From the
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
The mating response of haploid yeast Saccharomyces
cerevisiae cells is regulated by the action of the mating
pheromones a and
The pheromone signal is transmitted by the
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
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
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.
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) .
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 STE7
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.
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
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.
We have previously shown in
genetic experiments that the function of Ste20p within the pheromone
signaling pathway lies downstream of the
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
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
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
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
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 [
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
[
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).
Plasmid pVTU-STE20 (STE20 wild-type), the control vector
pVT102-U (ste20
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-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) .
-
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.
and
-subunits of G-proteins
might be involved(20, 21) .
-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) .
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.
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.
allele (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) .
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.
-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.
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.
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.
-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.
(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).
-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.
(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.
-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.
-
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.
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.
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
), 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.''
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.