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INTRODUCTION |
PHO2 (also known as GRF10, BAS2) (1, 2), a protein of 559 amino
acids (3), is currently known as a transcriptional activator of
PHO5 (4), PHO81 (5), HIS4 (2),
CYC 1 (6), TRP4 (7), HO (8), and
ADE1, ADE2, ADE5, 7,
and ADE8 genes (9) in Saccharomyces cerevisiae.
It was first identified as a positive regulatory factor that, together
with a second DNA binding factor PHO4 (4), activated the transcription
of PHO5, PHO11, PHO81, and
PHO84 in the so-called S. cerevisiae PHO system.
The transcription of PHO5 gene encoding a secreted acid
phosphatase in S. cerevisiae is regulated in response to the
extracellular concentration of inorganic phosphate through a system
consisting of the products of at least five genes, PHO2, PHO4
PHO80, PHO85, and PHO81 (10). A current model for the
acid phosphatase regulation proposes that the two positive regulatory
factors encoded by PHO4 and PHO2 are
indispensable for the transcription of PHO5 (10). In high
phosphate medium, the complex consisting of
cyclin-dependent kinase PHO85 and cyclin PHO80
phosphorylates and presumably inactivates the transcriptional factor
PHO4 (11). When the concentration of phosphate in the medium is
sufficiently low, PHO81 protein, which is a
cyclin-dependent kinase inhibitor, inhibits the kinase activity of the PHO80·PHO85 complex (12), thus allowing the hypophosphorylated PHO4 protein, together with PHO2 protein, to activate the transcription of PHO5 gene.
PHO2 has four conspicuous regions (see Fig. 1). The first one is a
glutamine-rich region (amino acids 23-52), the deletion of which was
showed to have no effect on PHO2 function (13). The other three regions
include the homeodomain (amino acids 77-136) (14), the acidic region
(amino acids 294-329), and the region (amino acids 241-258) with
homology to PHO80 protein. Homeo boxes were found in many genes of
higher eukaryotes involved in development (e.g.
Drosophila, mouse and human). The PHO2 homeodomain is
directly involved in DNA recognition. The acidic region of PHO2
contains continuous stretches of aspartic acid and asparagine. The
PHO80 homologous region may be an association domain of PHO2 with PHO4, the deletion of which inactivated the PHO2 protein (13). The deletions
at the C-terminal end of PHO2 protein may go up to residue 408 without
strongly affecting the derepression of PHO5 (15).
The results of the two-hybrid assay (16) argued that DNA binding by
PHO4 is dependent on the phosphate-sensitive interaction with PHO2. It
was also suggested that interaction with PHO2 increases the
accessibility of the activation domain of PHO4 (17). Recently, immunoprecipitation experiments and protein binding assays showed that
PHO2 and PHO4 form a complex with a DNA fragment and interacted with
each other directly in vivo (18).
Protein phosphorylation has been found to play an important role in the
control of diverse cellular processes, especially that of
transcriptional factors in the regulation of gene expression. PHO80-PHO85 cyclin-CDK (CDK, cyclin-dependent kinase)
complex can regulate the expression of PHO5 gene by
phosphorylating PHO4 (11). Cyclic AMP-dependent protein
kinase (protein kinase A) has also been observed to exert its function
in the synthesis of repressible acid phosphatase (19). As well,
cdc2/CDC28 type kinase can affect gene expression by phosphorylation.
The consensus motif of sites phosphorylated by cdc2/CDC28-type kinase
is (Ser/Thr)-Pro-X-(Lys/Arg) (20). This sequence is found
more frequently in nuclear proteins involved in transcriptional
regulation than in proteins generally. Many of these proteins also
contain DNA binding motif such as zinc fingers or helix-turn-helix
structures (21-24).
In this report we describe the identification of a potential
phosphorylation site near the PHO80 homologous region of PHO2 protein.
The mutation of the site can affect the function of PHO2 protein.
Moreover, the introduction of a negative charge to this residue seems
to be necessary for PHO2 functions. We also demonstrate that PHO2 can
be phosphorylated by CDC28 kinase in vitro.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Media--
The wild type S. cerevisiae strain used in this work was YPH499 (Stratagene) with
the following genotype: MAT
, ura3-52,
lys2-801amber,
ade2-101ochre, trp1
63,
his3200, leu21. YJ1 is a derivative of
YPH499, which contains a URA3 disruption of PHO2.
The strain SFY526 (MATa, ura3-52, his3-200, ade2-101, lys2-801,
trp1-901, leu3-2, 112, canr, gal4-542, gal80-538,
URA3::GAL1-lacZ) was used in two-hybrid assays. SFY526 (pho4::HIS3) contains a
HIS3 disruption of PHO4.
