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Originally published In Press as doi:10.1074/jbc.M300132200 on March 31, 2003

J. Biol. Chem., Vol. 278, Issue 23, 20673-20680, June 6, 2003
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Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase A*

Avula Sreenivas and George M. Carman {ddagger}

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.
   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in phospholipid synthesis in the yeast Saccharomyces cerevisiae. The phosphorylation of Opi1p by protein kinase A (cAMP-dependent protein kinase) was examined in this work. Using a maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase A activity was time- and dose-dependent and dependent on the concentrations of Opi1p and ATP. Protein kinase A phosphorylated Opi1p on multiple serine residues. The synthetic peptides SCRQKSQPSE and SQVRESLLNL containing the protein kinase A motif for Ser31 and Ser251, respectively, within Opi1p were substrates for protein kinase A. Phosphorylation of S31A and S251A mutant maltose-binding protein-Opi1p fusion proteins by protein kinase A was reduced when compared with the wild type protein, and phosphopeptides present in wild type Opi1p were absent from the S31A and S251A mutant proteins. In vivo labeling experiments showed that the extent of phosphorylation of the S31A and S251A mutant proteins was reduced when compared with the wild type protein. The physiological consequence of the phosphorylation of Opi1p at Ser31 and Ser251 was examined by measuring the effects of the S31A and S251A mutations on the expression of the UASINO-containing gene INO1. The {beta}-galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1{Delta} mutant cells expressing the S31A and S251A mutant Opi1p proteins was elevated 42 and 35%, respectively, in the absence of inositol and 55 and 52%, respectively, in the presence of inositol when compared with cells expressing wild type Opi1p. These data supported the conclusion that phosphorylation of Opi1p at Ser31 and Ser251 mediated the stimulation of the negative regulatory function of Opi1p on the expression of the INO1 gene.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phospholipids are essential molecules that contribute to the structure and function of membranes, and indeed, the synthesis of phospholipids is a major activity in which cells engage throughout growth. The yeast Saccharomyces cerevisiae serves as a model eukaryotic organism to study the regulation of phospholipid synthesis (1, 2, 3, 4, 5, 6). Almost all of the structural and regulatory genes involved in phospholipid synthesis have been cloned and characterized, and mutations in these genes have been isolated (1, 2, 3, 4, 5, 6, 7, 8). Moreover, several of the phospholipid synthetic enzymes have been purified and characterized (1, 2, 3, 4, 5, 6). The characterization of the wild type and mutant genes, as well as the gene products encoded by these alleles, has significantly advanced understanding of phospholipid synthesis. The regulation of phospholipid synthesis is complex and occurs by both genetic (e.g. transcriptional) and biochemical (e.g. phosphorylation) mechanisms (1, 2, 3, 4, 5, 6, 9, 10).

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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  		FIG. 1.
Domain structure of Opi1p. The diagram shows the positions of the protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites, and the Sin3p, leucine zipper, and glutamine-rich domains in the Opi1p sequence. The numbers at the top indicate the amino acid positions for each domain in the protein.

 

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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All chemicals were reagent grade. Growth medium supplies were purchased from Difco Laboratories. Restriction endonucleases, modifying enzymes, recombinant Vent DNA polymerase, amylose affinity chromatography resin, MBP, and anti-MBP antibodies were purchased from New England Biolabs. The plasmid DNA purification and DNA gel extraction kits were purchased from Qiagen. The oligonucleotides were prepared by Genosys Biotechnologies, Inc. The QuikChange site-directed mutagenesis kit was purchased from Stratagene. The Yeast Maker yeast transformation system was from Clontech. The DNA size ladder used for agarose gel electrophoresis was from Invitrogen. Radiochemicals were purchased from PerkinElmer Life Sciences. Phosphocellulose filters were purchased from Pierce. Phenylmethylsulfonyl fluoride, bovine serum albumin, histone, benzamidine, aprotinin, leupeptin, pepstatin, Nonidet P-40, polyvinylpyrrolidone (40 kDa), phosphoamino acids, and O-nitrophenyl-{beta}-D-galactopyranoside were purchased from Sigma. Protein assay reagents, electrophoretic reagents, immunochemical reagents, molecular mass protein standards, and isopropyl-{beta}-D-thiogalactoside were purchased from Bio-Rad. Mouse monoclonal anti-HA antibodies (12CA5) and goat anti-mouse IgG alkaline phosphatase conjugates were from Roche Applied Science and Pierce, respectively. Anti-Opi1p antibodies were prepared previously (39). Protein kinase A catalytic subunit (bovine heart) and protein kinase C (rat brain) were purchased from Promega. Protein A-Sepharose CL-4B, Hybond-P PVDF paper, and the enhanced chemifluorescence Western blotting detection kit were purchased from Amersham Biosciences. The cellulose thin layer glass plates were from EM Science. Scintillation counting supplies and acrylamide solutions were purchased from National Diagnostics. The peptides SCRQKSQPSE, ILDRVSNKII, KKRLVTCLHL, NARTTTGAGE, and SQVRESLLNL were synthesized and purified commercially by Bio-Synthesis, Inc.

