Phosphorylation and Regulation of Choline Kinase from Saccharomyces cerevisiae by Protein Kinase A*

Kee-Hong Kim and George M. CarmanDagger

From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The CKI1-encoded choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from Saccharomyces cerevisiae was phosphorylated in vivo on multiple serine residues. Activation of protein kinase A activity in vivo resulted in a transient increase in the phosphorylation of choline kinase. This phosphorylation was accompanied by a stimulation in choline kinase activity. In vitro, protein kinase A phosphorylated choline kinase on a serine residue with a stoichiometry (0.44 mol of phosphate/mol of choline kinase) consistent with one phosphorylation site/choline kinase subunit. The major phosphopeptide derived from the enzyme phosphorylated in vitro by protein kinase A was common to one of the major phosphopeptides derived from the enzyme phosphorylated in vivo. Protein kinase A activity was dose- and time-dependent and dependent on the concentrations of ATP (Km 2.1 µM) and choline kinase (Km 0.12 µM). Phosphorylation of choline kinase with protein kinase A resulted in a stimulation (1.9-fold) in choline kinase activity whereas alkaline phosphatase treatment of choline kinase resulted in a 60% decrease in choline kinase activity. The mechanism of the protein kinase A-mediated stimulation in choline kinase activity involved an increase in the apparent Vmax values with respect to ATP (2.6-fold) and choline (2.7-fold). Overall, the results reported here were consistent with the conclusion that choline kinase was regulated by protein kinase A phosphorylation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphatidylcholine (PC)1is the most abundant phospholipid in the yeast Saccharomyces cerevisiae (1, 2). It serves as a major structural component of cellular membranes and as a source of several lipid messengers (3). There are two pathways for PC synthesis in yeast, the CDP-choline pathway and the CDP-diacylglycerol (DAG) pathway (1, 2) (Fig. 1). Both pathways function to synthesize PC in wild-type cells (1, 2). The CDP-DAG pathway is used primarily by cells cultured in the absence of exogenous choline. The PC synthesized by this pathway is constantly metabolized to free choline and phosphatidate (PA) via the reaction catalyzed by phospholipase D (2) (Fig. 1). The free choline is then incorporated back into PC by the CDP-choline pathway, and the PA is recycled back into PC and into other phospholipids (e.g. phosphatidylinositol (PI)) by the CDP-DAG pathway (2). Mutants defective in the reactions leading from phosphatidylserine (PS) to PC in the CDP-DAG pathway require choline for growth to synthesize PC by the CDP-choline pathway (2).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   Pathways for the synthesis and turnover of PC in S. cerevisiae. The pathways shown for the metabolism of PC include the relevant steps discussed in the text. CK, choline kinase; PAP, PA phosphatase; PSS, PS synthase; PLD, phospholipase D; PDE, phosphatidyldimethylethanolamine; PME, phosphatidylmonomethylethanolamine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; CDP-DAG, CDP-diacylglycerol; PA, phosphatidate; PKA, protein kinase A.

Regulation of the CDP-choline pathway is important to overall lipid synthesis and to cell physiology. Activation of the CDP-choline pathway, brought about by the unregulated synthesis of CTP, causes significant increases in the synthesis of PC and PA and a decrease in the synthesis of PS (4). This activation is accompanied by an increase in total neutral lipid content at the expense of total phospholipids (4). These changes in lipid metabolism are characteristic of the stress-like condition of the stationary phase (5). Proper regulation of the CDP-choline pathway is further underscored by the fact that the synthesis of PC by this pathway is lethal in the absence of a functional PI/PC transfer protein (Sec14p) (6, 7). Sec14p, which plays an essential role in vesicle budding from the Golgi complex, functions to down-regulate the CDP-choline pathway (8). Apparently, too much PC synthesis by the CDP-choline pathway is detrimental to the secretory process.

The enzyme responsible for catalyzing the committed step in the CDP-choline pathway is choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) (9) (Fig. 1). The regulation of this enzyme should play an important role in PC synthesis by the CDP-choline pathway. Choline kinase is encoded by the CKI1 gene (10), and its expression is regulated by the growth phase of cells and by inositol and choline supplementation (11). The genetic regulation of choline kinase by these factors occurs in coordination with the regulation of other enzymes in the CDP-choline and CDP-DAG pathways (1, 2, 12). Much less is known about the biochemical regulation of choline kinase. The recent purification of the yeast CKI1-encoded choline kinase has facilitated defined studies on the regulation of its enzyme activity (13). Data indicate that choline kinase is allosterically regulated by ATP and ADP and that this regulation may be physiologically relevant (13).

To gain further insight into the regulation of choline kinase activity, we have examined the consequence of phosphorylation of the enzyme by cAMP-dependent protein kinase (protein kinase A). Protein kinase A is the principal mediator of signals transmitted through the RAS-cAMP pathway in S. cerevisiae (14, 15). In this paper we demonstrate that the activation of the RAS-cAMP pathway can mediate the phosphorylation and activation of choline kinase in vivo. In addition, pure choline kinase was a substrate for protein kinase A, and the phosphorylation of choline kinase resulted in the stimulation of choline kinase activity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All chemicals were reagent grade. Yeast growth medium supplies were from Difco Laboratories. Nucleotides, choline, phosphocholine, ammonium reinecke, phenylmethanesulfonyl fluoride, benzamide, aprotinin, leupeptin, pepstatin, bovine serum albumin, phosphoamino acids, TPCK-trypsin, and nitrocellulose paper were from Sigma. Protein kinase A catalytic subunit (bovine heart) was from Promega. Phosphocellulose papers were from Pierce. Radiochemicals, RennesanceTM immunoblotting kit and EN(3)-HANCE spray were from NEN Life Science Products. The IMMUNOcatcherTM immunoprecipitation kit was from CytoSignal Presearch Products. Scintillation counting supplies were from National Diagnostics. Protein assay reagent, electrophoresis reagents, molecular mass standards, and immunochemical reagents were from Bio-Rad. Alkaline phosphatase was from Amersham Pharmacia Biotech. Silica Gel G thin layer plates were from Analtech, and cellulose thin layer sheets were from EM Science.