Yeast culture were grown at 30 °C in either YPD (1% yeast
extract, 2% glucose, 2% peptone) or synthetic medium (0.67% yeast nitrogen base, 2% glucose) supplemented with the appropriate amino acids as required. All yeast transformations were done using the high
efficiency lithium acetate method of Gietz and Schiestl (24). For the
experiments involving high or low phosphate, Burkholder medium (25) was
used, but the content of KH2PO4 was changed as
described previously (26).
Plasmids--
pVA3 and pTD1 were obtained from
CLONTECH. pRS2 is a derivative of pRS415
(YCp)1 (Stratagene), with a
3.6-kb HindIII/HindIII fragment containing the
PHO2 whole gene. pGBT9-PHO2 (or mutants) and
pGAD424-PHO2 were constructed by preparing a 2.6-kb
EcoRI/XhoI fragment from pRS2 (or mutants) and
inserting into the EcoRI/SalI gap of pGBT9 and
pGAD424 (CLONTECH). pRS4 is a derivative of pRS415
with a HindIII/BclI fragment of PHO4
gene. pGBT9-PHO4 was constructed as follows: a 1.0-kb
NcoI/PstI fragment of PHO4 gene, whose
5'-end was blunted, was inserted into
SmaI-PstI-digested pGBT9.
Polymerase chain reaction was used with yeast genomic DNA to amplify a
0.9-kb fragment including the entire coding region of CDC28
gene. The 5' oligonucleotide primer was ATCC GGAT CCTG ATGA GCGG TGAA
TTAG CA, and the 3' oligonucleotide primer was GGAG CTGC AGTT ATGA TTCT
TGGA AGTA GG. Polymerase chain reaction was also used with yeast
genomic DNA to tag the CDC28 protein by adding a C-terminal extension
of GAYPYDVPDYASLG, which includes a hemagglutinin antigen (HA) epitope.
The same 5' oligonucleotide primer was used in conjunction with the 3'
oligonucleotide primer: AGTC TGCA GTCA TCCC AAGC TAGC GTAG TCAG GAAC
GTCA TATG GATA GGCG CCTG ATTC TTGG AAGT AGGG GTGG. Amplified fragments
were digested with BamHI and PstI, which cleaved
within the primers and, respectively, cloned into the
BamHI/PstI sites of pGBT9 and the yeast
expression vector pVTU102 (27) to create plasmids
pGBT9-CDC28 and pVTU102-CDC28HA. The absence of
polymerase chain reaction-introduced mutations was verified
by DNA sequencing.
For the construction of the expression plasmids for GST-PHO2 fusion
protein, a 12-mer BglII linker (Life Technologies, Inc.) was
inserted into the SmaI sites of pGEX-2T (Amersham Pharmacia Biotech). A 1480-bp BclI fragment of PHO2 gene
(wild type or mutants) coding region was ligated into the
BglII-cleaved pGEX-2T (SmaI 
BglII)
to create plasmids including pGEX2W (wild type), pGEX2M1(S230A), pGEX2M2 (P231S), pGEX2M3 (S230D). These constructs express a fusion protein that contains amino acids 35-528 (of 559 amino acids total) of
PHO2 fused to GST at the PHO2 N terminus.
To construct plasmid pBL-PHO4, a 1.0-kb
NcoI/HindIII fragment was prepared from
PHO4 gene and inserted into the
NcoI/HindIII sites of plasmid pBL. This plasmid
produces the entire PHO4 protein (312 amino acids).
Site-directed Mutagenesis of the PHO2 Gene--
Five synthetic
oligonucleotides were used for site-directed mutagenesis with a
U-DNA mutagenesis kit (Roche Molecular Biochemicals): 5'-ATCAGATCTGCTATTTTCTTT-3';
5'-GAGAAACCTAGCGCCAATAAA-3';
5'-GTTGAGAAACCTAGACCCAATAAAGAT-3'; 5'-AAACCTATCGTCAATAAAGAT-3';
5'-ATCGCCAATACAGATTAATAAC-3'. A 1.0-kb PvuI/HindII fragment of the PHO2
gene was cloned into the M13mp18 vector to produce single-stranded DNA.
The mutagenesis procedures were then conducted according to the kit
protocols. The mutants were isolated and sequenced to ensure that the
intended base changes were present. The fragment from the
replication form of M13mp18, containing the designed mutation,
was then cloned back to pRS2.
Activity Assay of Acid Phosphatase--
The strains to be tested
were grown in 3 ml of synthetic medium lacking Leu overnight. The cells
were harvested, washed twice, and used to inoculate into 3 ml of
Burkholder high or low phosphate medium to an
A600 of 0.05. Incubation was carried out
at 30 °C for 16 h with shaking. The cells were harvested,
washed with 0.05 M acetate buffer (pH 4.0), and resuspended
in 1 ml of 0.05 M acetate buffer. Acid phosphatase activity
was assayed according to the method described previously (16, 26).
