From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901
Received for publication, January 7, 2003
, and in revised form, March 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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The expression of genes encoding enzymes responsible for the synthesis of phosphatidylinositol (e.g. INO1) and phosphatidylcholine (e.g. CDS1, CHO1/PSS1, PSD1, CHO2/PEM1, OPI3/PEM2, CKI1, and CPT1), the two most abundant and essential phospholipids in S. cerevisiae, is regulated by inositol (1, 2, 4, 5, 6, 9). These genes are maximally expressed when inositol is absent from the growth medium and repressed when inositol is supplemented to the growth medium. Repression by inositol supplementation is enhanced by the inclusion of choline in the growth medium (1, 2, 4, 5, 6). Inositol-mediated regulation involves the transcriptional regulatory proteins Ino2p, Ino4p, and Opi1p (1, 2, 4, 5, 6). Ino2p (11) and Ino4p (12) are positive transcription factors, whereas Opi1p (13) is a negative transcription factor. Regulation of phospholipid synthesis by inositol is mediated by a UASINO1 (inositol/choline-responsive) cis-acting element (1, 14, 15, 16, 17) present in the promoters of the structural genes that code for phospholipid synthesis enzymes (1, 2, 4, 5, 6, 18). The UASINO element contains the binding site for an Ino2p-Ino4p heterodimer, which is required for maximum expression of the co-regulated UASINO-containing genes (4, 5, 6, 19, 20, 21). Repression of the co-regulated phospholipid synthesis genes depends on Opi1p (13, 22).
Opi1p contains a leucine zipper and two glutamine-rich domains (13) (Fig. 1) that are required for Opi1p repressor activity (23). Opi1p mediates its negative regulatory activity through the UASINO element (24) but not by direct interaction (23). Instead, in vitro data indicate that Opi1p interacts with DNA-bound Ino2p within the leucine zipper domain of Opi1p (25). In addition, the global repressor Sin3p interacts with the N-terminal region of Opi1p (25) (Fig. 1). Studies using mutant alleles of INO2, INO4, OPI1, and SIN3 support a model whereby these interactions play a role in the expression of UASINO-containing genes in vivo (25, 26, 27, 28, 29).
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Phosphorylation is a mechanism by which the activity of a regulatory protein may be controlled (30, 31, 32, 33, 34, 35), and indeed in vivo labeling experiments have shown that Opi1p is a phosphoprotein (36). Some of this phosphorylation is due to protein kinase C, and Ser26 has been identified as a major site of phosphorylation by this protein kinase (36) (Fig. 1). Phosphorylation of Ser26 attenuates the negative regulatory activity of Opi1p on the expression of the UASINO-containing INO1 gene (36). In this work, we demonstrated that Opi1p was phosphorylated by protein kinase A, the principal mediator of signals transmitted through the Ras-cAMP pathway in S. cerevisiae (37, 38). Ser31 and Ser251 were identified as major sites of protein kinase A phosphorylation, and phosphorylation of these sites played a role in stimulating Opi1p activity.
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EXPERIMENTAL PROCEDURES |
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Strains, Plasmids, and Growth ConditionsThe strains and
plasmids used in this work are listed in
Table I. Escherichia
coli strain DH5 was used for the propagation of plasmids and for
the production of MBP-Opi1p fusion proteins. The cells were grown in LB medium
(1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) at 37 °C. Ampicillin
(150 µg/ml) was added to cultures of DH5
-carrying plasmids. For the
expression of MBP-Opi1p fusion proteins, the cultures (250 ml) were grown to a
cell density of A600 nm = 0.40.6 at 37 °C, and
the cells were harvested by centrifugation at 5,000 x g for 5
min and resuspended in fresh medium containing 0.6 mM
isopropyl-
-D-thiogalactoside. After incubation for 3 h at 30
°C, the induced cells were harvested by centrifugation at 5,000 x
g for 5 min, washed with cold 10 mM Tris-HCl buffer (pH
7.4) containing 0.2 M NaCl, 10 mM 2-mercaptoethanol, and
1 mM EDTA, and then frozen at 70 °C. The induction was
carried out at 30 °C to reduce the degradation of the fusion proteins.
