vitro by cyclic AMP-dependent protein kinase

Proc. Natl. Acad. Sci. USA Vol. 85, pp. 7962-7966, November 1988 Biochemistry

Phosphorylation of yeast phosphatidylserine synthase in vivo and in vitro by cyclic AMP-dependent protein kinase ANTHONY J. KINNEY AND GEORGE M. CARMAN* Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, NJ 08903

Communicated by Eugene P. Kennedy, July 29, 1988 (received for review June 21, 1988)

PtdSer synthase activity by a noncompetitive inhibition mechanism (9). Studies using purified enzyme reconstituted into unilamellar phospholipid vesicles have shown that PtdSer synthase is regulated by the relative proportions of phospholipids in the membrane (19). Increases in the ratio of phosphatidylinositol to PtdSer in phospholipid vesicles result in a reduction in enzyme activity (19). PtdSer synthase activity is also regulated by the growth phase of cells, where activity (not enzyme) is reduced in the stationary phase of growth (20). In this communication we demonstrate that PtdSer synthase was phosphorylated in vivo and in vitro by cAMPdependent protein kinase. The phosphorylation of the enzyme resulted in reduced PtdSer synthase activity. These studies provide evidence of yet another level at which this important enzyme in phospholipid metabolism is regulated. To our knowledge, in vivo and in vitro regulation of a phospholipid biosynthetic enzyme by cAMP-dependent protein kinase from any organism has not been reported previously.

Evidence is presented that demonstrates that ABSTRACT phosphatidylserine synthase (CDPdiacylglycerol:L-serine 0phosphatidyltransferase, EC 2.7.8.8) from Saccharomyces cerevisiae is phosphorylated in vivo and in vitro by cAMPdependent protein kinase. Phosphatidylserine synthase activity in cell extracts was reduced in the bcyl mutant (which has high cAMP-dependent protein kinase activity) and elevated in the cyri mutant (which has low cAMP-dependent protein kinase activity) when compared with wild-type cells. The reduced phosphatidylserine synthase activity in the bcyl mutant correlated with elevated levels of a phosphorylated form of the phosphatidylserine synthase Mr 23,000 subunit. The elevated phosphatidylserine synthase activity in the cyri mutant correlated with reduced levels of the phosphorylated form of the enzyme. There was negligible phosphorylation of the phosphatidylserine synthase M, 23,000 subunit from stationary-phase cells. Pure phosphatidylserine synthase was phosphorylated by the cAMP-dependent protein kinase catalytic subunit, which resulted in a 60-70% reduction in phosphatidylserine synthase activity. The cAMP-dependent protein kinase catalytic subunit catalyzed the incorporation of 0.7 mol of phosphate per mol of phosphatidylserine synthase Mr 23,000 subunit. The specific cAMP-dependent protein kinase inhibitor prevented the phosphorylation of phosphatidylserine synthase and the inhibition of its activity by the catalytic subunit. Analysis of peptides derived from protease-treated labeled phosphatidylserine synthase showed only one labeled peptide. Phospho amino acid analysis of labeled phosphatidylserine synthase showed that the enzyme was phosphorylated at a serine residue.