Yeast Strains and Growth Conditions-- Choline kinase was overexpressed in S. cerevisiae strain W303-1A (MATa ade2-1, his3-11, 15, leu2-3, 112, trp1-1, ura3-1, can1-100) (16) by transformation (17) with the multicopy plasmid pCK1D (10). This plasmid contains the CKI1 gene and directs the overexpression of choline kinase (10). Cells were grown in complete synthetic medium (18) without leucine to the exponential phase of growth (1-2 × 107 cells/ml) at 30 °C. To examine the effect of the RAS-cAMP pathway on the phosphorylation of choline kinase in vivo, cells were grown in YEPA medium (1% yeast extract, 2% peptone, 2% acetate). Cell numbers were detected by microscopic examination with a hemacytometer.

In Vivo Labeling of Choline Kinase-- Exponential phase cells grown in YEPA medium were labeled in low phosphate (19) YEPA medium with 32Pi (0.3 mCi/ml) and L-14C(U)-amino acids (5 µCi/ml) for 4 h. Glucose was added to a final concentration of 5% to activate the RAS-cAMP pathway (20). At the indicated time intervals, labeled cells were harvested by centrifugation and washed with phosphate-buffered saline. Cells were disrupted with glass beads in radioimmune precipitation lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitors (0.5 mM phenylmethanesulfonyl fluoride, 1 mM benzamide, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin) and phosphatase inhibitors (10 mM NaF, 5 mM beta -glycerophosphate, and 1 mM sodium vanadate) (21). Choline kinase was immunoprecipitated from the cell lysate with purified anti-choline kinase antibodies (13) using the IMMUNOcatcherTM immunoprecipitation kit according to the manufacturer's instructions. Choline kinase was dissociated from the enzyme-antibody complex (21), and the amount of label incorporated into the enzyme was determined by scintillation counting. Immunoprecipitated enzyme was subjected to SDS-polyacrylamide gel electrophoresis. Gels were dried and subjected to autoradiography.

Preparation of Cell Extracts-- Yeast cells were disrupted with glass beads for seven 30-s bursts with a 1-min pause between bursts using a Mini-Bead-Beater (Biospec Products) in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM Na2EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol (22). Glass beads and cell debris were removed by centrifugation at 1,500 × g for 5 min at 4 °C. The supernatant (cell extract) was used for enzyme assays.

Purification of Choline Kinase-- CKI1-encoded choline kinase was expressed in Sf-9 insect cells and purified to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q (13). The specific activity of the pure enzyme was 128 µmol/min/mg.

Phosphorylation of Choline Kinase with Protein Kinase A-- Purified choline kinase was phosphorylated with protein kinase A using the bovine heart catalytic subunit. This enzyme is structurally and functionally similar to the S. cerevisiae protein kinase A catalytic subunit (23). The protein kinase A preparation used in our studies was judged to be electrophoretically pure and phosphorylated casein with the activity stated by the manufacturer under the assay conditions used here. Phosphorylation reactions were measured for 10 min at 30 °C in a total volume of 40 µl. Choline kinase (12 µg/ml) was incubated with 50 mM Tris-HCl (pH 7.5), 60 mM dithiothreitol, 15 µM [gamma -32P]ATP (4 µCi/nmol), 10 mM MgCl2, and the indicated concentrations of protein kinase A. At the end of the phosphorylation reactions, samples were treated with 2 × Laemmli's sample buffer (24) followed by SDS-polyacrylamide gel electrophoresis. Gels were dried and subjected to autoradiography. The incorporation of phosphate into choline kinase was determined by scintillation counting of phosphorylated enzyme excised from SDS-polyacrylamide gel slices. Alternatively, the protein kinase A phosphorylation reactions were performed with unlabeled ATP. After incubation with protein kinase A, the reaction mixtures were diluted 2-fold, and choline kinase activity was measured as described below.

Electrophoresis and Immunoblotting-- SDS-polyacrylamide gel electrophoresis (24) was performed with 9% slab gels. Molecular mass standards were phosphorylase b (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa). Immunoblot assays were performed with affinity-purified anti-choline kinase antibodies as described previously (13). The density of the choline kinase bands on immunoblots was quantified by scanning densitometry. Immunoblot signals were in the linear range of detectability.

Phosphoamino Acid Analysis-- Choline kinase was phosphorylated with protein kinase A and [gamma -32P]ATP for 10 min and then subjected to SDS-polyacrylamide gel electrophoresis. Gel slices containing 32P-labeled choline kinase were treated with 50 mM ammonium bicarbonate (pH 8.0) and 0.1% SDS at 37 °C for 30 h to elute the enzyme. 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, protein precipitates were collected by centrifugation. Proteins were washed three times with cold acetone and dried in vacuo. 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 (25). After electrophoresis, the plates were dried, sprayed with 0.25% ninhydrin in acetone to visualize carrier phosphoamino acids, and subjected to PhosphorImaging analysis.