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Methods for yeast growth were performed as described previously (40, 41). Yeast cultures were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in SC medium containing 2% glucose at 30 °C. For selection of cells bearing plasmids, appropriate amino acids were omitted from SC medium. The cell numbers in liquid media were determined spectrophotometrically at an absorbance of 600 nm. The media were supplemented with 2% agar for growth on plates. The inositol excretion phenotype was examined on SC medium plates (minus inositol) by using growth of the inositol auxotrophic mutant ino1 (42) as described previously (43).
DNA Manipulations, Amplification of DNA by PCR, and DNA SequencingPreparation of plasmid DNA, restriction enzyme digestions, and DNA ligations were performed using standard protocols (41). Transformation of yeast (44) and E. coli (41) were performed as described previously. Amplification of DNA by PCR was optimized as described previously (45). DNA sequencing reactions were performed by the dideoxy method using Vent (exo) polymerase (41).
Construction of PlasmidsPlasmid pMAL-OPI1 containing a malE-OPI1 fusion gene (36) was used for the expression of MBP-Opi1p fusion protein. The codons for Ser31 and Ser251 in Opi1p were changed to alanine codons by site-directed mutagenesis. The OPI1S31A (primers, 5'-CAATCATGCAGACAGAAGgCGCAGCCTTCAGAGGACGTC-3' and 5'-GACGTCCTCTGAAGGCTGCGcCTTCTGTCTGCATGATTG-3') and OPI1S251A (primers, 5'-GCAAGATCTCAGGTTCGGGAAgcTCTTCTAAACTTACCC-3' and 5'-GGGTAAGTTTAGAAGAgcTTCCCGAACCTGAGATCTTGC-3') mutations were constructed by PCR with a QuikChange site-directed mutagenesis kit using plasmid pMAL-OPI1 as the template. The lowercase letters in the primers refer to sequences used for the mutations. Clones containing the wild type and mutant OPI1 coding sequence were identified by restriction enzyme analysis. DNA sequencing of the wild type and mutant genes confirmed that the constructs were in frame with the malE gene and did not possess additional mutations. Plasmid pSA3 is a single-copy plasmid that contains the OPI1 gene with sequences for a HA epitope tag inserted after the start codon (36). Plasmids pSA5 and pSA6, which bear the HA-OPI1S31A and HA-OPI1S251A mutations, respectively, were derived from plasmid pSA3 after site-directed mutagenesis using the primers described above. Plasmid pSA1 is a multicopy plasmid that contains the OPI1 gene with sequences for a HA epitope tag inserted after the start codon (36). Plasmids pSA7 and pSA8 were constructed by subcloning HA-OPI1S31A and HA-OPI1S251A from pSA5 and pSA6, respectively, into the SacI/HindIII sites of plasmid YEp351. These plasmid constructions were confirmed by DNA sequencing.
Purification of Wild Type and Mutant MBP-Opi1p Fusion Proteins from E. coliWild type and mutant MBP-Opi1p fusion proteins were purified from E. coli by disruption of cells with a French press followed by amylose-agarose affinity chromatography as described by Sreenivas et al. (36).
Phosphorylation of MBP-Opi1p and Synthetic Peptides with Protein
Kinases A and CThe phosphorylation reactions were measured for 10
min at 30 °C in a total volume of 40 µl. The indicated concentrations
of MBP-Opi1p or synthetic peptides were phosphorylated with protein kinase A
in a reaction mixture that contained 50 mM Tris-HCl (pH 7.5), 10
mM MgCl2, 0.2 mM [-32P]ATP
(5,000 cpm/pmol), and protein kinase A (0.2 unit/ml). MBP-Opi1p (0.1 mg/ml)
was phosphorylated with protein kinase C in a reaction mixture that contained
50 mM Tris-HCl buffer (pH 8.0), 10 mM MgCl2,
10 mM 2-mercaptoethanol, 0.375 mM EDTA, 0.375
mM EGTA, 1.7 mM CaCl2, 20 µM
diacylglycerol, 50 µM phosphatidylserine, 50 µM
[
-32P]ATP (5,000 cpm/pmol), and protein kinase C (1
unit/ml). Samples containing 32P-labeled MBP-Opi1p were treated
with an equal volume of 2x Laemmli's sample buffer
(46), followed by
SDS-polyacrylamide gel electrophoresis and transfer to PVDF paper, and
visualized by phosphorimaging. The extent of phosphorylation was analyzed
using ImageQuant software. Phosphorylation signals were in the linear range of
detectability. The reactions containing synthetic peptides were terminated by
spotting an aliquot of the reaction mixture onto phosphocellulose filters. The
filters were washed with 75 mM phosphoric acid and subjected to
scintillation counting. The phosphorylation reactions were performed in
triplicate. A unit of protein kinase A activity was defined as the amount of
enzyme that catalyzed the formation of 1 nmol of product/min.