Preparation of Cell Extracts--
Cell cultures were first grown
to saturation in synthetic medium lacking uracil at 30 °C. The cells
were harvested by centrifugation, washed twice with sterilized water,
and then incubated into the Burkholder low phosphate medium at an
A600 of 0.05. These cultures were grown at
30 °C with shaking for 8 to 16 h to a final
A600 of 0.5-1.5 before analysis. 50 ml of these
cultures were washed with 10 ml of sterilized water. All subsequent
steps were carried out at 4 °C. Cells were resuspended in 450 µl of HSB buffer (45 mM HEPES-KOH (pH 7.5), 400 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5% Nonidet P-40, 2 mM dithiothreitol, 2 mM
benzamidine, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml
pepstatin A, 2 µg/ml leupeptin, 4 µg/ml antipain, 10 mM
NaF, 80 mM 
glycerol phosphate), and 0.8 mg of
acid-washed glass beads (425-600 µm, Sigma) were added. Cells were
lysed on a Vortex (Vortex-Genie2) for 30 s at the maximum setting,
followed by cooling in an ice bath for 30 s. This procedure was
repeated 10 times. Lysates were clarified by centrifugation at 4 °C
for 15 min at 14,000 rpm twice.
Expression in Escherichia coli and Purification of GST-PHO2
Fusion Protein and PHO4 Protein--
Purification of recombinant
GST-PHO2 protein and its mutants was performed essentially as described
(28). BL21(DE3)-plysS E. coli cells (29) harboring the
appropriate expression vector were grown in 50 ml of LB containing
ampicillin (50 µg/ml) overnight at 37 °C. This culture was diluted
into 500 ml of LB containing ampicillin (50 µg/ml) and grown at
30 °C until the A600 was approximately 0.5. Isopropyl--D-thiogalactopyranoside (Roche Molecular
Biochemicals) was added to a final concentration of 1 mM,
and the incubation was continued for 3 h at 30 °C. The
following procedures were performed at 4 °C with ice-cold buffers.
After the cells were centrifuged, the cell pellet was washed with 20 ml
of phosphate-buffered saline (PBS) (150 mM NaCl, 16 mM Na2HPO4, 4 mM
NaH2PO4, 10% glycerol (pH 7.3)). Cells were
resuspended in 10 ml of PBS +1% Triton X-100. Cell suspension was
frozen in liquid nitrogen for 15 min and then frozen in 70 °C for
1 h. Frozen cells were thawed as quickly as possible at room
temperature. Cell lysate was clarified by centrifugation at 14,000 rpm
for 15 min 3 times. Then the cell lysate was loaded on a 1-ml
Glutathione-Sepharose 4B (Amersham Pharmacia Biotech) column
equilibrated in PBS + 1% Triton X-100. After loading, the column was
washed with 2× 5 ml of PBS + 1% Triton X-100, then with 3× 5 ml of
PBS. The bound fusion protein was eluted with 5 ml of elution buffer
(10 mM glutathione in 50 mM Tris·HCl (pH
8.0), and 0.5-ml fractions were collected and analyzed by
SDS-polyacrylamide gel electrophoresis.
BL21(DE3)-plysS cells harboring the PHO4 expression vector
pBL-PHO4 were grown in 4 ml of LB containing ampicillin (50 µg/ml) overnight at 30 °C. The culture was diluted into 400 ml of
LB containing ampicillin (50 µg/ml) and grown to
A600 of 0.5 at 30 °C, then induced for 3 h at 42 °C. The following procedures were performed as described
(11).
In Vitro Phospho-labeling Assays--
For kinase reaction, 1 µl of YPH499 cell extract was added to a 19 µl of kinase mixture to
form a reaction mixture (20 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 4 µg GST-PHO2 (wild type or Pro-231 to Ser mutant), 50 µM ATP, 10 µCi of
[
-32P] ATP (Amersham Pharmacia Biotech)). The reaction
mixtures were incubated at 30 °C for 20 min. The reaction was
stopped by the addition of 20 µl 2× SDS sample buffer (30) and
heating to 95 °C for 5 min. Denatured samples were electrophoresed
on SDS, 10% polyacrylamide gel, vacuum dried, and autoradiographed.
Galactosidase Filter Assays--
The yeast two-hybrid
system was detailed in MatchMaker two-hybrid system protocol
(CLONTECH (PT1265-1)). pGBT9-CDC28 and
pGAD424-PHO2 as well as combinations of vectors and the
two-hybrid constructions were cotransformed into SFY526. Transformants
were grown on synthetic medium agar plates lacking Trp and Leu at
30 °C for 2-4 days. Some clones were spotted onto nitrocellulose
filters placed on synthetic medium (-Trp, -Leu) agar plate. The plate
was then placed at 30 °C for 2 days. The filter was subsequently
removed, snap-frozen on an aluminum foil case in a liquid nitrogen
bath, and placed on Whatman 3MM paper saturated with Z buffer
(31) containing 0.33 mg/ml X-gal (5-bromo-4-chloro-3-indolyl
-D-galactopyranoside). The filter was incubated at
30 °C for the appearance of blue clones.