Tryptic Digestion and Two-dimensional Peptide Mapping-- SDS-polyacrylamide gel slices containing 32P-labeled choline kinase were subjected to proteolysis using TPCK-trypsin (0.15 mg/ml) in 50 mM ammonium bicarbonate for 16 h at 37 °C (26). The mixture was subjected to centrifugation, and the supernatant was removed and retained. Fresh TPCK-trypsin was added to the gel slices for an additional 11 h of proteolysis. The digest was subjected to centrifugation, and the supernatant was again removed and retained. The gel slices were then incubated in water for 1 h at 37 °C. The mixture was centrifuged, and the supernatant was collected and added to the previously retained supernatants. The combined supernatants were dried in vacuo. Samples were resuspended in 1 ml of water and dried again. This process was repeated four times. The samples were resuspended in 10 µl of 1% ammonium carbonate, clarified by centrifugation, and spotted on cellulose thin layer chromatography plates (27). Separation of phosphopeptides was accomplished by electrophoresis in 1% ammonium bicarbonate at 1,000 volts for 35 min followed by ascending chromatography (n-butyl alcohol/glacial acetic acid/pyridine/water, 10:3:12:15) for 7 h (27). Dried plates were then subjected to PhosphorImaging analysis.

Alkaline Phosphatase Treatment of Choline Kinase-- Alkaline phosphatase was used to dephosphorylate choline kinase. Dephosphorylation reactions performed for 20 min in a total volume of 20 µl. Choline kinase (12 µg/ml) was incubated with 50 mM Tris acetate (pH 8.0), 10 mM magnesium acetate, 50 mM potassium acetate, and 100 µmol/min/mg alkaline phosphatase. The amount of alkaline phosphatase activity was based on the activity obtained using p-nitrophenyl phosphate as substrate. The alkaline phosphatase reaction was terminated by the addition of phosphatase inhibitors (10 mM NaF and 5 mM beta -glycerol phosphate). The dephosphorylated enzyme preparation was used for the measurement of choline kinase activity and for phosphorylation with protein kinase A.

Enzyme Assays, Protein Determination, and Analysis of Kinetic Data-- Choline kinase activity was measured for 10 min at 30 °C by following the formation of 3H-labeled phosphocholine from [methyl-3H]choline (500 cpm/nmol) as described previously (28). The reaction mixture contained 67 mM glycine-NaOH buffer (pH 9.5), 5 mM choline, 0.5 mM ATP, 10 mM MgSO4, 1.3 mM dithiothreitol, and enzyme protein in a final volume of 60 µl. Radiolabeled phosphocholine was separated from the radiolabeled substrate by the precipitation of the substrate as choline reineckate (28). The amount of labeled product in the supernatant was determined by scintillation counting. The product phosphocholine was identified by thin layer chromatography on silica gel plates using the solvent system of methanol, 0.5% sodium chloride, and ammonia hydroxide (50:50:1) (29). The position of the labeled phosphocholine on chromatograms was determined by fluorography using EN(3)-HANCE and compared with a standard. Protein kinase A activity was measured by following the phosphorylation of casein with 4 µCi/nmol [gamma -32P]ATP for 10 min at 30 °C. The reaction mixture contained 50 mM Tris-HCl buffer (pH 8.0), 15 µM ATP, 10 mM MgCl2, 60 mM dithiothreitol, and 25 µg/ml casein. Reactions were terminated by loading samples onto phosphocellulose filter paper. The filters were washed with 75 mM phosphoric acid and subjected to scintillation counting. All assays were performed in triplicate with an average S.D. of ± 3%. All assays were linear with time and protein concentration. A unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol of product/min. Specific activity was defined as units/mg of protein. Protein concentration was determined by the method of Bradford (30) using bovine serum albumin as the standard. Kinetic data were analyzed according to the Michaelis-Menten and Hill equations using the EZ-FIT enzyme kinetic model fitting program (31).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphorylation of Choline Kinase in Vivo-- We addressed the question of whether choline kinase was phosphorylated in vivo. Yeast cells were labeled with 32Pi followed by the immunoprecipitation of choline kinase from cell extracts with anti-choline kinase antibodies. SDS-polyacrylamide gel electrophoresis of the immunoprecipitate and autoradiographic analysis revealed that choline kinase was in fact phosphorylated in vivo. The identity of choline kinase in the immunoprecipitate was confirmed by immunoblot analysis. Immunoprecipitated 32P-labeled choline kinase was subjected to phosphoamino acid analysis. Choline kinase was phosphorylated on a serine residue(s) (Fig. 2A). TPCK-trypsin digestion of the enzyme phosphorylated in vivo yielded several phosphopeptides upon phosphopeptide mapping analysis (Fig. 3A).


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 2.   Phosphoamino acid analysis of 32P-labeled choline kinase phosphorylated in vivo and in vitro. Panel A, choline kinase was immunoprecipitated from cell extracts of yeast cells labeled with 32Pi and subjected to SDS-polyacrylamide gel electrophoresis. Panel B, pure choline kinase was phosphorylated with protein kinase A using [gamma -32P]ATP followed by SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel slices containing 32P-labeled choline kinase were subjected to phosphoamino acid analysis. The positions of the carrier standard phosphoamino acids are indicated in the figure. P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine.