EXPERIMENTAL PROCEDURES Materials. All chemicals were reagent grade. Serine, phospholipids, ATP, cAMP-dependent protein kinase catalytic subunit (bovine heart), protein kinase inhibitor (synthetic peptide) (21), protein A-Sepharose CL-4B, trypsin, chymotrypsin, phospho amino acids, and bovine serum albumin were purchased from Sigma. Radiochemicals were purchased from ICN, Amersham, and Du Pont/New England Nuclear. Scintillation counting supplies were purchased from National Diagnostics (Mannville, NJ). Triton X-100 was a gift from Rohm and Haas. Molecular weight standards, electrophoresis, immunochemical reagents, and Bio-Gel P-6 were purchased from Bio-Rad. Unmodified nitrocellulose paper (0.2 ,um) was obtained from Schleicher & Schuell. Cellulose thin-layer sheets were obtained from Eastman Kodak. CDPdiacylglycerol was prepared from soybean lecithin as described (22). Growth medium supplies were purchased from Difco. Strains and Growth Conditions. Strains R146-21B (MATa trpl ura3 BCYI::URA3, gene disruption mutant of the regulatory subunit of cAMP-dependent protein kinase), R14621C (MATa trpl ura3 lys2, wild type for the regulatory subunit of cAMP-dependent protein kinase), and T50-3A [MATa trpl ura3 his3 leu2 cyrl-2 (ts), mutant for adenylate cyclase] were obtained from K. Matsumoto (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA). Wild-type strain S288C (aga12) was used for the purification of PtdSer synthase. Strains were maintained on 1% yeast extract, 2% peptone, and 2% glucose medium plates containing 2% agar. Cells were precultured to the exponential phase at 25°C in complete synthetic medium (23, 24). From this preculture, 4 x 10' colony-forming units was inoculated

In the primary biosynthetic pathway of phospholipid biosynthesis in Saccharomyces cerevisiae, phosphatidylserine (PtdSer) is the precursor to the major phospholipids phosphatidylethanolamine and phosphatidylcholine (1). The biosynthesis of PtdSer plays a major role in overall phospholipid biosynthesis and cell growth in this organism (2-5). The enzyme responsible for PtdSer synthesis in S. cerevisiae is PtdSer synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8). The enzyme catalyzes the formation of PtdSer by displacing CMP from CDPdiacylglycerol with serine (6) by a sequential reaction mechanism (79). PtdSer synthase, which is encoded by the CHOI gene (2), is localized in the mitochondrial and microsomal fractions of the cell (10, 11), has been purified to homogeneity (7), and has a subunit Mr of 23,000 (7, 12). The primary product of the CHOI gene is an inactive protein with a subunit Mr of -30,000 (13, 14), which is converted by proteolysis to active PtdSer synthase with a subunit Mr of 23,000 (13). PtdSer synthase is a highly regulated enzyme in S. cerevisiae. The enzyme is regulated by inositol alone and in concert with other water-soluble phospholipid precursors, including serine, ethanolamine, and choline, by an enzymerepression mechanism (5, 15-18). Inositol also regulates The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: PtdSer, phosphatidylserine. *To whom reprint requests should be addressed. 7962