Immunoprecipitation and Protein Kinase Assay--
Yeast cell
extracts (in any given experiment all samples were normalized to
contain the same amount of total protein (32)) were immunoprecipitated
with anti-HA monoclonal antibody 12CA5 (Roche Molecular
Biochemicals) (2 µg in each reaction) for 1 h on ice. After
centrifugation at 12,000 rpm for 2 min, the supernatant was added to 30 µl of slurry of protein A-agarose (Roche Molecular Biochemicals)
equilibrated in HSB buffer followed by a 2-h rotation at 4 °C.
Immunoprecipitates were washed with HSB buffer three times. Histone H1
kinase assays were performed as described previously (33). For
phosphorylation of GST-PHO2, immunoprecipitates were washed twice with
kinase assay buffer (20 mM Tris·HCl (pH 7.5), 10 mM MgCl2) and resuspended in 20 µl of kinase
assay mixture (20 mM Tris·HCl (pH 7.5), 10 mM
MgCl2, 4 µg of GST-PHO2, 50 µM ATP, 10 µCi of [-32P]ATP (Amersham Pharmacia Biotech)).
These kinase reaction mixtures were incubated at 30 °C for 20 min.
Reactions were stopped by addition of 20 µl of 2× SDS sample buffer
and heating to 95 °C for 5 min. Denatured samples were
electrophoresed on SDS, 10% polyacrylamide gel, vacuum-dried, and autoradiographed.
Liquid Culture -Galactosidase Assay--
Cell cultures were
first grown to saturation in selective synthetic medium at 30 °C.
The cells were harvested and washed twice with sterilized water and
then incubated into 3 ml of Burkholder low (or high) phosphate medium
at an A600 of 0.2. Cultures were grown at
30 °C with shaking to a final A600 of 1.5. The cells were harvested and washed twice with Z buffer. Cells were
resuspended in 0.6 ml of Z buffer. 100 µl of cell suspension was
removed to a fresh microcentrifuge tube. Assays for -galactosidase
activity were then performed as described in the protocol described in the MATCHMAKER two-hybrid system (PT1265-1)
(CLONTECH).
BIAcore® Analysis--
Purified PHO4 protein (30 nM
in 10 mM sodium acetate, pH 4.5) was injected at 5 µl/min
on
N-hydroxysuccinimide/N-ethyl-N'-(3-diethylaminopropyl)carbodi-imide-activated CM chips. Unreacted groups on the chip were then inactivated by 1 M ethanol-amino-HCl, pH 8.5. A surface density of 2900 resonance units was generated (namely, the surface concentration
of the sensor chip was about 2.9 ng/nm2). GST-PHO2,
GST-PHO2SA (Ser-230 Ala), and GST-PHO2SD (Ser-230 Asp) at the
same concentration of 5 µM were injected over the surface. The sensorgrams obtained were used to decide their binding specificity.
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RESULTS |
Mutations in the Consensus Sequence (SPIK) Recognized by
cdc2/CDC28-type Kinase in PHO2 Led to Complete Loss of Its
Activity--
A survey of potential recognition sites in PHO2 protein
for cdc2/CDC28-type kinase or cyclic AMP-dependent protein
kinase (protein kinase A) revealed a SPIK (230) site near the
PHO80 homologous region and a RKKIS (107) site in the second helix
of the PHO2 homeodomain (Fig. 1). The
amino acid sequence surrounding serine residue 230 resembles the
consensus sequence of sites phosphorylated by cdc2/CDC28 protein kinase
(20-23). The consensus sequence R/KR/KXS is the optimal protein kinase
A recognition site (34). The serines in the two consensus sequences
were considered to be theoretical phospho-acceptors. We assumed that
protein kinase A phosphorylated Ser-111 and cdc2/CDC28-type kinase
phosphorylated Ser-230, thereby affecting PHO2 function. To verify our
hypothesis, we mutated Ser-111 and Ser-230 to Ala and transformed the
plasmids pRS2 (YCp containing a wild type PHO2 gene), pRS2
(Ser-111 Ala), and pRS2 (Ser-230 Ala) into
PHO2-disrupted strain YJ1, then determined the acid
phosphatase activity of the cell suspensions as described under "Experimental Procedures." As can be seen in Table
I, the substitution of Ala for Ser-111
had no significant effect on the acid phosphatase activity under high
or low phosphate conditions, whereas the mutation of PHO2 Ser-230 to
Ala resulted in the loss of its ability to activate PHO5
expression. The results suggested that Ser-230 might be a critical
residue for PHO2 to activate PHO5 expression. Since Ser-230
is located in the consensus sequence SPIK, which may be recognized and
phosphorylated by cdc2/CDC28 type kinase in vivo, its
phosphorylation may be necessary for PHO2 function. This possibility is
supported by the finding that another PHO2 mutant (Pro-231 Ser), in
which the consensus sequence has been changed, also showed no ability
to confer repressible acid phosphatase activity. Interestingly, the
PHO2 variant containing an acidic residue in place of Ser-230 (Ser-230
Asp) could activate PHO5 expression under low phosphate
conditions. It seems feasible that the hydroxyl group may serve as a
phospho-acceptor in vivo, and the introduction of a negative
charge at residue 230 may be necessary for PHO2 function. Intriguingly,
the alteration of Lys-233 to Ala showed no effect on PHO2 function to
activate PHO5 expression.