Phosphoamino Acid and Phosphopeptide Map AnalysesGel slices containing 32P-labeled MBP-Opi1p were treated with 50 mM ammonium bicarbonate (pH 8.0) and 0.1% SDS at 37 °C for 30 h to elute the protein. Bovine serum albumin (50 µg) was added to the samples as carrier protein, and trichloroacetic acid was added to a final concentration of 20%. After incubation for 30 min at 4 °C, the protein precipitates were collected by centrifugation. The proteins were washed three times with cold acetone and dried in vacuo. The samples were then subjected to acid hydrolysis with 6 N HCl at 100 °C for 4 h. The hydrolysates were dried in vacuo and applied to 0.1-mm cellulose thin layer chromatography plates with 2.5 µg of phosphoserine, 2.5 µg of phosphothreonine, and 5 µg of phosphotyrosine as carrier phosphoamino acids in water. Phosphoamino acids were separated by two-dimensional electrophoresis (47). Following electrophoresis, the plates were dried, sprayed with 0.25% ninhydrin in acetone to visualize carrier phosphoamino acids, and subjected to phosphorimaging analysis to identify the radiolabeled phosphoamino acid.
Pieces of PVDF paper containing 32P-labeled MBP-Opi1p were subjected to digestion with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin and two-dimensional peptide mapping analysis as described by MacDonald and Kent (48). Electrophoresis (1% ammonium bicarbonate buffer at 1000 volts for 20 min) and ascending chromatography (n-butyl alcohol/glacial acetic acid/pyridine/water, 10:3:12:15 for 7 h) were performed on cellulose thin layer glass plates. The dried plates were then subjected to phosphorimaging analysis.
Preparations of Yeast Cell Extracts, -Galactosidase
Assay, and Protein DeterminationExponential phase yeast cells were
harvested by centrifugation and were disrupted with glass beads using a
Mini-Bead Beater (Biospec Products) as described previously
(49). The cell disruption
buffer contained 50 mM Tris-maleate (pH 7.0), 1 mM
Na2EDTA, 0.3 M sucrose, 10 mM
2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1
mM benzamidine, and 5 µg/ml each of aprotinin, leupeptin, and
pepstatin. The glass beads and cell debris were removed by centrifugation at
1,500 x g for 5 min. The supernatant was used as the cell
extract.
-Galactosidase activity was measured in cell extracts at 25 °C by
following the conversion of
O-nitrophenyl-
-D-galactopyranoside to
O-nitrophenol (molar extinction coefficient of 3,500
M1 cm1) at 410 nm on
a recording spectrophotometer
(50). The reaction mixture
contained 100 mM sodium phosphate buffer (pH 7.0), 3 mM
O-nitrophenyl-
-D-galactopyranoside, 1 mM
MgCl2, 100 mM 2-mercaptoethanol, and enzyme protein in a
total volume of 0.1 ml. The enzyme reactions were linear with time and protein
concentration. The average standard deviation of the enzyme assays (performed
in triplicate) was ±5%. A unit of enzymatic activity was defined as the
amount of enzyme that catalyzed the formation of 1 µmol of product/min. The
protein concentration was determined by the method of Bradford
(51) using bovine serum
albumin as the standard.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblot AnalysisSDS-polyacrylamide gel electrophoresis (46) and immunoblotting (52) using PVDF paper were performed as described previously. Mouse monoclonal anti-HA antibodies (12CA5) were used at a final protein concentration of 0.8 µg/ml as a primary antibody, and goat anti-mouse Ig-G-alkaline phosphatase conjugate was used as a secondary antibody at a dilution of 1:5,000. The HA-tagged Opi1p proteins were detected on immunoblots using the enhanced chemifluorescence Western blotting detection kit as described by the manufacturer, and the images were acquired by fluorimaging analysis. The relative densities of the protein bands were analyzed using ImageQuant software. Immunoblotting signals were in the linear range of detectability.