Biochemistry: Kinney and Carman into 5-ml batches of complete synthetic medium and incubated at 250C to the exponential and stationary phases of growth. 32p; (200 ,Ci/ml; 1 Ci = 37 GBq) was then added to the cultures and incubated for 90 min. The labeled cells were harvested by centrifugation and washed with distilled water. Preparation of Cell Extracts, Pure PtdSer Synthase, and Anti-PtdSer Synthase Antibodies. Cell extracts were prepared by cell disruption with glass beads as described (18). PtdSer synthase was purified to homogeneity from wild-type S. cerevisiae S288C by Triton X-100 solubilization of microsomes, CDPdiacylglycerol-Sepharose affinity chromatography, and DE-52 chromatography as described by Bae-Lee and Carman (7). The specific activity of pure PtdSer synthase was 3000 units/mg. Polyclonal antibodies were prepared against purified PtdSer synthase as described (5). Immunoprecipitation and Analysis of PtdSer Synthase Labeled in Vivo. Cells labeled with 32Pi were disrupted by spinning in a Vortex with glass beads (0.05 g) for 90 s in 0.1 ml of 50 mM potassium phosphate buffer (pH 7.5) containing 100 mM NaCI, 1% sodium dodecyl sulfate (SDS), 2% Triton X-100, 50 mM NaF, and 10 mM 2-mercaptoethanol. The samples were heated at 100'C for 3 min and then centrifuged at 10,000 x g for 5 min. The resulting supernatant was precleared by incubation with 20 p1 of protein A-Sepharose CL-4B (10%o suspension, wt/vol) for 30 min. Labeled PtdSer synthase was immunoprecipitated from the cleared supernatant by incubation with 0.1 ml of PtdSer synthase antiserum for 4 hr followed by incubation with 20,u1 of protein ASepharose for 1 hr. The precipitate was collected by centrifugation, washed twice with 0.2 ml of 100 mM potassium phosphate buffer (pH 7.5) containing 0.5 M NaCl, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate, and washed twice with 0.2 ml of 50 mM Tris-HCI (pH 7.5) containing 10 mM NaCI. The precipitate was then suspended in 80 ,u1 of 50 mM Tris-HCI (pH 7.5) containing 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol and heated for 5 min at 90°C to dissociate the enzyme-antibody complex. The suspension was centrifuged in a Microfuge for 1 min to obtain the labeled PtdSer synthase (supernatant). A 10-,ul sample of labeled PtdSer synthase was precipitated onto Whatman GF/F filter discs pretreated with 20%o trichloroacetic acid and 20 mg of bovine serum albumin per ml. The extent of phosphorylation of the enzyme was measured as described (25, 26). A 50-,lI sample of the labeled enzyme was subjected to SDS/polyacrylamide gel electrophoresis (27) with 10% slab gels followed by electrophoretic transfer to 0.2-,um unmodified nitrocellulose papers (28, 29). The position of the labeled PtdSer synthase on the nitrocellulose papers was determined by autoradiography. The nitrocellulose blots were further probed with PtdSer synthase antiserum as described (5). The density of the PtdSer synthase bands on the immunoblots were quantitated with a Hoefer GS 300 scanning densitometer. The extent of in vivo phosphorylation (arbitrary units) was expressed as the ratio of cpm incorporated into immunoprecipitated PtdSer synthase to the relative density of the PtdSer synthase band on the immunoblot. The immunoblot signals were in the linear range of detectability. Phosphorylation and Analysis of Pure PtdSer Synthase. Purified PtdSer synthase (0.05 nmol/min) was incubated with 50 mM Tris-HCI, pH 8.0/60 mM dithiothreitol/60 ,uM ATP/5 mM MgCI2/protein kinase catalytic subunit (10 pmol/min) at 30°C for 10 min in a total volume of 0.1 ml. The reaction was terminated by the addition of 50 ng of protein kinase inhibitor followed by the assay of PtdSer synthase activity. Alternatively, pure enzyme (5 ,ug) was incubated under phosphorylation conditions with [-32P]ATP (2 ,uCi). The reaction was terminated by the addition of 0.1 ml of 0. 125 M Tris HCI, pH 6.8/4% SDS/20%o glycerol/10 mM 2-mercaptoethanol. Samples were heated at 90TC for 5 min and subjected to SDS/

Proc. Natl. Acad. Sci. USA 85 (1988)

7963

polyacrylamide gel electrophoresis, electrophoretic transfer to nitrocellulose paper, autoradiography, and immunoblotting as described above. The extent of PtdSer synthase phosphorylation was measured as described above. Analysis of Peptide Fragments and Phospho Amino Acids. Pure PtdSer synthase was phosphorylated as described above. The 32P-labeled PtdSer synthase was separated from unreacted [y-32P]ATP by Bio-Gel P-6 gel filtration chromatography. The sample was freeze-dried and suspended in 50 mM ammonium bicarbonate (pH 7.7). The suspension was treated with trypsin or chymotrypsin, at a final ratio of PtdSer synthase to protease of 20:1 (wt/wt), and mixed at 250C by end-over-end rotation for 20 hr. The resulting peptide fragments were analyzed by isoelectric focusing (30). Following isoelectric focusing, the gel was cut into 0.5-cm slices and each slice was quantitated by scintillation spectroscopy. 32P-labeled pure PtdSer synthase was subjected to partial acid hydrolysis and amino acids were separated by highvoltage electrophoresis on thin-layer cellulose sheets as described (31). Standard phospho amino acids were visualized with ninhydrin and labeled phospho amino acids were located by autoradiography. PtdSer Synthase Assay. PtdSer synthase activity was measured by following the incorporation of 0.5 mM L-[33H]serine (10,000 cpm/nmol) into PtdSer in the presence of 50 mM Tris-HCI, pH 8.0/1 mM MnCl2/0.2 mM CDPdiacylglycerol/3.2 mM Triton X-100/enzyme protein (32). The phospholipid product of the reaction was identified with standard PtdSer by thin-layer chromatography (32). All assays were conducted at 30°C for 20 min. A unit of enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product per min under the conditions described. Specific activity was defined as the units/mg of protein. Protein was determined by the method of Bradford (33) with bovine serum albumin as the standard.