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Fig. 1.
Diagrammatic representation of PHO2
and mutagenesis of amino acids in the protein kinase A or
cdc2/CDC28-type protein kinase phosphorylation consensus sequence in
PHO2 protein. Site-directed mutagenesis was performed using a
U-DNA mutagenesis kit (Roche Molecular Biochemicals) to mutate
the PHO2 Ser-111 to Ala, Ser-230 to Ala, Ser-230 to Asp, Pro-231 to
Ser, and Lys-233 to Ala as described under "Experimental
Procedures." All the mutations were verified by sequencing.
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Table I
Effects of PHO2 and PHO2 mutants on the repressive acid phosphatase of
S. cerevisiae
The wild type PHO2 gene and the PHO2 genes
mutated in the potential phosphorylation sites carried by pRS415 (YCp)
were introduced into the PHO2-disrupted host, YJ1 (YPH499
(pho2::URA3)). Acid phosphatase activities of the
transformants were measured as described under "Experimental
Procedures." Values are the means for triplate determinations. wt,
wild type.
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In Vitro Phosphorylation of GST-PHO2 Fusion Protein by YPH499 Cell
Extract--
To ascertain whether Ser-230 can be phosphorylated, the
E. coli-expressed GST-PHO2 and GST-PHO2 (Pro-231 Ser)
were subjected to in vitro phospho-labeling experiments. The
labeled proteins were separated by SDS-polyacrylamide gel
electrophoresis and visualized by autoradiography. When the cell
extract from a wild type YPH499 strain grown under low phosphate
conditions was used as the enzyme with the GST-PHO2 fusion protein as
the substrate, an approximate 90-kDa protein was observed to be
phosphorylated (Fig. 2B,
lane 2), but no phosphorylated band was found at the
corresponding position when GST-PHO2 (Pro-231 Ser) was used as the
substrate under identical labeling conditions (Fig. 2B,
lane 3) nor when fusion protein was absent (Fig.
2B, lane 1). These results inicated that the PHO2
portion (wild type but not PHO2 (Pro-231 Ser)) of the fusion
protein is the target of the protein kinase in cell extract from YPH499
strain grown under low phosphate conditions, and the Pro-231 to Ser
mutation diminished or prevented the phosphorylation of PHO2
protein. Furthermore, we also found that GST-PHO2 could be
phosphorylated by cell extract from YPH499 cells grown under high
phosphate conditions (data not shown). These data allowed us to suggest
that PHO2 protein can be phosphorylated in vitro by a
protein kinase(s) from YPH499 cells, and the Ser-230 may be the major
phosphorylation site.
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Fig. 2.
In vitro phosphorylation of
GST-PHO2 fusion proteins by YPH499 cell extract. A,
purified GST-PHO2 (lane 2) and the mutant GST-PHO2 (Pro-231
to Ser) (lane 3) were analyzed by 10% SDS, polyacrylamide
gel electrophoresis and Coomassie Blue staining. B, the wild
type PHO2 (lane 2) and the mutant PHO2 (Pro-231 to Ser)
(lane 3) fused with GST were subjected to in
vitro phospho-labeling experiments using cell extract of YPH499
grown under low phosphate conditions of enzyme as described
under "Experimental Procedures." Fusion protein was absent in
lane 1. Phosphorylation proteins were visualized by 10%
SDS, polyacrylamide gel electrophoresis and autography.
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Protein Kinase CDC28 Can Interact with PHO2 in the Two-hybrid
System--
Because the potential phosphorylation site of PHO2
resembles the consensus motif of sites phosphorylated by
cdc2/CDC28-type protein kinase, yeast CDC28 protein was fused to the
GAL4 binding domain (BD) (pGBT9-CDC28) and tested directly
for interaction with GAL4 activation domain (AD)-PHO2 (expressed by
pGAD424-PHO2) in the two-hybrid assay. Interaction of the
two proteins was determined for their ability to activate transcription
of a GAL1::lacZ reporter gene in SFY526 by
-galactosidase filter assays (Fig. 3).