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 3.   Phosphopeptide mapping analysis of 32P-labeled choline kinase phosphorylated in vivo and in vitro. Panel A, choline kinase was immunoprecipitated from cell extracts of yeast cells labeled with 32Pi and subjected to SDS-polyacrylamide gel electrophoresis. Panel B, pure choline kinase was phosphorylated with protein kinase A using [gamma -32P]ATP followed by SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel slices containing 32P-labeled choline kinase were digested with TPCK-trypsin. The resulting peptides were separated on cellulose thin layer sheets by electrophoresis (from left to right) in the first dimension and by chromatography (from bottom to top) in the second dimension. The phosphopeptide common to each map is indicated.

Effect of the RAS-cAMP Pathway on the Phosphorylation and Stimulation of Choline Kinase in Vivo-- Phosphorylation by protein kinase A is a major mechanism of cellular regulation in S. cerevisiae (14, 15). To examine whether the phosphorylation of choline kinase was mediated by protein kinase A in vivo, the extent of enzyme phosphorylation was measured in cells that were activated in the RAS-cAMP pathway. The RAS-cAMP pathway is activated by the addition of glucose to nonfermenting cells (15, 20). Glucose triggers a transient increase in cAMP levels, protein kinase A activity, and the phosphorylation of enzymes known to be substrates for protein kinase A (15, 20). Cells expressing the CKI1 gene were grown in YEPA medium to attenuate the RAS-cAMP pathway (20) and then incubated for 4 h with 32Pi to detect phosphorylated choline kinase and L-14C(U)-amino acids to normalize for the amount of choline kinase isolated. The ratio of cpm of 32P incorporated into choline kinase to the counts of 14C incorporated into choline kinase was used to examine the extent of phosphorylation. Glucose (5%) was added to the growth medium to activate protein kinase A activity (20), choline kinase was isolated by immunoprecipitation, and the amount of each label incorporated into the enzyme was determined. The addition of glucose to the growth medium resulted in a time-dependent and transient increase in the phosphorylation of choline kinase (Fig. 4). In a separate experiment, cell extracts were prepared from unlabeled cells activated in the RAS-cAMP pathway, and choline activity was measured. Activation of the pathway caused a transient increase in choline kinase activity which paralleled the increase in choline kinase phosphorylation (Fig. 4). The maximum increase in choline kinase phosphorylation (2.6-fold) and in choline kinase activity (3.2-fold) occurred 2 min after the addition of glucose to the medium.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of activating the RAS-cAMP pathway on the phosphorylation and activity of choline kinase. Cultures (10 ml) of yeast cells were grown in YEPA medium to the exponential phase of growth. Cells were harvested by centrifugation, washed with low phosphate YEPA medium, and resuspended in 1 ml of the same medium. Cells were then labeled with 32Pi and L-14C(U)-amino acids for 4 h. Glucose was added to a final concentration of 5% to activate the RAS-cAMP pathway. Choline kinase was immunoprecipitated from cells using anti-choline kinase antibodies. Choline kinase was dissociated from the enzyme-antibody complex, and the amount of the label incorporated into choline kinase was determined by scintillation counting. The values (open circle ) are reported as cpm of 32P incorporated into choline kinase relative to the cpm of 14C incorporated into choline kinase. In a separate experiment, choline kinase activity () was measured from cell extracts prepared from unlabeled cells that were activated in the RAS-cAMP pathway. The data are from an average of two independent growth studies.

Phosphorylation of Choline Kinase by Protein Kinase A in Vitro-- To determine whether choline kinase was a substrate for phosphorylation by protein kinase A in vitro, we determined if protein kinase A catalyzed the incorporation of the gamma  phosphate of 32P-labeled ATP into purified choline kinase. After the phosphorylation reaction, samples were subjected to SDS-polyacrylamide gel electrophoresis. Autoradiography of the SDS-polyacrylamide gels showed that choline kinase was indeed a substrate for protein kinase A. Protein kinase A activity was dependent on the concentration of protein kinase A (Fig. 5A) and the time of the reaction (Fig. 5B) using choline kinase as substrate. The dependence of protein kinase A activity on the concentration of ATP and choline kinase was examined. Protein kinase A activity followed typical saturation kinetics with respect to ATP (Fig. 6A). The Km value for ATP was 2.1 µM. Protein kinase A activity did not follow saturation kinetics with respect to choline kinase (Fig. 6B). Instead, the enzyme followed positive cooperative kinetics (32) as demonstrated by the double-reciprocal plot of the data, where the curve was not linear but concave upward. An analysis of the data according to the Hill equation yielded a Hill number and a Km value for choline kinase of 1.78 and 0.12 µM, respectively. The cooperative kinetic behavior of protein kinase A with respect to choline kinase may be a reflection of the fact that choline kinase exists in dimeric and other oligomeric forms (13).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Dose- and time-dependent phosphorylation of choline kinase by protein kinase A. Panel A, pure choline kinase (12 µg/ml) was incubated with the indicated amounts (U = pmol/min) of protein kinase A and 15 µM [gamma -32P]ATP for 10 min. Panel B, pure choline kinase (12 µg/ml) was incubated with protein kinase A (100 pmol/min/ml) and 15 µM [gamma -32P]ATP for the indicated time intervals. After the incubations, samples were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The incorporation of phosphate into choline kinase was determined by scintillation counting of phosphorylated enzyme excised from the SDS-polyacrylamide gel slices. The stoichiometry of the reaction was based on the monomeric form of the enzyme. A portion of the autoradiograms showing the position of choline kinase is shown above each panel.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Dependence of protein kinase A activity on the concentrations of ATP and choline kinase. Panel A, protein kinase A (100 pmol/min/ml) and choline kinase (12 µg/ml) were incubated with the indicated concentrations of [gamma -32P]ATP for 10 min. Panel B, protein kinase A (100 pmol/min/ml) and 15 µM [gamma -32P]ATP were incubated with the indicated concentrations of choline kinase for 10 min. After the incubations, samples were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The incorporation of phosphate into choline kinase was determined by scintillation counting of phosphorylated enzyme excised from SDS-polyacrylamide gel slices. The figure shows the double reciprocal plots of the data. The lines drawn in panel A were the result of a least squares analysis of the data.

