©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization of Ste20p, a Potential Mitogen-activated Protein or Extracellular Signal-regulated Kinase Kinase (MEK) Kinase Kinase from Saccharomycescerevisiae(*)

Cunle Wu , Malcolm Whiteway , David Y. Thomas , Ekkehard Leberer (§)

From the (1)Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Ste20p protein kinase was immunopurified from yeast cells and analyzed in an in vitro assay system. Ste20p immune complexes exhibited autophosphorylating activity at serine and threonine residues and specifically phosphorylated a bacterially expressed glutathione S-transferase (GST) fusion of Ste11p (a mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase homologue) at serine and threonine residues. In contrast, GST fusions either of Ste7p (a MEK homologue) or the -subunit of the mating response G-protein and immunoprecipitated Ste5p were not phosphorylated by the Ste20p immune complexes. Myelin basic protein was identified as an excellent in vitro substrate, whereas histone H1 was only poorly phosphorylated. Evidence was obtained that autophosphorylation might play a regulatory role for the in vitro kinase activity. The in vitro activity was found to be Ca-independent. Both the in vivo and in vitro activities were abolished by mutational changes of either the conserved lysine residue 649 within the ATP binding site or threonine 777 between the catalytic subdomains VII and VIII. Wild-type Ste20p and the catalytically inactive T777A mutant were identified as phosphoproteins in vivo. The phosphorylation occurred at serine and threonine residues independent of pheromone stimulation. Based on the genetically determined significance of Ste20p in pheromone signal transduction and on our in vitro studies, we propose the model that Ste20p represents a yeast MEK kinase kinase whose function is to link G-protein-coupled receptors through G to a mitogen-activated protein kinase module.


INTRODUCTION

The mating response of haploid yeast Saccharomyces cerevisiae cells is regulated by the action of the mating pheromones a and -factor released from MATa and MAT cells, respectively. The cellular responses to these polypeptides include growth arrest of the cells in G of the cell cycle, morphological and biochemical alterations in the cell surface to enhance the selective binding to cells of the opposite mating type, and transcriptional activation of genes whose products are required for the fusion of the two conjugating cells and ultimately the fusion of the two nuclei(1) . These responses are triggered by binding of the pheromones to cell type-specific receptors coupled to a heterotrimeric G-protein that is common to both cell types(1) .

The pheromone signal is transmitted by the - and -subunits of the mating response G-protein (2, 3) to a protein kinase cascade assembled by the Ste11p, Ste7p, Fus3p, and Kss1p protein kinases. Genetic and biochemical experiments suggest that these protein kinases function in the order of Ste11p, Ste7p, and Fus3p/Kss1p(4, 5, 6, 7, 8, 9) and carry the signal to a pheromone-responsive transcription factor encoded by the STE12 gene and to Far1p, a protein required for pheromone-inducible growth arrest(1) . Sequence comparisons indicate that the functionally redundant Fus3p and Kss1p protein kinases represent yeast homologues of MAP()kinases (10-12), whereas the Ste7p and Ste11p protein kinases are yeast homologues of MEK (13, 14) and MEK kinase(15, 16) , respectively.

MAP kinases are a family of protein-serine/threonine kinases that become stimulated in response to a large variety of extracellular signals and are regulated via a protein kinase cascade, referred to as a MAP kinase module(1, 17) . MAP kinases are activated by the dual specificity protein kinase MEK through phosphorylation of two clustered threonine and tyrosine residues in the kinase domain. MEK in turn is regulated by phosphorylation either through Raf, which links the MAP kinase module to receptor tyrosine kinases by a mechanism that involves the small GTP-binding protein Ras(18, 19) , or through MEK kinase, which is believed to link the MAP kinase module to G-protein-coupled receptors(15) . The mechanism by which MEK kinase receives signals from G-protein-coupled receptors is still unclear, although increasing evidence suggests that, as in the mating response pathway in yeast, the and -subunits of G-proteins might be involved(20, 21) .

The STE20 gene encodes a protein with homology to protein-serine/threonine kinases and was first identified in a genetic screen that searched for gene dosage suppressors of defective G-protein -subunit mutants(22) . Genetic experiments indicate that Ste20p is essential for the transmission of the pheromone signal from G to the downstream MAP kinase module(22) . This step also requires the function of Ste5p (22, 23) and, through an unknown mechanism, the function of Ste50p(24) . The Ste5p function has been genetically placed between Ste20p and Ste11p within the signaling pathway(22) . It has recently been shown that Ste5p forms a complex with the Ste11p, Ste7p, and Fus3p protein kinases and it has been proposed that Ste5p might be a scaffolding protein required for the function of the pheromone response MAP kinase module(25, 26, 27) .

Here we provide a biochemical characterization of the Ste20p protein kinase and demonstrate that Ste20p is a protein-serine/threonine kinase. Intriguingly, we found that the Ste11p protein kinase provides a good in vitro substrate for Ste20p. Based on this finding and the genetically demonstrated point of the in vivo function of Ste20p within the pheromone signaling pathway(22) , we propose the model that Ste20p represents a yeast homologue of a MEK kinase kinase whose role is to transmit a signal to a MAP kinase module in response to G-protein activation.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases and DNA-modifying enzymes were obtained from Boehringer Mannheim, Life Technologies, Inc., Pharmacia Biotech Inc., and New England Biolabs. Taq thermostable DNA polymerase was purchased from Cetus. [-P]dATP, [-P]ATP and [P]phosphate were obtained from ICN. Acid-washed glass beads (450-600 mm), synthetic -factor, myelin basic protein, potato acidic phosphatase, protease inhibitors, phosphotyrosine, phosphoserine, phosphothreonine, and bovine serum albumine were purchased from Sigma. -Factor was dissolved in 90% methanol at a concentration of 1.0 mg/ml and stored at -20 °C. Histone H1 and calf intestine phosphatase were obtained from Boehringer Mannheim. Plasmid pGEX-2T, glutathione-Sepharose beads, GST-Sepharose beads, and glutathione were obtained from Pharmacia Biotech Inc. Alkaline phosphatase-conjugated and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad. Protein phosphatase 2A was from Upstate Biotechnology Inc. The anti-Myc monoclonal antibody 9E10 was supplied by Santa Cruz Biotechnology Inc. Nitrocellulose membranes were from Xymotech. Thin-layer chromatography plates (100 µm) were obtained from EM Science. All other reagents were of the highest purity grade commercially available.