Clones containing pGBT9-CDC28 and pGAD424 or pGBT9 and
pGAD424-PHO2 were white, whereas clones containing
pGBT9-CDC28 and pGAD424-PHO2 displayed a distinct
blue color (Fig. 3). These results showed that CDC28 protein and PHO2
protein can interact with each other in the two-hybrid system.
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Fig. 3.
Interaction between CDC28 and PHO2 in yeast
two-hybrid system. pGBT9 contains a GAL4 DNA binding domain.
pGAD424 contains a GAL4 activation domain. pGBT9-CDC28
contains the fusion of CDC28 and GAL4 DNA binding domain.
pGAD424-PHO2 expresses GAL4AD-PHO2 chimera. pVA3, murine p53
in pGBT9. pTD1 contains the fusion of SV40 large T-antigen and GAL4
activation domain. The cotransformant of pTD1 and pVA3 was a positive
control. -Galactosidase activity of SFY526 transformants was assayed
as described under "Experimental Procedures." Each was repeated
twice as shown.
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Immunoprecipitate of CDC28HA from Low Phosphate Cell Extract Can
Phosphorylate GST-PHO2--
The interaction between CDC28 and PHO2 in
the two-hybrid system suggested that CDC28 may be the protein kinase of
the transcriptional factor PHO2. We fused an HA tag to the C terminus
of CDC28 by polymerase chain reaction for the facility of
immunoprecipitation. To ascertain whether CDC28 can phosphorylate PHO2
in vitro, we used anti-HA to immunoprecipitate CDC28 from
the strain YPH499 bearing CDC28HA on a multicopy expression
plasmid (pVTU102-CDC28HA) grown under low phosphate
conditions. The immunoprecipitated CDC28HA could phosphorylate GST-PHO2
(Fig. 4A, lane 1),
whereas the immunoprecipitate from control strain YPH499 containing
vector pVTU102 had very low kinase activity (Fig. 4A,
lane 2). The immunoprecipitate derived from cell extract
containing CDC28HA phosphorylated histone H1 (Fig. 4B,
lane 1), whereas the control had only background kinase activity (Fig. 4B, lane 2). These results showed
that PHO2 could be phosphorylated in vitro by CDC28 kinase
complex from YPH499 (pVTU102-CDC28HA) grown under low
phosphate conditions.
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Fig. 4.
CDC28 phosphorylates PHO2 in
vitro. A, phosphorylation of GST-PHO2 assay.
The phosphorylation of GST-PHO2 was assayed by immunoprecipitates from
the strain YPH499, bearing CDC28HA on a multicopy expression
plasmid (pVTU102-CDC28HA) (lane1), and a control
strain YPH499 containing the empty plasmid (pVTU102) (lane
2). B, histone H1 kinase assay. The immunoprecipitates
from the strain YPH499 containing CDC28HA (lane 1) and the
control strain (lane 2) was used in histone H1 kinase
assay.
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Phosphate Switch Regulates the Transcriptional Activation Activity
of PHO2 and Mutations of the (SPIK) Site Affect the Transcriptional
Activation Activity of PHO2 and the Interaction between PHO2 and
PHO4--
PHO2 was fused to the DNA BD of yeast transcriptional factor
GAL4. The plasmid expressing GAL4 BD-PHO2 chimera,
pGBT9-PHO2, was transformed into a yeast strain, SFY526,
containing an integrated GAL1::lacZ
reporter gene. By measuring the expression of the reporter gene
(lacZ) under high and low phosphate conditions (Table
II), it was found that the chimera alone
could activate the expression of lacZ, and the
-galactosidase activity was regulated by the phosphate switch. The
activity under low phosphate condition was 8-fold more than that under
high phosphate conditions. In contrast, when pVA3 (containing the
fusion of murine p53 and GAL4 DNA binding domain) and pTD1 (containing
a fusion of SV40 large T-antigen and GAL4 activation domain) were
cotransformed into SFY526, the expression of lacZ was also
activated, but the -galactosidase activity was not affected by the
phosphate switch. These results demonstrated that the transcriptional
activation activity of PHO2 was regulated by the phosphate switch,
which was repressed under high phosphate condition and was derepressed
under low phosphate conditions.
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Table II
Analysis of transcriptional activation activity of yeast PHO2 protein
PHO2 and its mutants, PHO2 (S230A) and PHO2 (S230D), were fused to the
GAL4 DNA binding domain (in pGBT9) and expressed in yeast strain
SFY526. -Galactosidase activity derived from activation of the
GAL1-lacZ reporter were measured as described under "Experimental
Procedures." Values are the means for triplate determinations. pVA3,
murine p53 in pGBT9. pTD1 contains the fusion of SV40 large T-antigen
and GAL4 activation domain.