In Vivo Labeling of HA-tagged Opi1p ProteinsThe cells
(opi1 mutant) bearing multicopy plasmids containing the
HA-tagged wild type and S31A and S251A mutant OPI1 alleles were used
to examine the phosphorylation of Opi1p in vivo. Exponential phase
cells grown in SC medium containing 75 µM inositol were labeled
with 32Pi (0.25 mCi/ml) for 3 h. Following the
incubation, the labeled cells were harvested by centrifugation, washed, and
disrupted with glass beads in 50 mM Tris-HCl (pH 7.4) containing
protease (0.5 mM phenylmethylsulfonyl fluoride, 1 mM
benzamide, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin)
and phosphatase (10 mM NaF, 5 mM
-glycerophosphate, 1 mM sodium vanadate) inhibitors. The
HA-tagged Opi1p proteins were immunoprecipitated from cell extracts (0.5 mg of
protein) using 4 µg of anti-HA antibodies in 0.5 ml of
radioimmunoprecipitation buffer (50 mM Tris-HCl (pH 8.0), 150
mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS)
(53). The HA-tagged Opi1p
proteins were dissociated from enzyme-antibody complexes
(53), subjected to
SDS-polyacrylamide gel electrophoresis, and transferred to PVDF paper. The
relative amounts of the 32P-labeled proteins were quantified using
ImageQuant software after phosphorimaging analysis.
Analysis of DataThe kinetic data were analyzed according to the Michaelis-Menten equation using the EZ-FIT enzyme kinetic model-fitting program (54). Statistical analyses, including the t test for significance, were performed with SigmaPlot 5.0 software.
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RESULTS |
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Phosphoamino Acid Analysis and Two-dimensional Phosphopeptide Mapping of MBP-Opi1p Phosphorylated by Protein Kinase AProtein kinase A (56, 57, 58) is a serine/threonine-specific protein kinase. To examine which amino acid residue(s) of Opi1p was a target for phosphorylation, MBP-Opi1p was phosphorylated with protein kinase A, and the 32P-labeled fusion protein was subjected to phosphoamino acid analysis. Protein kinase A phosphorylated Opi1p on a serine residue (Fig. 4). 32P-Labeled MBP-Opi1p was also subjected to digestion with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin followed by thin layer electrophoresis and chromatographic analysis. The protease digestion yielded several phosphopeptides (Fig. 5A). This indicated that Opi1p was phosphorylated on multiple serine residues by protein kinase A. Some of the peptides shown in the map also resulted from incomplete proteolysis of the MBP-Opi1p (see below).
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Previous studies have shown that Opi1p is phosphorylated by protein kinase C at Ser26 as well as at other unidentified sites (36). We questioned whether sites phosphorylated by protein kinase C were the same as the sites phosphorylated by protein kinase A. To address this question, purified MBP-Opi1p was phosphorylated with protein kinase C, and the 32P-labeled protein was subjected to two-dimensional phosphopeptide mapping analysis. The phosphopeptide map of the protein kinase C-phosphorylated MBP-Opi1p fusion protein was distinctly different from the map of the protein kinase A-phosphorylated protein. This indicated that the two protein kinases phosphorylated different sites of Opi1p
Opi1p Synthetic Peptides Containing a Protein Kinase A Sequence Motif Are Substrates for Protein Kinase AAnalysis of the deduced sequence of Opi1p revealed that the protein has five potential phosphorylation sites (Ser31, Ser60, Thr141, Thr183, and Ser251) within a protein kinase A sequence motif. Five peptides (SCRQKSQPSE, ILDRVSNKII, KKRLVT- CLHL, NARTTTGAGE, and SQVRESLLNL, respectively) containing the potential target sites were synthesized based on the deduced protein sequence of Opi1p. The ability of these peptides to serve as substrates for protein kinase A was examined. Of the five peptides, the SCRQKSQPSE and SQVRESLLNL peptides, which contain sequences for Ser31 and Ser251, were substrates for protein kinase A (Fig. 6). None of the other potential sites proved to be targets for protein kinase A phosphorylation by this assay.