Strains, Plasmids, and Growth Conditions—The strains and plasmids used in this work are listed in Table I. Escherichia coli strain DH5{alpha} 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{alpha}-carrying plasmids. For the expression of MBP-Opi1p fusion proteins, the cultures (250 ml) were grown to a cell density of A600 nm = 0.4–0.6 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-{beta}-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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  		TABLE I
Strains and plasmids used in this work

 

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 Sequencing—Preparation 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 Plasmids—Plasmid 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. coli—Wild 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 C—The 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 [{gamma}-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 [{gamma}-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 Analyses—Gel 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, {beta}-Galactosidase Assay, and Protein Determination—Exponential 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.

{beta}-Galactosidase activity was measured in cell extracts at 25 °C by following the conversion of O-nitrophenyl-{beta}-D-galactopyranoside to O-nitrophenol (molar extinction coefficient of 3,500 M—1 cm—1) at 410 nm on a recording spectrophotometer (50). The reaction mixture contained 100 mM sodium phosphate buffer (pH 7.0), 3 mM O-nitrophenyl-{beta}-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 Analysis—SDS-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 Proteins—The cells (opi1{Delta} 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 {beta}-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 Data—The 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of Opi1p by Protein Kinase A in Vitro—We examined the hypothesis that Opi1p was a substrate for protein kinase A in vitro. To facilitate well defined studies, purified MBP-Opi1p was utilized as a protein kinase A substrate. Protein kinase A catalytic subunit from bovine heart was used as the source of kinase enzyme. This kinase is structurally and functionally similar to the S. cerevisiae protein kinase A catalytic subunit (55). To determine whether Opi1p was a target for phosphorylation by protein kinase A, we examined whether the kinase catalyzed the incorporation of the {gamma} phosphate of 32P-labeled ATP into MBP-Opi1p. After the phosphorylation reaction, the samples were subjected to SDS-polyacrylamide gel electrophoresis and transfer to PVDF paper. Phosphorimaging analysis of the PVDF paper showed that Opi1p was a substrate for protein kinase A (Fig. 2, lane 3). The position of 32P-labeled MBP-Opi1p on the PVDF paper was confirmed by immunoblot analysis using antibodies to MBP and to Opi1p. The MBP itself was not a substrate for protein kinase A (Fig. 2, lane 2). The autophosphorylation of protein kinase A is also shown in Fig. 2. Immunoblot analysis with anti-MBP antibodies showed that the phosphorylated protein that was not labeled in lane 3 of Fig. 2 is a proteolysis product of MBP-Opi1p. The dependence of protein kinase A activity on MBP-Opi1p and on ATP was examined. Protein kinase A followed saturation kinetics with respect to MBP-Opi1p (Fig. 3A) and with respect to ATP (Fig. 3B). An analysis of the data according to the Michaelis-Menten equation yielded Km values for MBP-Opi1p and ATP of 70 µg/ml and 75 µM, respectively. Under standard phosphorylation conditions, protein kinase A activity was linear with time and with kinase protein concentration.



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  		FIG. 2.
Phosphorylation of MBP-Opi1p by protein kinase A. MBP-Opi1p (0.1 mg/ml) was incubated with protein kinase A (0.2 unit/ml) and 0.2 mM [{gamma}-32P]ATP (5,000 cpm/pmol) for 10 min. Following the incubation, the samples were subjected to SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and phosphorimaging. Lane 1 did not contain MBP-Opi1p. Lane 2 contained MBP (0.1 mg/ml) instead of MBP-Opi1p. The positions of MBP-Opi1p, MBP, protein kinase A, and molecular mass standards are indicated in the figure. The phosphorylated protein that was not labeled in the figure is a proteolysis product of MBP-Opi1p. The data shown are representative of two independent experiments.