At the point of maximum phosphorylation, protein kinase A catalyzed the incorporation of 0.11 mol of phosphate/mol of choline kinase subunit (Fig. 5). This relatively low stoichiometry may be explained if the enzyme purified from Sf-9 insect cells was already partially phosphorylated by protein kinase A. As described above, choline kinase in yeast was phosphorylated in vivo. The purified choline kinase was treated with alkaline phosphatase to dephosphorylate the enzyme. The alkaline phosphatase-treated enzyme was then phosphorylated with protein kinase A, and the stoichiometry of the reaction was examined again. Protein kinase A catalyzed the incorporation of 0.44 mol of phosphate/mol of the alkaline phosphatase-treated choline kinase (Fig. 7). This stoichiometry was consistent with one phosphorylation site/choline kinase subunit.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of alkaline phosphatase treatment of choline kinase on the stoichiometry of the phosphorylation by protein kinase A. Pure choline kinase (Native) and alkaline phosphatase-treated choline kinase (Dephosphorylated) were incubated with protein kinase A (100 pmol/min/ml) and 15 µM [gamma -32P]ATP for 20 min. After the incubations, samples were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The incorporation of phosphate into choline kinase was determined by scintillation counting of phosphorylated enzyme excised from SDS-polyacrylamide gel slices. The stoichiometry of the reaction was based on the monomeric form of the enzyme.

To examine which amino acid residue(s) of choline kinase was a target for phosphorylation in vitro, choline kinase was phosphorylated with protein kinase A, and the 32P-labeled enzyme was subjected to phosphoamino acid analysis. Protein kinase A phosphorylated choline kinase on a serine residue (Fig. 2B). This result was consistent with the observation that choline kinase was phosphorylated at a serine residue in vivo (Fig. 2A). 32P-Labeled choline kinase was also subjected to digestion with TPCK-trypsin followed by thin layer electrophoresis and chromatographic analysis. The protease digestion yielded one major phosphopeptide and some additional minor phosphopeptides (Fig. 3B). The major phosphopeptide derived from choline kinase phosphorylated in vitro was also presented in the enzyme phosphorylated in vivo (Fig. 3A).

Effect of Protein Kinase A and Alkaline Phosphatase on Choline Kinase Activity-- If the phosphorylation of choline kinase by protein kinase A was physiologically relevant, it might be expected that phosphorylation by protein kinase A would affect choline kinase activity. This question was examined by first phosphorylating choline kinase with protein kinase A followed by the measurement of choline kinase activity. In these experiments, choline kinase activity was measured with subsaturating concentrations (13) of ATP and choline. In this manner we could simultaneously monitor for stimulatory or inhibitory effects of phosphorylation on choline kinase activity. Phosphorylation of choline kinase by protein kinase A resulted in a dose-dependent activation of choline kinase activity (Fig. 8A). Incubation of choline kinase with 100 pmol/min/ml protein kinase A resulted in a 1.9-fold stimulation of choline kinase activity.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of protein kinase A and the effect of alkaline phosphatase on choline kinase activity. Panel A, pure choline kinase (12 µg/ml) was incubated with the indicated amounts (U = pmol/min) of protein kinase A for 20 min. Panel B, pure choline kinase (12 µg/ml) was incubated with the indicated amounts (U = µmol/min) of alkaline phosphatase for 20 min. After the incubations, samples were diluted 2-fold, and choline kinase activity was measured using 0.1 mM ATP and 0.3 mM choline.

We questioned whether the dephosphorylation of choline kinase by treatment with alkaline phosphatase would result in an inactivation of enzyme activity. Choline kinase was incubated with alkaline phosphatase for 20 min. After this incubation, the phosphatase activity was inhibited by the addition of phosphatase inhibitors, and then choline kinase activity was measured using subsaturating concentrations of ATP and choline. The alkaline phosphatase treatment of choline kinase resulted in a dose-dependent decrease in choline kinase activity (Fig. 8B). Incubation of choline kinase with 100 µmol/min/ml phosphatase resulted in a 60% decrease in choline kinase activity