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Effect of the S31A and S251A Mutations on the Phosphorylation of Opi1p by Protein Kinase A in VitroOPI1S31A and OPI1S251A alleles were constructed by site-directed mutagenesis and expressed in E. coli as MBP-Opi1p fusion proteins. The S31A and S251A mutant fusion proteins were purified by amylose-agarose affinity chromatography. The purification procedure resulted in nearly homogeneous preparations of the mutant fusion proteins as evidenced by SDS-polyacrylamide gel electrophoresis (Fig. 7). The fusion protein size (95 kDa) was consistent with the combined sizes of MBP and Opi1p. Immunoblot analysis with anti-MBP and anti-Opi1p antibodies confirmed the identity of MBP and Opi1p, respectively, in the fusion protein. The S31A and S251A mutant proteins were examined for their ability to be phosphorylated by protein kinase A using 32P-labeled ATP. The extent of phosphorylation of the S31A and S251A mutant MBP-Opi1p fusion proteins was reduced by 57 and 65%, respectively, when compared with the wild type control protein (Fig. 8). The protein kinase A-phosphorylated S31A and S251A mutant fusion proteins were digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin and subjected to two-dimensional phosphopeptide mapping analysis. The phosphopeptide shown in box 1 in wild type MBP-Opi1p (Fig. 5A) was absent in the phosphopeptide map of the S251A mutant MBP-Opi1p (Fig. 5C), whereas several of the phosphopeptides in box 2 in wild type (Fig. 5A) were absent in the map of the S31A mutant (Fig. 5B). These data indicated that Ser251 was contained in phosphopeptide box 1 and that Ser31 was contained in the phosphopeptides present in box 2. The multiple phosphopeptides in box 2 are likely results of partial cleavage by the protease. The identity of the amino acid residue(s) contained in the remaining phosphopeptide in box 2 and the phosphopeptides to the left of box 2 in the map remains to be determined.
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Effect of the S31A and S251A Mutations on the Phosphorylation of Opi1p
in VivoThe effects of the S31A and S251A mutations on the
phosphorylation of Opi1p in vivo was examined using an HA-tagged
version of the protein. As described previously
(36), HA-tagged wild type
Opi1p was functional in vivo (i.e. it suppressed the
inositol excretion phenotype of the opi1 mutant) and was
recognized in opi1
mutant cells by anti-HA antibodies at the
expected molecular mass of 50 kDa (Fig.
9A). HA-tagged OPI1S31A and
OPI1S251A alleles were constructed and used for the
expression of the S31A and S251A mutant Opi1p proteins in opi1
mutant cells. The mutant and wild type OPI1 alleles were expressed in
opi1
mutant cells to obviate any effects caused by Opi1p
encoded by the genomic wild type copy of the OPI1 gene. A multicopy
plasmid was used to increase expression of Opi1p to facilitate isolation of
the phosphorylated forms of the protein from cell extracts. Like wild type
Opi1p, the S31A and S251A mutant proteins migrated on SDS-polyacrylamide gels
with a subunit molecular mass of 50 kDa
(Fig. 9A). Scanning
densitometry showed that the levels of the wild type and mutant Opi1p on the
immunoblots were essentially the same, indicating that the mutations did not
affect the expression of the protein. Moreover, the S31A and S251A mutant
Opi1p proteins suppressed the inositol excretion phenotype of the
opi1
mutant, indicating that OPI1S31A and
OPI1S251A alleles were functional in vivo.
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opi1 mutant cells bearing plasmids with the wild type and
mutant OPI1HA alleles were labeled with
32Pi followed by the immunoprecipitation of the Opi1p
proteins from cell extracts with anti-HA antibodies. SDS-polyacrylamide gel
electrophoresis of the immunoprecipitates, transfer to PVDF paper, and
phosphorimaging analysis showed that the mutations caused a decrease in the
extent of Opi1p phosphorylated in vivo
(Fig. 9B). The S31A
and S251A mutations caused a decrease in the extent of phosphorylation of
Opi1p by 60 and 70%, respectively. Immunoblot analysis showed that the wild
type and mutant Opi1p proteins were present at similar amounts. In these
experiments, the effects of the mutations on Opi1p phosphorylation were
examined in cells grown in the presence of inositol.