Recombinant DNA Techniques and Yeast Manipulations

Standard protocols were used for all recombinant DNA techniques(28) . DNA sequencing was performed by the dideoxy chain termination method (29) with the Klenow fragment of DNA polymerase I and [-P]dATP.

Yeast media, culture conditions, and manipulations of yeast strains were as described previously(30) . Yeast transformations with circular or linearized plasmid DNA were carried out after treatment of yeast cells with lithium acetate(30) . Plasmid DNA was isolated from yeast cells as described previously(30) .

Strains and Plasmids

The S. cerevisiae strains used in this study were either the STE20 wild-type strain UC100 (MATaleu2 trp1 ura3 pep4 prb1 GAL) (31) or the ste20 deleted strain YEL206 (MATaste20::TRP1 ade2 his3 leu2 trp1 ura3 can1). For construction of strain YEL206, the complete coding sequence of the STE20 gene in strain W303-1A (R. Rothstein, Columbia University) was replaced by the TRP1 gene as follows. An NsiI-KpnI fragment extending from nucleotide positions -977 to +3,075 of the STE20 gene (22) was subcloned into the PstI and KpnI sites of the Bluescript KS(+) vector (Stratagene) to yield plasmid pDH106. A plasmid that contained STE20 flanking sequences from nucleotides -68 to -977 and from nucleotides +2,679 to +3,075, flanked by NsiI sites, was amplified by polymerase chain reaction(32) . For this purpose, Taq thermostable DNA polymerase was used with the divergent oligodeoxynucleotide primers 5`-TGCATGCATTTGGGTTGCTTGCTACCTCGC-3` and 5`-TGCATGCATAGCTGAGAACATGGATGCTGA-3` (the NsiI sites are underlined). The amplified DNA was cleaved with NsiI and ligated with a 0.9-kilobase PstI to PstI fragment of TRP1 derived from pJJ246 (33) to yield plasmid pDH104. This plasmid was linearized with BamHI and KpnI and transformed into yeast cells to replace the complete coding sequence of STE20 with TRP1 by homologous recombination. Replacement of the STE20 gene was confirmed by polymerase chain reaction using oligodeoxynucleotide primers corresponding to the nucleotide sequences from positions -238 to -218 and from +2821 to +2844. Amplification of genomic yeast DNA gave rise to fragments of 3 kilobases for the wild-type and 1 kilobase for the TRP1-disrupted STE20 gene.

Escherichia coli strain MC1061 was used for the propagation of plasmids. Plasmid pVTU-STE20 is multicopy plasmid pVT102-U carrying STE20 under control of the ADH1 promoter and URA3 as selectable marker(22) . The GST-STE20 fusion genes were constructed as follows. The STE20 fragments A (from nucleotide positions 1 to 1,485), B (from nucleotide positions 1,489 to 1,849) and C (from nucleotide positions 2,455 to 2,839) were amplified by polymerase chain reaction (32) using following pairs of oligodeoxynucleotide primers: 5`-TCAGATCTATGAGCAATGATCCATCT-3` (a BglII site that is added immediately upstream of the initiation codon is underlined) and 5`-AGAATTCATGACGGGTGA-3` (the intrinsic EcoRI site at nucleotide position 1,485 is underlined) for fragment A; 5`-CTGGATCCTCTGCCGCCAATGTTTCG-3` and 5`-CGGAATTCTTGGGCTACCGTCTGAGC-3` for fragment B (the newly created BamHI and EcoRI sites, respectively, are underlined); and 5`-CTGGATCCCCGCTAAGAGCACTGTAT-3` and 5`-CGGAATTCTGTACCCTGCTTGCTACG-3` for fragment C (the newly created BamHI and EcoRI sites, respectively, are underlined). The fragments were then subcloned into plasmid pGEX-KT (kindly provided by J. Dixon) to yield plasmids pGEX-KT-S20A, pGEX-KT-S20B, and pGEX-KT-S20C, respectively.

In order to construct a GST-STE4 fusion gene, a 2.0-kilobase BamHI to EcoRI fragment from plasmid pL19 (34) was subcloned into the BamHI and EcoRI sites of plasmid pGEX-2T (Pharmacia Biotech Inc.) to yield plasmid pGST-STE4. Plasmid pNC358 is plasmid pGEX-2T carrying a GST fusion of the Myc epitope-tagged STE7allele (5) and was kindly provided by B. Errede. Plasmid pGA1970 (kindly provided by B. Errede and G. Ammerer) is plasmid pGEX-2T carrying a GST fusion of the Myc epitope-tagged STE11 truncation allele, which carries a truncation of 341 amino acids at the amino terminus and a replacement of threonine residue 596 by isoleucine.

Oligodeoxynucleotide-directed Mutagenesis of STE20

E. coli strain CJ236 (dutung) was used to prepare single-stranded, uracil-containing template DNA of plasmid pVTU-STE20(35) . The following oligodeoxynucleotides were then used to direct DNA synthesis by T4 DNA polymerase(35) : 5`-GTGGCCATTAGGCAAATGAATCTC-3` to change lysine residue 649 to an arginine residue; 5`-GTGGCCATTGCGCAAATGAATCTC-3` to change this lysine residue to an alanine residue; 5`-CTTGAAAAGAGCTGCTATGGTG-3` to change the threonine residues at positions 772 and 773 to alanine residues; and 5`-ATGGTGGGAGCGCCTTATTGG-3` to change the threonine residue at position 777 to an alanine residue. The mutations were confirmed by DNA sequencing.

Preparation of GST Fusion Proteins

The GST fusion proteins were expressed in E. coli strain MC1061, bound to glutathione-Sepharose beads, and eluted with glutathione as described previously(36) , with the exception that expression was induced with 0.4 mM isopropyl-1-thio--D-galactopyranoside for 4 h at 30 °C at a cell density of A = 1.0.