RESULTS Phosphorylation of PtdSer Synthase in Wild-Type Cells. The reversible covalent modification of enzymes by phosphorylation-dephosphorylation is a major mechanism of cellular regulation (34, 35). To examine if PtdSer synthase could be phosphorylated in vivo, wild-type cells were grown in the presence of 32p;. When PtdSer synthase was immunoprecipitated from exponential-phase cells with antiserum directed against pure PtdSer synthase, the Mr 23,000 subunit was phosphorylated. The PtdSer synthase antiserum also precipitated the PtdSer synthase Mr 30,000 subunit; however, it was not phosphorylated (Fig. 1). The levels of the PtdSer synthase Mr 23,000 and Mr 30,000 subunits precipitated from stationary-phase cells were slightly greater than those from

Autoradiogram

Immunoblot

- 30,000

VA

- 23,000

FIG. 1. Immunoprecipitation of phosphorylated PtdSer synthase

Mr 23,000 subunit from wild-type cells. Wild-type strain S288C was grown in the presence of 32Pi and harvested in the exponential phase of growth (4 x 107 colony-forming units/ml), and PtdSer synthase

was immunoprecipitated from cells and analyzed. A portion of an autoradiogram and the corresponding immunoblot are shown. The positions of the PtdSer synthase Mr 23,000 and Mr 30,000 subunits are indicated. No other phosphorylated proteins were immunoprecipitated by the PtdSer synthase antibody.

7964

Biochemistry: Kinney and Carman

Proc. Nati. Acad. Sci. USA 85

exponential-phase cells. However, the phosphorylation of the Mr 23,000 subunit was barely detectable (data not shown). Phosphorylation of PtdSer Synthase in Yeast Mutants Deficient in cAMP-Dependent Protein Kinase and Adenylate Cyclase. Several enzymes from S. cerevisiae are regulated by phosphorylation by way of cAMP-dependent protein kinase (36-38). To examine if the in vivo phosphorylation of PtdSer synthase was catalyzed by cAMP-dependent protein kinase, the extent of PtdSer synthase phosphorylation and PtdSer synthase activity was measured in yeast mutants defective in cAMP-dependent protein kinase and adenylate cyclase. The bcyl (BCYI::URA3 gene disruption) mutant has elevated cAMP-dependent protein kinase activity due to a defect in the regulatory subunit of cAMP-dependent protein kinase (39, 40). The cyri mutant when grown at the restrictive temperature (350C) has no cAMP-dependent protein kinase activity due to a defect in adenylate cyclase and is arrested in the G1 phase of the cell cycle (41). At the permissive temperature (250C) used in this study the mutant produces low levels of cAMP and thus has low levels of cAMPdependent protein kinase activity (41). These cells are not arrested in G1 phase at the permissive temperature (41). PtdSer synthase was immunoprecipitated from exponentialphase mutant and wild-type cells labeled with 32Pi and analyzed as described under Experimental Procedures. Cell extracts were also prepared from mutant and wild-type cells and assayed for PtdSer synthase activity. As in wild-type cells, only the PtdSer synthase Mr 23,000 subunit was phosphorylated in mutant cells (data not shown). The extent of phosphorylated PtdSer synthase from the bcyl mutant and the cyri mutant was higher by a factor of -2 and lower by a factor of '4, respectively, when compared with wild-type cells (Fig. 2). PtdSer synthase activity from the bcyl mutant and the cyrl mutant was lower by a factor of =2 and higher by a factor of -2, respectively, when compared with wildtype cells (Fig. 2). Thus, PtdSer synthase activity is regulated in vivo by cAMP-dependent protein kinase phosphorylation, which resulted in an inhibition of activity. Phosphorylation of Pure PtdSer Synthase by cAMPDependent Protein Kinase. The phosphorylation of PtdSer synthase was examined in vitro by using pure PtdSer synthase and bovine cAMP-dependent protein kinase catalytic subunit. The bovine catalytic subunit is structurally and functionally identical to the yeast catalytic subunit (42). PtdSer synthase activity was inhibited 60-70% when the pure enzyme was preincubated under phosphorylation conditions. The inhibition of PtdSer synthase activity by the cAMPdependent protein kinase catalytic subunit was dependent on >~~~~,