 


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  		FIG. 3.
Dependence of protein kinase A activity on the concentrations of MBP-Opi1p and ATP. A, protein kinase A (0.2 unit/ml) and 0.2 mM [{gamma}-32P]ATP (5,000 cpm/pmol) were incubated with the indicated concentrations of MBP-Opi1p for 10 min. B, protein kinase A (0.2 unit/ml) and MBP-Opi1 (0.1 mg/ml) were incubated with the indicated concentrations of [{gamma}-32P]ATP for 10 min. Following the phosphorylation incubations, the samples were subjected to SDS-polyacrylamide gel electrophoresis, transferred to PVDF paper, and subjected to phosphorimaging analysis. The data shown are representative of two independent experiments.

 

Phosphoamino Acid Analysis and Two-dimensional Phosphopeptide Mapping of MBP-Opi1p Phosphorylated by Protein Kinase A—Protein 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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  		FIG. 4.
Phosphoamino acid analysis of MBP-Opi1p phosphorylated by protein kinase A. MBP-Opi1p (0.1 mg/ml) was phosphorylated with protein kinase A (0.2 unit/ml) and 0.2 mM [{gamma}-32P]ATP (5,000 cpm/pmol) for 10 min. Following the phosphorylation incubation, the sample was subjected to SDS-polyacrylamide gel electrophoresis. Gel slices containing 32P-labeled MBP-Opi1p were subjected to phosphoamino acid analysis. The positions of the carrier standard phosphoamino acids are indicated in the figure. The data shown are representative of two independent experiments. P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine.

 


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  		FIG. 5.
Phosphopeptide mapping analysis of wild type and mutant MBP-Opi1p fusion proteins phosphorylated by protein kinase A. Wild type (WT) MBP-Opi1p (A) and S31A (B) and S251A (C) mutant MBP-Opi1p fusion proteins (0.1 mg/ml each) were phosphorylated with protein kinase A (0.2 unit/ml) and 0.2 mM [{gamma}-32P]ATP (5,000 cpm/pmol) for 10 min. Following the phosphorylation incubation, the samples were subjected to SDS-polyacrylamide gel electrophoresis followed by transfer to PVDF paper. Paper pieces containing 32P-labeled MBP-Opi1p fusion proteins were digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin. The resulting peptides were separated on cellulose thin layer plates by electrophoresis (from left to right) in the first dimension and by chromatography (from bottom to top) in the second dimension. The positions of the phosphopeptides that were absent in the S31A and S251A mutant MBP-Opi1p fusion proteins, respectively, that were present in wild type MBP-Opi1p are indicated in the figure. The data shown are representative of two independent experiments.

 

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 A—Analysis 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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  		FIG. 6.
Opi1p synthetic peptides containing a protein kinase A sequence motif are substrates for protein kinase A. Protein kinase A activity was measured as a function of the concentration of the indicated synthetic peptides. The values reported are the averages of three separate experiments ± S.D.

 

Effect of the S31A and S251A Mutations on the Phosphorylation of Opi1p by Protein Kinase A in Vitro—OPI1S31A 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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  		FIG. 7.
SDS-polyacrylamide gel electrophoresis of purified S31A and S251A mutant MBP-Opi1p fusion proteins. S31A and S251A mutant MBP-Opi1p proteins were expressed in E. coli and purified by amylose-agarose affinity chromatography. The purified proteins were subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue. The positions of the protein molecular mass standards and the mutant MBP-Opi1p fusion proteins are indicated in the figure.

 


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  		FIG. 8.
Effects of the S31A and S251A mutations on the phosphorylation of MBP-Opi1p by protein kinase A. Protein kinase A (0.2 unit/ml) and 0.2 mM [{gamma}-32P]ATP (5,000 cpm/pmol) were incubated with the indicated MBP-Opi1p fusion proteins (0.1 mg/ml each) for 10 min. Following the phosphorylation incubations, the samples were subjected to SDS-polyacrylamide gel electrophoresis, transferred to PVDF paper, and subjected to phosphorimaging analysis. The values reported are the averages of three separate experiments ± S.D. WT, wild type.

 

Effect of the S31A and S251A Mutations on the Phosphorylation of Opi1p in Vivo—The 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{Delta} mutant) and was recognized in opi1{Delta} 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{Delta} mutant cells. The mutant and wild type OPI1 alleles were expressed in opi1{Delta} 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{Delta} mutant, indicating that OPI1S31A and OPI1S251A alleles were functional in vivo.