Preparation and Characterization of Antibodies

Two different polyclonal antibodies against Ste20p were raised in rabbits. A GST fusion of the Ste20p fragment A was used to produce the polyclonal antibodies 317, and a mixture of GST fusions of the Ste20p fragments B and C was used to produce the polyclonal antibodies 251. For the primary injection, 100 µg of fusion protein were dissolved in 0.5 ml of 10 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl, emulsified in complete Freund's adjuvant, and then injected intramuscularly. Subsequently, the rabbits were boosted at 4-week intervals with the same amounts of protein emulsified in incomplete Freund's adjuvant. Three boosts were performed; 10 days after the final boost, the rabbit was bled and the serum was collected. The antibodies 317 were affinity purified with a nitrocellulose strip containing immobilized GST-Ste20p fragment A as described by Ref. 37. For affinity purification of the antibodies 251, the anti-serum was passed over an affinity column containing GST cross-linked to glutathione-Sepharose(38) . The flow-through was then applied to a glutathione-Sepharose column to which the GST fusions of the Ste20p fragments B and C were bound(38) . After extensive washing, specific Ste20p antibodies were then eluted with 0.2 M glycine buffer, pH 2.2, and quickly neutralized with 1 M Tris base(39) .

For immunoblot analyses, 50-100 µg of total yeast cell extracts were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (40) and transferred electrophoretically to nitrocellulose membranes(41) . The membranes were blocked with 5% nonfat milk powder in buffer A (50 mM Tris-HCl buffer, pH 7.5, containing 150 mM sodium chloride and 0.05% Tween 20) for 30 min and, after washing in buffer A, incubated for 1 h with 1:1,000 dilutions of the Ste20p antibodies in buffer A containing 1% bovine serum albumin. The membranes were washed 3 times in buffer A and then incubated for 1 h with 1:3,000 dilutions of either alkaline phosphatase-conjugated or horseradish peroxidase-conjugated goat anti-rabbit IgG in buffer A. The membranes were washed again as described above and developed using 5-bromo-4-chloro-3-indolyl phosphate and p-nitro tetrazolium chloride blue when alkaline phosphatase-conjugated secondary antibodies were used or the ECL system from Amersham when horseradish peroxidase-conjugated antibodies were used.

Anti-Ste5p antibodies were raised against a GST fusion with amino acids 716-917 of Ste5p(23) . Preparation and characterization of these antibodies will be described elsewhere.

Preparation of Yeast Cell Extracts

Cultures of 100 ml of yeast cells were grown in selective media to an A of 0.5-0.8, harvested by centrifugation at 3,000 g and resuspended in 1 ml of buffer B (50 mM Tris-HCl buffer, pH 7.5, containing 100 mM sodium chloride, 50 mM sodium fluoride, 5 mM EDTA, 1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 5 µg/ml antipain, and 1% Triton X-100). Cells were then disrupted by vortexing with glass beads for 6 pulses of 30 s each and kept on ice for 1 min between pulses. The extracts were clarified by centrifugation in a microcentrifuge at top speed for 15 min at 4 °C, and total protein concentration was determined by the Bradford reaction using the Bio-Rad protein determination kit following the manufacturer's instructions (Bio-Rad). The supernatants were supplemented with 10% glycerol, and stored at -80 °C in aliquots containing approximately 200 µg of total protein.

Protein Kinase Assays

Aliquots of cell extracts containing approximately 200 µg of total cellular protein were thawed on ice and incubated with 5 µl of anti-Ste20p antibodies for 1 h at 4 °C in a final volume of 0.5 ml of buffer B supplemented with 0.1% bovine serum albumine. The antibody-antigen complexes were then incubated with 30 µl of protein A-Sepharose beads in buffer B (50% (v/v)) for 1 h at 4 °C. After washing 4 times in buffer B and twice in kinase buffer (50 mM Tris-HCl buffer, pH 7.5, containing 20 mM magnesium chloride, 1 mM dithiothreitol, 0.5 mM sodium orthovanadate, 5 µg/ml aprotinin, and 5 µg/ml leupeptin), the adsorbed immune complexes were prewarmed at 30 °C for 10 min. The kinase reactions were then started by the addition of 30 µl of prewarmed (30 °C) kinase buffer supplemented with 1 µM ATP and 1 µl of [-P]ATP (4,500 Ci/mmol, 10 µCi/µl). The reaction mixture was incubated for 5-60 min at 30 °C and then boiled for 5 min after the addition of 30 µl of 2 Laemmli buffer (40). Aliquots (10-20 µl) of the solubilized immune complexes were then separated by SDS-PAGE(40) . Gels were either dried and autoradiographed directly, or the samples were transferred to nitrocellulose (41) and then autoradiographed.

Phosphatase treatments of Ste20p immune complexes prior to the kinase reactions were performed as follows. The Ste20p immune complexes were first washed 3 times with buffer B and then twice with phosphatase buffer (10 mM MES, pH 5.5, 1 mM magnesium chloride, 50 mM sodium chloride, 0.1 mM dithiothreitol, 5 µg/ml aprotinin, and 5 µg/ml leupeptin for incubation with potato acidic phosphatase; 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 50 mM -mercaptoethanol, 1 mM manganese chloride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin for incubation with protein phosphatase 2A; and 50 mM Tris-HCl, pH 8.8, 1 mM magnesium chloride, 0.1 mM zinc chloride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin for incubation with calf intestine phosphatase). The Ste20p immune complexes were then incubated for 30 min at 37 °C in 100 µl of phosphatase buffer containing either 2.5 units of potato acidic phosphatase, 0.2 units of protein phosphatase 2A or 20 units of calf intestine phosphatase. Control incubations were performed in the presence of phosphatase inhibitors (5 mM sodium orthovanadate to inhibit either potato acidic phosphatase or calf intestine phosphatase, or 30 nM okadaic acid to inhibit protein phosphatase 2A) or in the absence of phosphatases. After washing 3 times with kinase buffer, the kinase reaction was then performed with myelin basic protein as a substrate as described above.

