Ira2 in vitro. This inhibition may be caused by competitive sequestration of Ira2 from Ras2 by association of the Byr2 N-terminal polypeptide with Ras2.

GTP-dependent Binding of Ras Proteins to Byr2 N Terminus

Since Ras proteins are considered to be active in vivo in a GTP-bound form, we examined whether Ras bound to Byr2 N-terminal polypeptide in a GTP-dependent manner. The posttranslationally modified forms of Ras2 and H-Ras were loaded with GDP or GTPS and examined for binding with purified GST-Byr2(1-236) immobilized on glutathione-Sepharose 4B resin as described under ``Experimental Procedures.'' A great increase in binding was observed for the GTPS-bound Ras2 and H-Ras compared with their corresponding GDP-bound forms (Fig. 3A). Next the effect of a mutation in the effector domain of Ras2 on the binding activity to Byr2 was examined. We used the Ras2 mutant bearing a substitution of the critical Thr-42 for alanine, which had been shown to be inactive for binding with human Raf-1(7) . As shown in Fig. 3B, this mutant had negligible activity of binding to GST-Byr2(1-236). These results indicate that Ras proteins bind to the Byr2 N terminus in a GTP-dependent manner and that the binding site encompasses at least a part of the effector domain of Ras.


Figure 3: Binding of Ras to GST-Byr2(1-236). A, Ras2 and H-Ras, either in a GDP-bound or GTPS-bound form, were visualized by Western immunoblotting with an anti-Ras2 polyclonal antibody for Ras2 (17) and with a monoclonal antibody Y13-259 for H-Ras. B, Ras2 and its mutants in a GTPS-bound form were visualized by Western immunoblotting with the anti-Ras2 polyclonal antibody used in A. In each experiment, a aliquot of Ras protein used for the binding assay (shown by an arrowhead in the lower panel) and a aliquot of Ras protein bound to GST-Byr2(1-236)-bound resin (shown by an arrow in the upper panel) were applied onto each lane. Measurement of Ras binding was carried out as described under ``Experimental Procedures.''




DISCUSSION

We have demonstrated for the first time that S. pombe Byr2 binds directly to the GTP-bound conformation of Ras but not to its GDP-bound form. The binding was competitive with that of yeast adenylyl cyclase and abolished by an effector mutation of Ras, suggesting that the region of Ras recognized by Byr2 includes the effector domain. Measurement of a K(d) of the Byr2 N terminus for Ras2 yielded a value of as low as 1 nM, which was comparable with those of the other effectors, human Raf-1 and S. cerevisiae adenylyl cyclase, for their homologous Ras proteins (10) . Furthermore, Byr2 inhibited the GTPase stimulatory action of Ira2 on Ras2. This may provide other evidence that the interaction between Ras and Byr2 is direct considering that Ira2 interacts directly with Ras. Similar inhibition of Ras GAP activity was observed for human Raf-1 (5, 6) and presumably was ascribable to overlapping of the binding sites between GAP and the effector molecules. Although the physiological significance of the inhibition of GAP activity remains to be clarified, it may have a facilitating role in the signal transduction. Considering the observed genetic epistasis of the BYR2 over the RAS1(15) , the data indicate that Byr2 is an immediate downstream target of Ras1 in S. pombe.

S. pombe cells bearing the ras1 defect fail to conjugate and sporulate and are round in shape, unlike an elongated appearance of the wild-type cells(11) . In contrast, cells defective in Byr2 retain the normal elongated shape even though they possess deficiency in both conjugation and sporulation(15) . This may suggest that Ras1 has another effector molecule than Byr2 that affects the cell shape in S. pombe. Recently a candidate for this effector has been identified(21) . In mammalian cells, phosphatidylinositol 3-OH-kinase was proposed as a target of Ras(22) . Ras protein was shown to interact directly with the catalytic subunit of the enzyme in a GTP-dependent manner. In addition, a putative MAP kinase kinase kinase distinct from Raf-1 was shown to be regulated by Ras in mammalian cells although their mode of interaction remains to be clarified(23) .

We have shown that the N-terminal domains of both human Raf-1 and S. pombe Byr2 can effectively compete for Ras proteins with yeast adenylyl cyclase ( (10) and this paper). Strangely, the three targets of Ras, Byr2, Raf-1, and adenylyl cyclase, do not share any detectable homology among amino acid sequences of their Ras-binding domains even though all of them can bind mutually competitively to Ras proteins and discriminate between the GTP-bound and GDP-bound forms of Ras. In contrast, primary structures of both the effector region and its immediately flanking region of Ras proteins are unusually well conserved from yeasts to mammals. This raises the following two possibilities: 1) the three target molecules share a common tertiary structure for Ras binding that is presently unpredictable from their primary amino acid sequences and 2) they possess considerable differences in their binding recognition sequences of Ras. A further binding study using a set of Ras mutants and determination of the three-dimensional structure of the Ras-target complexes will be needed to solve these fundamental problems.


FOOTNOTES

*
This investigation was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture, Japan and by grants from the Yamanouchi Foundation for Research on Metabolic Disease and the Senri Bioscience Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-78-341-7451; Fax: 81-78-341-3837.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; GST, glutathione S-transferase; MES, 2-(N-morpholino)ethanesulfonic acid; DTT, dithiothreitol; GTPS, guanosine 5`-O-(3-thiotriphosphate); GAP, GTPase-activating protein.


ACKNOWLEDGEMENTS

We thank K. Tanaka (Osaka University) for kindly providing pKT10-IRA2.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.