Effect of the S31A and S251A Mutations in Opi1p on the Regulation of
INO1 ExpressionExpression of several UASINO-containing
genes, including INO1, is negatively regulated by Opi1p
(1,
4,
5,
6). Indeed,
opi1 mutant cells exhibit elevated expression of the
INO1 gene in cells grown in the absence and presence of inositol
(1,
4,
5,
6). The effect of the S31A and
S251A mutations in Opi1p on INO1 expression was examined in
opi1
mutant cells using an INO1-CYC1-lacZ reporter
gene (15). In these
experiments, we used HA-tagged wild type OPI1 and the
OPI1S31A and OPI1S251A alleles in the
single-copy plasmids pSA3, pSA5, and pSA6, respectively. Immunoblot analysis
using anti-HA antibodies showed that these alleles were expressed at similar
levels in opi1
mutant cells. The cells were grown in the
absence of inositol to the exponential phase, the extracts were prepared, and
-galactosidase activity was measured. As described previously
(36), the HA-tagged wild type
OPI1 allele suppressed the elevated (9-fold) expression of the
INO1 gene in opi1
mutant cells grown in the absence
of inositol (Fig.
10A). The
-galactosidase activity in
opi1
mutant cells bearing wild type Opi1p was similar to that
found in cells with the chromosomal copy of the OPI1 gene
(Fig. 10A). In
contrast, the
-galactosidase activity in opi1
mutant
cells bearing the S31A and S251A mutant Opi1p proteins was 42 and 35% higher
(p < 0.001), respectively, than that found in opi1
mutant cells bearing the wild type Opi1p protein
(Fig. 10A). Thus,
under the derepressed condition (i.e. absence of inositol), the S31A
and S251A mutations blunted the negative regulatory function of Opi1p.
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INO1, as well as other UASINO-containing genes, is
repressed by inositol supplementation
(1,
4,
5,
6). As described previously
(36), the -galactosidase
activity directed by the INO1-CYC1-lacZ reporter gene was not
repressed by inositol supplementation in opi1
mutant cells
(Fig. 10B). This
defect in the regulation of INO1 was suppressed by the wild type
HA-tagged OPI1 allele (Fig.
10B). Moreover, the INO1 gene was repressed
(3.5-fold) by inositol supplementation in opi1
mutant cells
bearing the wild type HA-tagged OPI1 allele
(Fig. 10, compare A
with B). The INO1 gene was also repressed by inositol in
opi1
mutant cells bearing the HA-tagged
OPI1S31A (2.7-fold) and OPI1S251A
(2.6-fold) alleles (Fig. 10,
compare A with B). However, the levels of
-galactosidase activity in opi1
mutant cells with the
S31A and S251A mutant Opi1p proteins were 55 and 52% higher (p <
0.001), respectively, when compared with cells containing wild type Opi1p
(Fig. 10B).
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DISCUSSION |
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Identification of protein kinase A target sites in Opi1p was addressed to gain information on the effects of phosphorylation on the regulatory activity of Opi1p. A combination of biochemical and molecular approaches was used to identify protein kinase A phosphorylation sites in Opi1p. The peptides SCRQKSQPSE and SQVRESLLNL, which contained protein kinase A sequence motifs at Ser31 and Ser251, respectively, were substrates for protein kinase A in vitro. These data provided support that Ser31 and Ser251 in Opi1p might be targets for protein kinase A phosphorylation. S31A and S251A mutations in Opi1p were constructed and used to support this hypothesis. The extent of phosphorylation of the S31A and S251A mutant MBP-Opi1p fusion proteins was reduced when compared with wild type MBP-Opi1p. Moreover, phosphopeptide mapping analysis of protein kinase A-phosphorylated MBP-Opi1p fusion proteins showed that distinct phosphopeptides present in the wild type protein were absent from the S31A and S251A mutant proteins. These data confirmed that Ser31 and Ser251 were specific targets for protein kinase A phosphorylation.