Relative phosphorylation levels were determined either by densitometric analyses of the autoradiographs with the LKB Ultroscan XL Laser Densitometer (Pharmacia) or by excising the labeled protein bands after SDS-PAGE from the dried gel and measuring their radioactivity by using the Beckman LS3801 scintillation counter. The relative protein amounts in the immune complexes were determined by immunoblot analyses using the ECL system (Amersham Corp.) and subsequent densitometric evaluation of immunoblots with the laser scanner.

Phosphoamino Acid Analysis

Phosphoproteins were separated by SDS-PAGE(40) , transferred electrophoretically to Immobilon P membranes(41) , and autoradiographed. The band of interest was excised, and the phosphoprotein was hydrolyzed with 6 M HCl for 60 min at 110 °C. The hydrolysate was dried by centrifugation under vacuum, resuspended in 300 µl of water, and dried again. After repeating this procedure, the sample was dissolved in 5-20 µl of buffer C (5.9% glacial acetic acid, 0.8% formic acid, 0.3% pyridine, and 0.3 mM EDTA) at an activity of 200-500 cpm/µl(42) . Aliquots with a radioactivity of 500 cpm were mixed with a mixture of equal amounts of phosphotyrosine, phosphoserine, and phosphothreonine (1 µg each) and spotted onto cellulose thin-layer chromatography plates. Electrophoresis was performed in buffer C at 800 V for 75 min(43) . The plates were then air dried, and the amino acids were visualized with 0.25% ninhydrin in acetone. Phosphoamino acids were identified by autoradiography.

Other Methods

In vivo phosphate labeling of yeast cells was performed with 1 mCi of [P]phosphate as described previously(9) . Quantitative mating assays were performed by a filter mating assay(23) . Briefly, aliquots of exponentially growing experimental cells were mixed with a 4-fold excess of exponentially growing tester cells and filtered through nitrocellulose filters. The filters were placed onto nonselective plates, and mating was allowed to proceed for 3 h at 30 °C. The cells were then washed off of the filters and titered on selective media to determine the number of diploid cells, while the numbers of input experimental cells and tester cells were determined from unmated aliquots of the cultures. The mating efficiency was defined as the number of diploid cells divided by the number of input experimental haploid cells.


RESULTS

Immunochemical Isolation of Functional Ste20p Protein Kinase from Yeast Cells

Affinity-purified polyclonal antibodies directed against GST fusions of various Ste20p protein fragments were found to specifically detect a protein with an apparent molecular mass of 110 kDa in immunoblot analyses of total cell extracts from STE20 wild-type cells (Fig. 1A) and to specifically immunoprecipitate a 110-kDa protein (Fig. 1B). This molecular mass was in good agreement with the predicted size of 102 kDa for Ste20p(22) . No protein was detected by the antibodies in ste20-deleted cells (Fig. 1), suggesting that the 110-kDa protein corresponds to Ste20p.


Figure 1: Immunoblot analysis (panel A) and immune precipitation (panel B) with anti-Ste20p antibodies. In panel A, yeast extracts were prepared from ste20 deleted cells (YEL206) carrying either the STE20 plasmid pVTU-STE20 (+) or the control vector pVT102-U (-). Sixty µg of the extracts were then subjected to SDS-PAGE in 8% gels, transferred electrophoretically to nitrocellulose, and incubated with a 1:1,000 dilution of the affinity-purified anti-Ste20p antibodies 251 directed against fragments B and C of Ste20p. Immune staining was performed with alkaline phosphatase-conjugated secondary antibodies as described under ``Experimental Procedures.'' In panel B, Ste20p immune complexes were isolated with anti-Ste20p antibodies 251 from ste20 deleted cells (YEL206) carrying either the STE20 plasmid pVTU-STE20 (+) or the control vector pVT102-U (-) as described under ``Experimental Procedures'' and subjected to immunoblot analysis as described in panel A. The positions of standard proteins in kDa, of Ste20p and the immunoglobulin heavy chain (IgG) are indicated. Similar results were obtained with the anti-Ste20p antibodies 317 directed against fragment A of Ste20p (data not shown).



Immune complexes isolated from cell extracts of STE20 wild-type cells contain protein kinase activity resulting in the phosphorylation of the 110-kDa protein (Fig. 2). This protein kinase activity was not observed in immune complexes from cell extracts of ste20-deleted cells, nor was it observed in cells expressing mutant versions of Ste20p in which the highly conserved lysine residue at position 649 was replaced with either arginine or alanine by site-directed mutagenesis (Fig. 2). Both mutant versions failed to complement the mating defect of ste20-deleted cells, suggesting that lysine 649 is essential for the in vivo function of Ste20p (). These results suggest that the phosphorylation of the 110-kDa protein observed in Ste20p immune complexes is derived from a functional Ste20p protein kinase rather than from an associated protein kinase and reflects the autophosphorylation activity of Ste20p. It is also evident from Fig. 3B, that this capacity for autophosphorylation does not require stimulation of the cells with pheromone prior to the isolation of the protein. Moreover, addition of 5 mM of the Ca chelator EGTA into the kinase assay medium had no effect on the autophosphorylation reaction performed in the presence of Mg suggesting that the Ste20p protein kinase activity is Ca-independent (data not shown).


Figure 2: Autophosphorylation of Ste20p in immune complexes. Ste20p immune complexes were isolated from ste20-deleted cells (YEL206) carrying either the control vector pVT102-U, the STE20 plasmid pVTU-STE20, or STE20 mutant plasmids expressing mutant versions of STE20 with changes of lysine residue 649 to arginine (K649R) or alanine (K649A). The phosphorylation assays were performed for 10 min and analyzed as described under ``Experimental Procedures.'' Autophosphorylated Ste20p was visualized by SDS-PAGE and autoradiography (panel a), and Ste20p protein was quantified by immunoblot analysis using the ECL system as described under ``Experimental Procedures'' (panel b). The positions of Ste20p are indicated on the right side of the panels.