We also examined the effect of protein kinase A on choline kinase that was dephosphorylated with alkaline phosphatase. As indicated above, dephosphorylation of the enzyme resulted in 60% loss in activity (Fig. 9). The rephosphorylation of the dephosphorylated enzyme with protein kinase A resulted in a 1.9-fold increase in choline kinase activity. The magnitude of this stimulation was the same as that observed when the native enzyme was phosphorylated with protein kinase A (Fig. 9). Although the rephosphorylation of the dephosphorylated enzyme resulted in a stimulation of activity, only 82% of the native enzyme activity was recovered after rephosphorylation with protein kinase A. This suggested that the alkaline phosphatase treatment of choline kinase resulted in the dephosphorylation of sites that are phosphorylated by other protein kinases. Thus, other protein kinase phosphorylations may be necessary for the full activation of choline kinase.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of dephosphorylation on the stimulation of choline kinase activity by protein kinase A. Pure choline kinase (Native), native choline kinase phosphorylated with protein kinase A (Phosphorylated), alkaline phosphatase-treated choline kinase (Dephosphorylated), and dephosphorylated choline kinase rephosphorylated with protein kinase A (Rephosphorylated) were measured using 0.1 mM ATP and 0.3 mM choline.

Effect of Phosphorylation on the Kinetics of Choline Kinase Activity-- Kinetic analyses were performed to characterize further the effects of protein kinase A phosphorylation of choline kinase on its activity in vitro. As indicated above, the choline kinase that we purified was already partially phosphorylated by protein kinase A. Therefore, the alkaline phosphatase-treated enzyme was used as a control to accentuate the effects of protein kinase A phosphorylation. The dependence of the phosphorylated and dephosphorylated forms of the enzyme on ATP was examined using a saturating concentration of choline. Both forms of the enzyme exhibited typical saturation kinetics with respect to ATP (Fig. 10A). The native purified choline kinase exhibits positive cooperative kinetics with respect to ATP at subsaturating concentrations of choline (13). Phosphorylation of choline kinase resulted in a 2.6-fold increase in the apparent Vmax value of the reaction with respect to ATP but did not affect the apparent Km value for ATP (Table I). The dependence of the phosphorylated and dephosphorylated forms of choline kinase on choline was examined using a saturating concentration of ATP (Fig. 10B). Phosphorylation of the enzyme resulted in a 2.7-fold increase in the apparent Vmax value of the reaction with respect to choline but did not affect the apparent Km value for choline (Table I).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 10.   Effect of phosphorylation on the kinetics of choline kinase activity with respect to ATP and to choline. Panel A, phosphorylated () and dephosphorylated (open circle ) choline kinase activities were measured as a function of the concentration of ATP using 2 mM choline. Panel B, phosphorylated () and dephosphorylated (open circle ) choline kinase activities were measured as a function of the concentration of choline using 0.5 mM ATP. The figure shows the double reciprocal plots of the data. The lines drawn in the figure were the result of a least squares analysis of the data.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Kinetic constants for the phosphorylated and dephosphorylated forms of choline kinase


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of the CDP-choline pathway is important to overall lipid metabolism and cell physiology in S. cerevisiae (4, 8). The fact that choline kinase catalyzes the committed step in the CDP-choline pathway suggests that this enzyme should play a pivotal role in its regulation. Data indicate that choline kinase activity is regulated in vivo by biochemical mechanisms; however, these mechanisms have yet to be defined. Phosphorylation is a major mechanism by which the activity of an enzyme is regulated (33, 34). In this work we showed that choline kinase was phosphorylated in vivo and in vitro. Results of phosphoamino acid analysis and phosphopeptide mapping experiments indicated that the enzyme was phosphorylated on multiple serine residues in vivo. The experiments using yeast cells that were activated in the RAS-cAMP pathway indicated that protein kinase A mediated the phosphorylation of choline kinase in vivo. Moreover, the increase in the phosphorylation of choline kinase in response to the activation of the RAS-cAMP pathway was accompanied by an increase in choline kinase activity.

In vitro, protein kinase A phosphorylated pure choline kinase on a serine residue with a stoichiometry consistent with one phosphorylation site/choline kinase subunit. This reaction was dose- and time-dependent and dependent on the concentrations of ATP and choline kinase. These results indicated that choline kinase was a substrate for protein kinase A. In addition, the in vitro phosphorylation of choline kinase resulted in a stimulation in choline kinase activity. Phosphorylation stimulated choline kinase activity by increasing the apparent Vmax of the reaction with respect to ATP and with respect to choline. Phosphorylation did not have a significant effect on the apparent Km values for ATP and choline. The major phosphopeptide derived from the enzyme phosphorylated in vitro was common to one of the major phosphopeptides derived from choline kinase phosphorylated in vivo. This suggested that choline kinase was a substrate for protein kinase A in vivo. Sequencing of the proteolytic fragments from the choline kinase labeled in vivo and in vitro will be necessary to confirm whether the site(s) phosphorylated by protein kinase A were in fact identical. The presence of the additional phosphopeptides derived from the enzyme phosphorylated in vivo indicated that additional protein kinases are also responsible for the phosphorylation of choline kinase.2 This hypothesis is also supported by the studies using the alkaline phosphatase-treated choline kinase where the full activation of the dephosphorylated enzyme was not achieved after rephosphorylation with protein kinase A.

Choline kinase is also an important enzyme in mammalian systems where the CDP-choline pathway is the primary route for PC synthesis (35-37). Genes encoding mammalian forms of choline kinase have been isolated (38-40), and various forms of the enzyme have been purified (41-43). The COOH-terminal region of the choline kinases from yeast and mammalian cells share some amino acid sequence similarity and contain a phosphotransferase consensus sequence (44), presumed to be involved in catalytic function (45, 46). However, the yeast and mammalian choline kinases differ significantly in their NH2-terminal regions (45, 46), and they also differ with respect to their catalytic properties (13). The mammalian forms of choline kinase have not been shown to be regulated by phosphorylation as was shown here for the yeast enzyme.