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The SPIK (230) site near PHO80 homologous region is a potential
phosphorylation site of PHO2, and the phosphorylation of Ser-230 is
necessary to activate PHO5 transcription. The mutation of
Ser-230 to Ala resulted in the loss of ability to be phosphorylated, so GAL4BD-PHO2 (Ser-230 Ala) chimera could not activate the expression of lacZ irrespective of phosphate concentration (Table II).
The substitution of Asp for Ser-230, which mimics the phosphorylation state by providing a negative charge at residue 230, had no significant effect on the activation activity of PHO2. These results showed that
the phosphorylation of PHO2 Ser-230 is necessary for the activation
activity of PHO2. The phosphorylation site-containing negative charge
may resemble an acidic activation domain. However, the activity of
GAL4BD-PHO2 (S230D) chimera was also regulated by the phosphate switch.
The activity under low phosphate condition was 5-fold more than that
under high phosphate condition.
The expression of lacZ could also be activated by
GAL4BD-PHO2 chimera in another strain, SFY526
(pho4::HIS3). The activity under low
phosphate conditions was 3-fold more than that under high phosphate
condition (Table III), although it was
less than that in SFY526. When pGBT9-PHO2 was cotransformed
with pRS4 (YCp containing a PHO4 gene), it had no
significant effect on the -galactosidase activity under high
phosphate condition, but the activity under low phosphate conditions
was 1-fold more than that using the GAL4BD-PHO2 chimera alone (Table
III). The results demonstrated that these two proteins may interact
with each other. When pGBT9-PHO4 (for expression of
GAL4BD-PHO4 chimera) was cotransformed into SFY526 (pho4::HIS3) with pGAD424-PHO2 (for
expression of GAL4 AD-PHO2 chimera), the -galactosidase activity was
40% more than that using pGBT9-PHO4 and pGAD424. The result
also indicated that PHO4 and PHO2 may interact in vivo.
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Table III
Two hybrid assay of PHO2 and PHO4 protein
Yeast strain SFY526 (pho4::HIS3) was transformed with
pGBT9-PHO2 alone or plasmid 1 and plasmid 2 as shown in the
table. The level of -galactosidase activity derived from a
GAL1-lacZ reporter was determined as described under
"Experimental Procedures." Values are the means for triplate
determinations. pGBT9-PHO2 and pGBT9-PHO2 (S230A)
contain the fusions of PHO2 or PHO2 (S230A) mutant and GAL4 DNA-binding
domain. pGAD424-PHO2 contains the fusion of PHO2 and GAL4
activation domain. pGBT9-PHO4 and pGAD424-PHO4
contain the fusions of PHO4 and GAL4 DNA binding domain or activation
domain. pVA3, murine p53 in pGBT9. pTD1 contains the fusion of SV40
large T-antigen and GAL4 activation domain.
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Another result showed that the phosphorylation of PHO2 is necessary for
the interaction between PHO2 and PHO4. When GAL4BD-PHO2 (S230A) was
coexpressed with GAL4AD-PHO4 chimera in SFY526
(pho4::HIS3), the galactosidase activity could not
be detected irrespective of the phosphate switch (Table III). The
result indicated that the phosphorylation site may be essential not
only for the transcriptional activation activity but also for the
interaction between PHO2 and PHO4, which may play a key role in
maintaining the transcriptional activity of PHO2.
BIAcore® Analysis of the Interaction between PHO2 and PHO4 in
Vitro--
The data above showed that the phosphorylation of PHO2
Ser-230 is necessary for the interaction between PHO2 and PHO4. To investigate whether the phosphorylation of Ser-230 is sufficient to
mediate interaction between PHO2 and PHO4, we used purified GST-PHO2,
GST-PHO2SA (Ser230 Ala), GST-PHO2SD (Ser-230 Asp), and PHO4 for
BIAcore® analysis.
PHO4 was covalently linked to a sensor chip (CM5). GST-PHO2,
GST-PHO2SA, and GST-PHO2SD, at the same concentration of 5 µM, were injected over the surface. As shown by the
sensorgrams obtained (Fig. 5), a strong
signal was detected when GST-PHO2SD was injected (Fig. 5B),
whereas signals were very weak when GST-PHO2 (Fig. 5A) or
GST-PHO2SA (Fig. 5C) was used. The sensorgram of GST-PHO2SD showed a very typical interaction. The effect of impurities and the
fused GST was eliminated for the results of GST-PHO2 and GST-PHO2SA. The results indicated the negative charge of PHO2 (Ser-230 Asp), which mimics the phosphorylation of Ser-230, may help the interaction between PHO4 and GST-PHO2SD.