Figure 3: Substrate analysis of Ste20p immune complexes. A, phosphorylation of myelin basic protein and histone H1. Ste20p immune complexes were isolated from STE20 wild-type cells (UC100). Phosphorylation reactions were performed for 10 min in the presence of either 1 µg myelin basic protein or 1 µg histone H1, subjected to SDS-PAGE in 12% gels and autoradiographed. The positions of standard proteins, Ste20p, myelin basic protein (MBP) and histone H1 are indicated. B, phosphorylation of Ste11p by Ste20p immune complexes. Ste20p immune complexes were isolated from ste20-deleted cells (YEL206) carrying either the STE20 plasmid pVTU-STE20 (Ste20 (IP)) (+) or the control vector pVT102-U (Ste20 IP) (-). The cells were either induced for 40 min with 2 µM -factor (F) (+) or uninduced (-). The immune complexes were then analyzed for protein kinase activity (reaction time 10 min) in the presence (+) or absence (-) of 1 µg of Myc epitope-tagged GST-Ste11p fusion protein isolated from E. coli, subjected to SDS-PAGE in 8% gels, and autoradiographed. Relative Ste20p and Ste11p protein amounts were determined by immunoblot analyses using anti-Ste20p antibodies and the anti-Myc monoclonal antibody 9E10, respectively (data not shown). The autoradiographs and immunoblots were densitometrically analyzed with a laser densitometer. The relative phosphorylation levels were calculated in relation to the relative protein amounts and are shown on the bottom of the panel. The positions of Ste20p, Ste11p, and standard proteins (in kDa) are indicated on the right side of the panel. Panel C, time dependence of Ste20p (upper panel) and Ste11p phosphorylation (lower panel). Ste20p immune complexes were isolated from ste20 deleted cells (YEL206) carrying either the STE20 plasmid pVTU-STE20 (, STE20+), a STE20 mutant plasmid expressing the K649R mutant version of Ste20p (, ste20-K649R) or the control vector pVT102-U (, ste20). Phosphorylation reactions were then performed after addition of 1 µg GST-Ste11p fusion protein isolated from E. coli and stopped at the indicated time points. Relative phosphorylation levels were determined by measuring the radioactivity of the labeled protein bands after SDS-PAGE as described under ``Experimental Procedures.'' The relative amounts of Ste20p and Ste11p were determined by immunoblot analyses as described under B. The phosphorylation levels are given in relation to the protein amounts and represent mean values ± S.D. of three independent experiments.



In Vitro Phosphorylation of Ste11p by Ste20p

We have examined the ability of Ste20p immune complexes to phosphorylate various substrates in vitro. Myelin basic protein was heavily phosphorylated by immune complexes isolated from STE20 wild-type cells, whereas histone H1 was found to be a very poor substrate (Fig. 3A). The ability to phosphorylate these two substrates was completely blocked by replacing lysine residue 649 of Ste20p with either arginine or alanine (data not shown). In order to exclude the possibility that the in vitro capacity of the Ste20p immune complexes to phosphorylate myelin basic protein depends on the copurification of known components of the pheromone signaling pathway, which might serve as regulators of the kinase activity, we have examined the activity of Ste20p immune complexes isolated from STE20 wild-type cells that were disrupted either in the STE4, STE18, STE5, FUS3/KSS1, or STE11 genes. We found that none of the disruptions had any effect on the kinase activity of the Ste20p immune complexes (data not shown), suggesting that none of these components are required for the in vitro activity of Ste20p.

We have previously shown in genetic experiments that the function of Ste20p within the pheromone signaling pathway lies downstream of the -subunit of the G-protein encoded by the STE4 gene and upstream of Ste5p and the pheromone response MAP kinase module(22) . We therefore investigated whether any of these proteins might serve as in vitro substrates of Ste20p.

We have found that a bacterially expressed GST-Ste11p fusion protein, which exhibited weak autophosphorylation activity as judged by the incorporation of low levels of phosphate in the absence of Ste20p immune complexes (data not shown) or after the addition of Ste20p immune complexes isolated from ste20 deleted cells (Figs. 3, B and C), revealed 5-8-fold higher levels of phosphorylation when Ste20p immune complexes isolated from STE20 wild-type cells were added (Fig. 3, B and C). These elevated phosphorylation levels were not found after the addition of Ste20p immune complexes isolated from STE20 (Fig. 3C) or STE20 cells (data not shown), strongly suggesting that the elevated levels result from phosphorylation through Ste20p. This conclusion is corroborated by the finding that the time course of increased Ste11p phosphorylation in the presence of functional Ste20p was concomitant with the time course of Ste20p autophosphorylation (Fig. 3C). We also found that the ability of Ste20p immune complexes to phosphorylate the Ste11p fusion protein did not require stimulation of the yeast cells with pheromone prior to isolation of the Ste20p immune complexes (Fig. 3B).

We found in several experiments that Ste5p immunoprecipitated from yeast cells did not serve as a substrate of Ste20p in our assay system, although the Ste20p immune complexes were found to be functional, as indicated by autophosphorylation of the Ste20p protein (data not shown). Moreover, GST fusions of the -subunit of the G-protein (Ste4p) or the Ste7p protein kinase purified from E. coli and analyzed in our kinase assay system under the same conditions as the GST-Ste11p fusion protein, did not serve as in vitro substrates of Ste20p, although autophosphorylation of Ste20p could be measured (data not shown). These results suggest that, among the pheromone signaling components that we analyzed, Ste11p is the only good in vitro substrate of Ste20p.

Autophosphorylation of Ste20p

We investigated whether autophosphorylation of Ste20p might play a regulatory role. We preincubated Ste20p immune complexes with Mg and ATP prior to starting the kinase reaction by the addition of [-P]ATP and myelin basic protein as a substrate. We then analyzed the phosphate incorporation into Ste20p and myelin basic protein. As shown in Fig. 4A, we found that preincubation of Ste20p with Mg and ATP resulted in reduced labeling of Ste20p and elevated phosphorylation of myelin basic protein. These results suggest that preincubation of Ste20p with unlabeled ATP results in elevated autophosphorylation of Ste20p, thereby stimulating its capacity to phosphorylate myelin basic protein as a substrate, and point to the possibility that Ste20p autophosphorylation could be an important step in the regulation of Ste20p activity.