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  		FIG. 9.
Effects of the S31A and S251 mutations on the phosphorylation of Opi1p in vivo. Cultures (50 ml) of the opi1{Delta} mutant bearing the multicopy plasmid pSA1 with the HA-tagged OPI1 gene or with plasmids pSA7 and pSA8 with the HA-tagged OPI1S31A and OPI1S251A genes, respectively, were grown to the exponential phase of growth in SC medium containing 75 µM inositol. A, cells were harvested, cell extracts were prepared, and 50-µg samples were subjected to immunoblot analysis using a 1:500 dilution of anti-HA antibodies. B, cells were harvested and resuspended in 5 ml of fresh medium containing 32Pi (0.25 mCi/ml) and incubated for 3 h. Following the incubation, the HA-tagged Opi1p proteins were immunoprecipitated from cell extracts (1 mg) with anti-HA antibodies and then subjected to SDS-polyacrylamide gel electrophoresis and transfer to PVDF paper. The 32P-labeled Opi1p proteins were visualized by phosphorimaging analysis, and the relative density of the 32P-labeled proteins was quantified using ImageQuant software. Immunoblot analysis using anti-HA antibodies showed that the wild type (WT) and mutant Opi1p proteins were present at similar amounts in the immunoprecipitates. The data shown are representative of two independent experiments.

 

opi1{Delta} 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 Expression—Expression of several UASINO-containing genes, including INO1, is negatively regulated by Opi1p (1, 4, 5, 6). Indeed, opi1{Delta} 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{Delta} 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{Delta} mutant cells. The cells were grown in the absence of inositol to the exponential phase, the extracts were prepared, and {beta}-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{Delta} mutant cells grown in the absence of inositol (Fig. 10A). The {beta}-galactosidase activity in opi1{Delta} 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 {beta}-galactosidase activity in opi1{Delta} mutant cells bearing the S31A and S251A mutant Opi1p proteins was 42 and 35% higher (p < 0.001), respectively, than that found in opi1{Delta} 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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  		FIG. 10.
Effects of the S31A and S251A mutations in Opi1p on the expression of the INO1 gene. Wild type cells (WT), opi1{Delta} mutant cells, and opi1{Delta} cells expressing either the wild type OPI1 gene or the mutant OPI1S31A and OPI1S251A genes from the single-copy plasmids pSA3, pSA5, and pSA6, respectively, were transformed with plasmid pJH359, which contains the INO1-CYC1-lacZ reporter gene. The cells were grown in SC medium in the absence (A) and presence (B) of 75 µM inositol. The cells were harvested at the exponential phase of growth; the cell extracts were prepared and used for the measurement of {beta}-galactosidase activity. The {beta}-galactosidase activities presented in A and B were relative to the activity (0.1 µmol/min/mg) derived from opi1{Delta} mutant cells bearing the wild type OPI1 gene. The values reported were determined from triplicate determinations from four independent growth studies ± S.D.

 

INO1, as well as other UASINO-containing genes, is repressed by inositol supplementation (1, 4, 5, 6). As described previously (36), the {beta}-galactosidase activity directed by the INO1-CYC1-lacZ reporter gene was not repressed by inositol supplementation in opi1{Delta} 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{Delta} 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{Delta} 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 {beta}-galactosidase activity in opi1{Delta} 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).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in membrane phospholipid synthesis in S. cerevisiae (1, 2, 4, 5, 6). We previously demonstrated that Opi1p is phosphorylated in vivo and that some of this phosphorylation is mediated by protein kinase C (36). In the present work, we showed that Opi1p phosphorylation is also mediated by protein kinase A. In vitro, protein kinase A phosphorylated MBP-Opi1p on multiple serine residues. The phosphorylation of Opi1p by protein kinase A was time- and dose-dependent and dependent on the concentrations of MBP-Opi1p and ATP. These results indicated that Opi1p was a substrate for protein kinase A. To our knowledge, this is the first report of the posttranslational modification of a phospholipid synthesis regulatory protein by phosphorylation via protein kinase A.

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{Delta} 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{Delta} 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.


   FOOTNOTES
 
* This work was supported in part by United States Public Health Service Grant GM-50679 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} 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. Back


   ACKNOWLEDGMENTS
 
We acknowledge Gil-Soo Han for helpful suggestions during the course of this work.



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