Regulation of the S. cerevisiae choline kinase by protein kinase A phosphorylation may be a mechanism by which choline kinase is coordinately regulated with other enzymes in the CDP-choline and CDP-DAG pathways for PC synthesis. PA phosphatase catalyzes the dephosphorylation of PA to form DAG (47). The DAG derived from this reaction can be used in the last step in the CDP-choline pathway (Fig. 1). PS synthase catalyzes a highly regulated step in the CDP-DAG pathway (1, 2) (Fig. 1). The 45-kDa Mg2+-dependent form of PA phosphatase (48) and the PS synthase (49) enzymes are both phosphorylated and regulated by protein kinase A. Like the choline kinase enzyme, the activity of PA phosphatase is stimulated by protein kinase A phosphorylation (48). On the other hand, the activity of PS synthase is inhibited by protein kinase A phosphorylation (49). The activation of the RAS-cAMP pathway in S. cerevisiae results in a number of changes in overall lipid metabolism (48, 50). These changes include an increase in PI synthesis at the expense of PS synthesis and an increase in the synthesis of DAG (48, 50). The decrease in PS synthesis has been attributed to the phosphorylation and inhibition of PS synthase (49, 50). The increase in DAG synthesis has been attributed to the phosphorylation and stimulation of PA phosphatase (48). The increase in PI synthesis is not caused by the phosphorylation of PI synthase (50). Instead, PI synthesis increases because of a loss of competition of PI synthase and PS synthase for their common substrate CDP-DAG (Fig. 1) as a result of down-regulation of PS synthase by phosphorylation (50). Although the PS synthase reaction in the CDP-DAG pathway is down-regulated by protein kinase A phosphorylation, the overall synthesis of PC is not affected by the activation of the RAS-cAMP pathway (50). The phosphorylation and stimulation of choline kinase and PA phosphatase would be consistent with the increased utilization of the CDP-choline pathway for PC synthesis under this condition. We acknowledge that these explanations are only a first approximation for the complex regulation by phosphorylation that occurs in vivo.

Studies are currently in progress to define the protein kinase A phosphorylation sequence(s) of choline kinase. With this information we plan to construct by site-directed mutagenesis mutant forms of choline kinase which will be useful in assessing the in vivo roles of the phosphorylated and dephosphorylated forms of choline kinase on PC synthesis and on cell physiology.

    ACKNOWLEDGEMENTS

We thank Satoshi Yamashita for providing the CKI1 clone used for the expression of choline kinase in yeast and Dennis R. Voelker for providing us with Sf-9 insect cells expressing the CKI1-encoded choline kinase.

    FOOTNOTES

* This work was supported in part by United States Public Health Service, National Institutes of Health Grant GM-50679. This is New Jersey Agricultural Experiment Station Publication D-10581-1-99.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 732-932-9611 (ext. 217); Fax: 732-932-6776; E-mail: carman{at}aesop.rutgers.edu.

2 Preliminary data indicate that choline kinase is also phosphorylated by protein kinase C (K.-H. Kim and G. M. Carman, unpublished data).