Figure 4: Autophosphorylation of Ste20p stimulates protein kinase activity in vitro. A, preincubation of Ste20p with ATP. Ste20p immune complexes were isolated from STE20 wild-type cells (UC100) that were induced with 2 µM -factor for 40 min. The Ste20p immune complexes were first incubated at 30 °C for 15 min with kinase buffer (see ``Experimental Procedures'') (lanes 1) or with kinase buffer containing 100 µM ATP (lanes 2), washed 4 times with kinase buffer and then analyzed in the in vitro protein kinase assay for 15 min in the presence of 1 µg of myelin basic protein as described under ``Experimental Procedures.'' The reaction mixtures were subjected to SDS-PAGE in 12% (a) and 7% (b) gels and transferred to nitrocellulose membranes. Labeled proteins were visualized by autoradiography (a). The amount of Ste20p protein was quantified by immunoblot analysis as described in b). The autoradiographs were densitometrically analyzed with a laser scanner. The relative phosphorylation levels of myelin basic protein are given by the numbers at the bottom of a. The positions of Ste20p and myelin basic protein (MBP) are indicated. B, preincubation of Ste20p with phosphatases. Prior to the kinase assay, the Ste20p immune complexes were incubated at 30 °C for 30 min with potato acidic phosphatase (PAP), protein phosphatase 2A (PP2-A) and calf intestine phosphatase (CIP) as described under ``Experimental Procedures.'' Controls were performed in the presence of phosphatase inhibitors (5 mM sodium orthovanadate for potato acidic phosphatase and calf intestine phosphatase, and 30 nM okadaic acid for protein phosphatase 2A) or without phosphatase. Kinase assays were performed for 15 min with myelin basic protein (MBP) as substrate and quantitatively evaluated by SDS-PAGE and counting the radioactivity of the excised protein band as described under ``Experimental Procedures.''



Preincubation of Ste20p immune complexes with potato acidic phosphatase prior to the kinase reaction was found to result in an approximately 90% loss of Ste20p-dependent phosphorylation of myelin basic protein (Fig. 4B). This finding supports the view that autophosphorylation might play a role in the activation of the kinase. We found, however, that incubation with calf intestine phosphatase or protein phosphatase 2A failed to have an effect on the kinase activity (Fig. 4B) although a band shift in the SDS-PAGE indicated that phosphate residues were removed from the protein (data not shown). It is conceivable that this differential effect is caused by variable substrate specificities of the various phosphatases. It should also be noted that the alkaline buffer conditions for incubation with calf intestine phosphatase were inhibitory (Fig. 4B).

Ste20p Phosphorylates Serine and Threonine Residues

Phosphoamino acid analysis established that the in vitro autophosphorylation of Ste20p occurred exclusively at serine and threonine residues (Fig. 5), confirming the sequence-derived prediction that STE20 encodes a serine/threonine protein kinase(22) . This conclusion was further supported by the finding that the Ste20p-dependent in vitro phosphorylation of the GST-Ste11p fusion protein also occurred only at serine and threonine residues (Fig. 5).


Figure 5: Phosphoamino acid analyses. Ste20p immune complexes were isolated from STE20 wild-type cells (UC100) and analyzed in the in vitro protein kinase assay in the presence of 1 µg bacterially expressed GST-Ste11p fusion protein. Phosphoamino acid analyses of Ste20p and GST-Ste11p were then performed as described under ``Experimental Procedures.'' The positions of standard phosphoamino acids are shown on the top.



Mutational Analysis of Conserved Threonine Residues in the Kinase Domain

The Ste20p protein kinase contains, in the catalytic domain between subdomains VII and VIII, threonine residues at positions 772, 773, and 777. The threonine residues 773 and 777 are conserved among related protein-serine/threonine kinases (Fig. 6A). Phosphorylation in this region has been shown to be regulatory for many protein kinases, and in some cases the homologous sites were found to be required for protein kinase activity (for reviews see Refs. 44-46).


Figure 6: Mutational analysis of conserved threonine residues in the loop between subdomains VII and VIII of Ste20p. A, comparison of amino acid sequences of Ste20p and homologous protein kinases between kinase subdomains VII and VIII. Amino acid sequences of STE20 (2), STE11 (16), Byr2 (55), STE7 (14), Byr1 (44), MKK1 (56), MAP kinase kinase (MAPKK1) (53), cAMP-dependent protein kinase (CAPK) (44), S6 protein kinase (RSK) (44), and cyclin-dependent protein kinase (CDK2) (57) were from published sources. Conserved serine or threonine residues are indicated by asterisks. Serine or threonine residues, which have been demonstrated to represent activating phosphorylation sites (7, 45, 46, 53, 58-60) are underlined. B, protein kinase assays. Ste20p immune complexes were isolated from ste20-deleted cells (YEL206) transformed with either the control vector pVT102-U or STE20 plasmids expressing Ste20p wild-type protein (STE20), the Ste20p mutant allele (T772/3A) or the Ste20p mutant allele (T777A). The phosphorylation assays were performed for 20 min as described under ``Experimental Procedures.'' Autophosphorylated Ste20p was visualized by SDS-PAGE and autoradiography (upper panel), and Ste20p protein was quantified by immunoblot analyses (lower panel). The autradiographs and immunoblots were densitometrically analyzed with a laser scanner. The relative amounts of Ste20p phosphorylation in relation to anti-Ste20p antibody binding are shown at the bottom of the upper panel. The positions of Ste20p are indicated on the right side of the panels.