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Fig. 5.
Sensorgrams of GST-PHO2SD protein binding to
immobilized PHO4 protein in BIAcore® experiments. PHO4 protein
was covalently linked to a sensor chip (CM5). 5 µM concentrations of GST-PHO2
(A), GST-PHO2SD (Ser-230 Asp) (B), and
GST-PHO2SA (Ser-230 Ala) (C) were injected over the
surface of the sensor chip (see "Experimental Procedures" for
details). RU, resonance units.
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DISCUSSION |
The functions of many transcriptional factors are regulated by
phosphorylation and dephosphorylation, which may act in several aspects. First, phosphorylation can change the cellular location of
some transcriptional factors. An S. cerevisiae
transcriptional factor, SWI5, which is localized in the cytoplasm at
the G2 phase of cell cycle due to its phosphorylation by
the cyclin-CDC28 protein kinase, enters the nucleus at Start in the
G1 phase and activates HO gene expression
(35). Similarly, PHO4 is localized in the cytoplasm under high
phosphate conditions (36) due to hyperphosphorylation by the
PHO80-PHO85 cyclin-CDK (CDK, cyclin-dependent kinase) (11). Second, some transcriptional activation factors, such as GAL4 and GCN4
(37, 38), which have acidic transcriptional activation domains, may
have a higher transcriptional activation activity upon point mutations
to increase negative charges. It is conjectured that phosphorylation
and dephosphorylation may regulate the transcriptional activation
activity by the increase or decrease of negative charges in response to
the change of the circumstance. Third, phosphorylation may affect the
interaction between transcriptional factors. Here we found that
phosphorylation of PHO2 can affect the transcriptional activation
activity. Only when PHO2 is phosphorylated, the secreted acid
phosphatase, PHO5, can be expressed under low phosphate conditions. Further investigations indicated that the phosphorylation of PHO2 not
only improve the transcriptional activation activity but facilitate the
interaction between PHO2 and PHO4. These results suggested the
pleiotropy of PHO2 phosphorylation. How the critical phosphorylation contributes to transcriptional activation activity and interaction is
unclear. Because the previous deletion analysis showed that the
possible PHO4 binding domain of PHO2 does not include the SPIK site
(13, 17) and our results indicated that the negative charge of PHO2
(Ser230 Asp) is sufficient to help the interaction between PHO2 and
PHO4 in vitro suggests that the phosphorylation of the
Ser-230 residue in PHO2 may alter the conformation of PHO2, making the
PHO4 binding domain of PHO2 suitable for interaction with PHO4 but not
participating in the interaction directly. The phosphorylation may also
increase the ability of PHO2 to activate transcription by altering the
three-dimensional conformation of PHO2. Furthermore, the phosphate
group may be directly involved in transcriptional activation by
increasing the negative charges of activation domain of PHO2.
PHO2 has a potential phosphorylation site, SPIK (230), that
resembles the consensus sequence of site recognized and phosphorylated by cdc2/CDC28-type kinase (20-23). We found that GST-PHO2 fusion protein expressed in E. coli could be
phosphorylated by yeast cell extract from YPH499 strain grown under low
phosphate conditions. Further investigation showed that CDC28HA
immunoprecipitate derived from yeast grown under low phosphate
conditions could phosphorylate GST-PHO2 in vitro. These
results indicate that CDC28 may be the kinase of PHO2. CDC28 is an
essential regulator of cell cycle progression, whereas PHO2 is required
for several metabolism processes. Another yeast
cyclin-dependent kinase, PHO85, has provided a link between
cell cycle regulation and phosphate metabolism (11, 39, 40). By
association with different cyclin subunits, PHO85 may be directed to
distinct biological functions. If CDC28 could be proved to be the
physiological kinase of PHO2, another link between cell cycle
progression and metabolism would be found. Perhaps a new cyclin would
be found to help CDC28 participate in metabolism. In addition to PHO85,
CDC28 might also serve to coordinate nutritional state and cell cycle progression.
Homeo box-encoding genes appear to be associated with developmental
control or cell type regulation. Previous investigations carried out by
Gilliquet and Berben (41) reveals the involvement of PHO2 that
contains a homeo domain in the life cycle. Homozygous PHO2
null diploids show a deficiency to progress through meiosis, which can
be corrected when the cells are transformed with
PHO2-bearing plasmid. Many nuclear factors involved in the
cell cycle have been observed to be phosphorylated and regulated by
cdc2/CDC28-type protein kinase, which is activated only at specific
cell cycle stages (42). Our present findings show that PHO2 may be
phosphorylated by CDC28. These evidences also suggest that there is
some association between metabolism and cell cycle machinery.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 86-21-64374430 (ext. 256); Fax: 86-21-64338357; E-mail: aosz@sunm.shcnc.ac.cn.
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