We addressed the physiological relevance of the phosphorylation of Opi1p on
Ser31 and Ser251 using HA-tagged versions of Opi1p
expressed in opi1 mutant cells. The S31A and S251A mutant
HA-tagged proteins were expressed at the same levels as that of wild type
Opi1pHA. Moreover, the mutant HA-tagged proteins were functional
in vivo as evidenced by the suppression of the characteristic
inositol excretion phenotype
(22) of the
opi1
mutant. The S31A and S251A mutant HA-tagged proteins were
phosphorylated in vivo, but the extent of their phosphorylation was
reduced by 60 and 70%, respectively, when compared with the wild type control
protein. The effects of the phosphorylation site mutations on Opi1p regulatory
activity were examined in vivo by the analysis of INO1
expression using a sensitive INO1-CYC1-lacZ reporter gene assay. This
analysis indicated that INO1 expression reached higher derepressed
(i.e. absence of inositol) levels in cells carrying the S31A (42%)
and S251A (35%) mutations when compared with cells carrying wild type Opi1p.
The mutations did not have a major effect on the inositol-mediated regulation
of INO1 expression (1,
2,
4,
5,
6). However, the
inositol-repressed levels of INO1 were elevated in cells carrying the
S31A (55%) and S251A (52%) mutant forms of Opi1p when compared with the
control. These results, together with the evidence that the extent of
phosphorylation of the S31A and S251A mutant proteins were reduced when
compared with wild type Opi1p, indicated that phosphorylation of
Ser31 and Ser251 resulted in the stimulation of Opi1p
repressor activity in cells grown in the absence and presence of inositol. The
fact that the phosphorylation sites in question were targets for protein
kinase A in vitro supported the conclusion that protein kinase A was
involved in the stimulation of Opi1p repressor activity in vivo.
Protein kinase A activity is required for proper regulation of growth, progression through the cell cycle, and development in response to various nutrients (37, 38). The enzyme consists of two catalytic subunits (encoded by TPK1, TPK2, and TPK3) and two regulatory subunits (encoded by BCY1). Elevated cAMP levels, which are controlled by adenylate cyclase (encoded by CYR1) via the Ras-cAMP pathway, promote dissociation of subunits allowing the catalytic subunit to phosphorylate a variety of substrates (37, 38). Some of these substrates are enzymes responsible for the synthesis of phospholipids. For example, the activities of CTP synthetase (59, 60), choline kinase (61, 62), and phosphatidate phosphatase (63) are stimulated by protein kinase A phosphorylation, whereas phosphatidylserine synthase (64) activity is inhibited by phosphorylation. These enzymes play key regulatory roles in phospholipid synthesis (1, 2, 4, 5, 6). Studies using phosphorylation site mutants and mutants defective in the Ras-cAMP pathway have shown that the phosphorylation of these enzymes by protein kinase A plays a role in regulating phospholipid synthesis (62, 63, 65). Thus, in the short term, protein kinase A may regulate phospholipid synthesis by modulating the activity of enzymes that are already expressed in the cell, whereas in the long term protein kinase A may regulate phospholipid synthesis by controlling the expression of enzymes via the phosphorylation of the transcription factor Opi1p.
The effect of protein kinase A phosphorylation on Opi1p regulatory activity was opposite to that of protein kinase C. Protein kinase C phosphorylates Opi1p at Ser26, and this phosphorylation mediates the attenuation of Opi1p regulatory activity (36). Thus, signals transmitted through the Ras-cAMP and the protein kinase C signaling pathways appear to regulate expression of phospholipid synthesis UASINO-containing genes by opposing mechanisms. The precise mechanism by which phosphorylation via protein kinases A and C mediate Opi1p regulatory activity is not yet known. Phosphorylation of a regulatory protein can control its cellular location, ability to bind DNA, or interaction with other proteins (30, 31, 32, 33, 34, 35). The precise target of Opi1p has been an enigma (23, 27). However, the recent work of Wagner et al. (25) has shown that Opi1p interacts with the pleiotropic repressor Sin3p and with the phospholipid synthesis positive transcription factor Ino2p, and indeed these interactions may be physiologically relevant (25, 26, 27, 28, 29). The availability of the phosphorylation site mutants will permit further studies on the role of phosphorylation by protein kinases A and C on Opi1p interactions with Sin3p and Ino2p and allow us to understand its repressor function in regulating phospholipid synthesis.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Food Science, Rutgers
University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext.
217); Fax: 732-932-6776; E-mail:
carman{at}aesop.rutgers.edu.
1 The abbreviations used are: UAS, upstream activating sequence; SC,
synthetic complete; MBP, maltose-binding protein; PVDF, polyvinylidene
difluoride; HA, hemagglutinin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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