We used oligodeoxynucleotide directed mutagenesis to create the T772A/T773A double mutant allele, in which threonine residues 772 and 773 were substituted by alanine residues, and the T777A single mutant allele, in which threonine residue 777 was replaced by alanine. The T772A/T773A double mutant allele was capable of fully complementing the mating defect of ste20 deleted cells, whereas the T777A single mutant allele was completely defective in the in vivo assay (). These results were confirmed in the in vitro protein kinase assays. The immunoprecipitated Ste20p mutant protein was found to be able to autophosphorylate to wild-type levels, whereas the Ste20p mutant protein was drastically reduced in its ability to autophosphorylate in the in vitro protein kinase assay (Fig. 6B). These observations demonstrate that threonine residue 777 is crucial for the catalytic activity of Ste20p in vivo and in vitro.

In Vivo Phosphorylation of Ste20p at Serine and Threonine Residues

Since the above described experiments indicated that autophosphorylation might play an important role for the regulation of Ste20p activity, we examined the in vivo phosphorylation of Ste20p. We labeled yeast cells with [P]phosphate and analyzed the phosphorylation levels of Ste20p immune complexes isolated from STE20 wild-type and STE20 mutant cells. As illustrated in Fig. 7a, we found in vivo phosphorylation of both alleles. This phosphorylation was observed in pheromone-induced as well as uninduced cells. It is noteworthy that the stimulation of cells with pheromone induced the appearance of a slightly slower migrating phosphoprotein in the SDS-PAGE (Fig. 7a). As revealed by phosphoamino acid analysis of the catalytically enfeebled T777A allele, the in vivo phosphorylation occurred exclusively at serine and threonine residues (Fig. 7b).


Figure 7: In vivo phosphorylation of Ste20p. STE20 plasmids expressing either the Ste20p mutant allele or Ste20p wild-type protein were introduced into ste20-deleted cells (YEL206). Cells were labeled with [P]phosphate as described under ``Experimental Procedures.'' When indicated (+), cells were induced with -factor for 40 min. Ste20p immune complexes were subjected to SDS-PAGE in 8% gels, transferred to nitrocellulose membranes, and analyzed by autoradiography (panel a). The position of Ste20p is indicated on the right side of the panel. The phosphoamino acid analysis of the Ste20p mutant protein (panel b) was performed as described under ``Experimental Procedures.'' The positions of standard phosphoamino acids is indicated on the top of the panel.




DISCUSSION

We have raised polyclonal antibodies against Ste20p and used an in vitro protein kinase assay to study the biochemical properties of the immunoprecipitated protein. We have shown that immunoprecipitated Ste20p autophosphorylates at serine and threonine residues and is able to phosphorylate a recombinant GST fusion construct of the Ste11p protein kinase at serine and threonine residues. These findings confirm our sequence-derived prediction that Ste20p belongs to the family of protein-serine/threonine kinases(22) .

The in vitro phosphorylation of Ste11p appears to be specific since immunopurified Ste5p protein and GST fusions of Ste7p and G (Ste4p) are not phosphorylated under the same assay conditions. Mutational changes of lysine 649, which is conserved within all protein kinases and essential for the catalytic activity of the protein kinases(47) , block both the in vivo and in vitro functions of Ste20p, strongly suggesting that the kinase activity observed in Ste20p immune complexes is derived from Ste20p and not from an associated protein kinase that might copurify with Ste20p.

The function of Ste20p had been genetically placed downstream of the mating response G-protein but upstream of the pheromone response MAP kinase module(22) . Two major questions concerning the in vivo function of Ste20p remain to be resolved. First, how is the protein kinase activity modulated by the -subunits of the G-protein in response to stimulation of cells with pheromone? Second, how does the activated Ste20p protein kinase carry the signal to the downstream MAP kinase module? We have found that normal levels of protein kinase activity were present when Ste20p immune complexes were isolated from yeast cells that carried disruptions in the genes encoding the - and -subunits of the G-protein and constituents of the MAP kinase module, suggesting that none of these components are required for the in vitro kinase activity observed in Ste20p immune complexes. We have also observed that Ste20p immune complexes exhibited protein kinase activity independent of the stimulation of the cells with pheromone prior to the isolation of Ste20p. These findings indicate that Ste20p is not regulated in vitro and can be best explained by the assumption that regulatory mechanisms that normally control the in vivo function of Ste20p have been lost during the isolation procedure. It is conceivable, for example, that the large amino-terminal domain of Ste20p has an inhibitory role and is not properly folded in vitro. This kind of interpretation is supported by the finding that truncation of the amino terminus generates a hyperactive Ste20p mutant(48) . Alternatively, it is possible that inhibitory factors have been lost during isolation of the protein. The in vivo mechanisms that control Ste20p function might involve the - and -subunits of the G-protein, as suggested by genetic experiments(22) , and the small GTP-binding protein Cdc42p, as suggested by the finding that Cdc42p binds to Ste20p and its mammalian homologue isolated from rat brain(49) . It should now be possible to use our in vitro system to study the regulatory role of these components.

Autophosphorylation might represent a crucial step in the activation of Ste20p. We have found that incubation of Ste20p immune complexes with ATP prior to the addition of [-P]ATP and the substrate stimulates the capacity to phosphorylate myelin basic protein that we have identified as an excellent in vitro substrate for the detection of Ste20p activity. In this respect, Ste20p resembles its mammalian homologue p65, which has also been shown to be activated in vitro by an autophosphorylation mechanism(49) . Consistent with this view is the finding that preincubation of Ste20p immune complexes with acidic phosphatase prior to the kinase reaction inhibits the kinase activity. Moreover, it is conceivable that intracellular localization might play a crucial role in the in vivo regulation of Ste20p activity. The availability of specific antibodies that are able to recognize the native Ste20p protein will allow investigation of this question in future immunocytochemical studies.

Specific serine and threonine residues in the region between the conserved DFG and (A/S)PE sequence motifs have been shown to be crucial for the regulation of a variety of protein kinases, either as autophosphorylation sites as in the case of cAMP-dependent protein kinase (for a review see Ref. 46) or as activating phosphorylation sites, which are phosphorylated by regulatory protein kinases as in the case of MAP kinases(50, 51) , the MEK kinase family (7, 52-54), and cyclin-dependent protein kinases. Ste20p contains three threonine residues in this region. We have shown by replacing these threonine residues with alanine that threonine 777 is crucial for both the in vivo and in vitro function of Ste20p, whereas the threonine residues at positions 772 and 773 are dispensable. It is interesting to note that the in vivo function of Ste20p was completely abolished by the T777A mutation (), although this mutant showed some weak autophosphorylating activity in the in vitro kinase assay. It is conceivable that threonine 777 is crucial for the catalytic activity and that the mutational change therefore generates an enfeebled kinase. It is also possible, however, that this residue represents an important regulatory phosphorylation site required for the in vivo activation of the kinase.