    ABBREVIATIONS

The abbreviations used are: PC, phosphatidylcholine; DAG, diacylglycerol; CDP-DAG, CDP-diacylglycerol; PA, phosphatidate; PI, phosphatidylinositol; PS, phosphatidylserine; protein kinase A, cAMP-dependent protein kinase; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Carman, G. M., and Zeimetz, G. M. (1996) J. Biol. Chem. 271, 13293-13296[Free Full Text]
  2. Henry, S. A., and Patton-Vogt, J. L. (1998) Prog. Nucleic Acids Res. 61, 133-179[Medline] [Order article via Infotrieve]
  3. Exton, J. H. (1994) Biochim. Biophys. Acta 1212, 26-42[Medline] [Order article via Infotrieve]
  4. Ostrander, D. B., O'Brien, D. J., Gorman, J. A., and Carman, G. M. (1998) J. Biol. Chem. 273, 18992-19001[Abstract/Free Full Text]
  5. Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669[CrossRef][Medline] [Order article via Infotrieve]
  6. Cleves, A. E., McGee, T. P., Whitters, E. A., Champion, K. M., Aitkin, J. R., Dowhan, W., Goebl, M., and Bankaitis, V. A. (1991) Cell 64, 789-800[Medline] [Order article via Infotrieve]
  7. McGee, T. P., Skinner, H. B., Whitters, E. A., Henry, S. A., and Bankaitis, V. A. (1994) J. Cell Biol. 124, 273-287[Abstract]
  8. Fang, M., Rivas, M. P., and Bankaitis, V. A. (1998) Biochim. Biophys. Acta 1404, 85-100[CrossRef][Medline] [Order article via Infotrieve]
  9. Wittenberg, J., and Kornberg, A. (1953) J. Biol. Chem. 202, 431-444[Free Full Text]
  10. Hosaka, K., Kodaki, T., and Yamashita, S. (1989) J. Biol. Chem. 264, 2053-2059[Abstract/Free Full Text]
  11. Hosaka, K., Murakami, T., Kodaki, T., Nikawa, J., and Yamashita, S. (1990) J. Bacteriol. 172, 2005-2012[Medline] [Order article via Infotrieve]
  12. Greenberg, M. L., and Lopes, J. M. (1996) Microbiol. Rev. 60, 1-20[Free Full Text]
  13. Kim, K.-H., Voelker, D. R., Flocco, M. T., and Carman, G. M. (1998) J. Biol. Chem. 273, 6844-6852[Abstract/Free Full Text]
  14. Broach, J. R., and Deschenes, R. J. (1990) Adv. Cancer Res. 54, 79-139[Medline] [Order article via Infotrieve]
  15. Thevelein, J. M. (1994) Yeast 10, 1753-1790[Medline] [Order article via Infotrieve]
  16. Thomas, B., and Rothstein, R. (1989) Cell 56, 619-630[Medline] [Order article via Infotrieve]
  17. Ito, H., Yasuki, F., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168[Medline] [Order article via Infotrieve]
  18. Culbertson, M. R., and Henry, S. A. (1975) Genetics 80, 23-40[Abstract/Free Full Text]
  19. Warner, J. R. (1991) Methods Enzymol. 194, 423-428[Medline] [Order article via Infotrieve]
  20. Thevelein, J. M., and Beullens, M. (1985) J. Gen. Microbiol. 131, 3199-3209[Medline] [Order article via Infotrieve]
  21. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Klig, L. S., Homann, M. J., Carman, G. M., and Henry, S. A. (1985) J. Bacteriol. 162, 1135-1141[Medline] [Order article via Infotrieve]
  23. Toda, T., Cameron, S., Sass, P., Zoller, M., and Wigler, M. (1987) Cell 50, 277-287[Medline] [Order article via Infotrieve]
  24. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  25. Boyle, W. J., Van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201, 110-149[Medline] [Order article via Infotrieve]
  26. Kawamoto, S., and Adelstein, R. S. (1987) J. Biol. Chem. 262, 7282-7288[Abstract/Free Full Text]
  27. Watkins, J. D., and Kent, C. (1991) J. Biol. Chem. 266, 21113-21117[Abstract/Free Full Text]
  28. Porter, T. J., and Kent, C. (1992) Methods Enzymol. 209, 134-146[Medline] [Order article via Infotrieve]
  29. Teegarden, D., Taparowsky, E. J., and Kent, C. (1990) J. Biol. Chem. 265, 6042-6047[Abstract/Free Full Text]
  30. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  31. Perrella, F. (1988) Anal. Biochem. 174, 437-447[Medline] [Order article via Infotrieve]
  32. Segel, I. H. (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems., John Wiley and Sons, New York
  33. Chock, P. B., Rhee, S. G., and Stadtman, E. R. (1980) Annu. Rev. Biochem. 49, 813-843[CrossRef][Medline] [Order article via Infotrieve]
  34. Krebs, E. G., and Beavo, J. A. (1979) Annu. Rev. Biochem. 48, 923-959[CrossRef][Medline] [Order article via Infotrieve]
  35. Ishidate, K. (1997) Biochim. Biophys. Acta Lipids Lipid Metab. 1348, 70-78[Medline] [Order article via Infotrieve]
  36. Vance, D. E. (1996) in Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D. E., and Vance, J., eds), pp. 153-181, Elsevier Science Publishers, Amsterdam
  37. Walkey, C. J., Yu, L., Agellon, L. B., and Vance, D. E. (1998) J. Biol. Chem. 273, 27043-27046[Abstract/Free Full Text]
  38. Hosaka, K., Tanaka, S., Nikawa, J., and Yamashita, S. (1992) FEBS Lett. 304, 229-232[CrossRef][Medline] [Order article via Infotrieve]
  39. Uchida, T., and Yamashita, S. (1992) J. Biol. Chem. 267, 10156-10162[Abstract/Free Full Text]
  40. Uchida, T. (1994) J. Biochem. 116, 508-518[Abstract]
  41. Ishidate, K., Nakagomi, K., and Nakazawa, Y. (1984) J. Biol. Chem. 259, 14706-14710[Abstract/Free Full Text]
  42. Porter, T. J., and Kent, C. (1990) J. Biol. Chem. 265, 414-422[Abstract/Free Full Text]
  43. Uchida, T., and Yamashita, S. (1990) Biochim. Biophys. Acta 1043, 281-288[Medline] [Order article via Infotrieve]
  44. Brenner, S. (1987) Nature 329, 21-21[Medline] [Order article via Infotrieve]
  45. Yamashita, S., and Hosaka, K. (1997) Biochim. Biophys. Acta 1348, 63-69[Medline] [Order article via Infotrieve]
  46. Ishidate, K. (1997) Biochim. Biophys. Acta 1348, 70-78[Medline] [Order article via Infotrieve]
  47. Carman, G. M. (1997) Biochim. Biophys. Acta 1348, 45-55[Medline] [Order article via Infotrieve]
  48. Quinlan, J. J., Nickels, J. T., Jr., Wu, W.-I., Lin, Y.-P., Broach, J. R., and Carman, G. M. (1992) J. Biol. Chem. 267, 18013-18020[Abstract/Free Full Text]
  49. Kinney, A. J., and Carman, G. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7962-7966[Abstract]
  50. Kinney, A. J., Bae-Lee, M., Singh Panghaal, S., Kelley, M. J., Gaynor, P. M., and Carman, G. M. (1990) J. Bacteriol. 172, 1133-1136[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.