We have labeled yeast cells with [P]phosphate and made the interesting observation that Ste20p is in vivo phosphorylated at serine and threonine residues in unstimulated as well as stimulated cells. In pheromone-stimulated cells, we observed the occurrence of an additional, electrophoretically slower migrating phosphoprotein, which we have shown, by phosphatase treatment, to represent a higher phosphorylated form of Ste20p.()The same phosphorylation pattern was observed in the catalytically enfeebled T777A mutant version, suggesting that the in vivo phosphorylation pattern is not predominantly caused by autophosphorylation. These results point to the possibility that Ste20p is phosphorylated by other protein kinases, which might have regulatory functions. Ste20p has been shown to exert an overlapping function with the homologous protein kinase Cla4p during cytokinesis.()Disruptions of the genes encoding both protein kinases generate a lethal phenotype. We therefore expect that the Ste20p in vivo function is regulated by two independent mechanisms: one during the cell cycle to regulate cytokinesis and another during mating to regulate pheromone responses. The observed basal phosphorylation in unstimulated cells might play a role during cytokinesis, whereas the additional phosphorylation after pheromone stimulation might be involved in pheromone signal transduction.

Like immunoprecipitated Ste20p, immunoprecipitated Ste11p or a GST-Ste11p fusion protein isolated form yeast cells is not regulated in vitro as judged by autophosphorylation activity and the capacity to phosphorylate Ste7p(7, 16) . We therefore have used in our in vitro Ste20p kinase assays a GST-Ste11p truncation mutant that was purified from E. coli and exhibited only a low autophosphorylation activity. This truncation mutant also carried a replacement of threonine 596 by isoleucine, which has been shown to generate a hyperactive in vivo phenotype(8) . We have also found that this bacterially expressed protein was unable to phosphorylate a bacterially expressed GST-Ste7p fusion protein (data not shown). We have hoped that Ste20p-dependent phosphorylation of the GST-Ste11p protein could reconstitute its ability to phosphorylate Ste7p. We have been unable, however, to detect any stimulatory effect of Ste20p on the activity of Ste11p (data not shown). It therefore remains to be determined in future experiments whether the observed Ste11p phosphorylation is of functional significance and leads to a stimulation of Ste11p kinase activity in vitro. The genetically determined point of the in vivo function of Ste20p within the pheromone signaling pathway (22) is an indirect indication, however, that activation of the MAP kinase module in pheromone-induced cells might occur through phosphorylation of Ste11p by Ste20p (Fig. 8).


Figure 8: Proposed model of the pheromone signaling pathway. We propose in our model that the Ste20p protein kinase carries the signal from the - and -subunits of the G-protein to the mating response MAP kinase module by phosphorylation of the Ste11p protein kinase. The Ste5p protein is proposed to act as a scaffolding protein for the MAP kinase module (25-27). Stimulation of the kinase cascade ultimately leads to phosphorylation of Ste12p to induce transcriptional activation and of Far1p, a protein required for pheromone-inducible growth arrest (for a review see Ref. 1). Our model does not exclude the involvement of not yet identified components. Lipid modifications of G and G that are thought to serve as membrane anchors for the G-protein are indicated.



Genetic experiments had indicated that the Ste5p protein plays an essential role in transmitting the pheromone signal from Ste20p to the MAP kinase module and acts immediately downstream of Ste20p and upstream of Ste11p(4, 8, 22, 23) . The Ste5p protein was therefore a strong candidate for a substrate of Ste20p. Here we have identified Ste11p instead of Ste5p as an in vitro substrate. The Ste5p protein, which does not contain any specific structural features that would allow to predict enzymatic activity(23) , has recently been shown to form a complex with the protein kinases that constitute the pheromone response MAP kinase module and has been suggested to form a scaffold required for the function of the MAP kinase cascade(25, 26, 27) . Based on these observations and our finding that Ste11p is specifically phosphorylated by Ste20p in vitro, we propose the model (Fig. 8) that G activates directly or indirectly the Ste20p protein kinase that in turn stimulates the MAP kinase module through phosphorylation of the Ste11p protein kinase. Thus we propose that Ste20p acts as a MEK kinase kinase whose function is to link G-protein-coupled receptors to a MAP kinase module.

  
Table: Mating efficiencies of ste20 mutant cells

Plasmid pVTU-STE20 (STE20 wild-type), the control vector pVT102-U (ste20), and the pVTU-STE20 plasmids carrying the indicated STE20 alleles were transformed into the ste20-deleted strain YEL206. Mating efficiencies were determined at 30 °C with the tester strain DC17 (MAT his1) as described under ``Experimental Procedures.''



FOOTNOTES

*
This work was supported in part by the National Research Council (NRC) of Canada and by a collaborative agreement with Glaxo Group Research Limited and Glaxo (Canada) Ltd. The NRC publication number for this work is 38534. 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: Eukaryotic Genetics Group, Biotechnology Research Inst., National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Tel.: 514-496-6358; Fax: 514-496-6213.

The abbreviations used are: MAP, mitogen-activated protein; MEK, MAP or extracellular signal-regulated kinase (Erk) kinase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electorphoresis; MES, 2-(N-morpholino)ethanesulfonic acid.

T. Leeuw, C. Wu and E. Leberer, unpublished observations.

F. Cvrckova and K. Nasmyth, personal communication.


ACKNOWLEDGEMENTS

We thank G. Ammerer, J. Dixon, E. A. Elion, and B. Errede for generous gifts of plasmids and yeast strains. We wish to thank D. Dignard for DNA sequencing and D. Harcus for excellent technical assistance in plasmid constructions.


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