60

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FIG. 3. Effect of ATP on the inhibition of PtdSer synthase activity by cAMP-dependent protein kinase. Pure PtdSer synthase (0.05 nmol/min) was preincubated under phosphorylation conditions with cAMP-dependent protein kinase catalytic subunit (10 pmol/min) with the indicated concentrations of ATP. Following incubation, PtdSer synthase activity was measured.

ATP (Fig. 3) and time (Fig. 4). In the presence of 60 ,uM ATP, maximum inhibition of PtdSer synthase activity was obtained after 10 min of preincubation with the catalytic subunit. Additional time of preincubation did not result in a further inhibition of activity. The time-dependent inhibition of PtdSer synthase activity correlated with an increase in the amount of 32P-labeled PtdSer synthase Mr 23,000 subunit (Fig. 5). At maximum inhibition of PtdSer synthase activity, the cAMP-dependent protein kinase catalytic subunit catalyzed the incorporation of 0.7 mol of phosphate per mol of PtdSer synthase Mr 23,000 subunit. The phosphorylation of PtdSer synthase was cAMP-dependent protein kinase specific as the Mr 23,000 was not phosphorylated when the catalytic subunit was omitted from the phosphorylation preincubation mixture (Fig. 5). Although the purified enzyme preparation contained the PtdSer synthase Mr 30,000 subunit, the Mr 30,000 subunit was not phosphorylated (Fig. 5). No label was observed in the Mr 30,000 subunit even when the specific activity of the [y32P]ATP used in the phosphorylation mixture was increased 100-fold (data not shown). Protein kinase inhibitor is a synthetic peptide that contains the protein kinase subsite Arg-Arg found in the Arg-Arg-XaaSer sequence (21). This inhibitor binds to the cAMPdependent protein kinase catalytic subunit but is not phos-

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FIG. 2. Immunoprecipitation of phosphorylated PtdSer synthase Mr 23,000 subunit and PtdSer synthase activity from wild-type and mutant cells. Wild-type strains S288C (wtl) and R146-21C (wt2) and mutant strains R146-21B (bcyl) and T50-3A (cyrl) were grown in the presence of 32Pi and harvested in the exponential phase of growth, and PtdSer synthase was immunoprecipitated from cells and analyzed. PtdSer synthase activity was measured in cell extracts of wild-type and mutant cells grown in the absence of 32p;.

Time, min FIG. 4. Time-dependent inhibition of PtdSer synthase activity after preincubation under phosphorylation conditions with cAMPdependent protein kinase. Pure PtdSer synthase (0.05 nmol/min) was preincubated in the phosphorylation mixture with (e) and without (o) cAMP-dependent protein kinase catalytic subunit (10 pmol/min) for the indicated time intervals. Following incubation, PtdSer synthase activity was measured.

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1C

0

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i _ _mt M& _AW

1

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FIG. 5. Time-dependent phosphorylation of the PtdSer synthase Mr 23,000 subunit by cAMP-dependent protein kinase. Pure PtdSer synthase (5 /ig) was incubated under phosphorylation conditions with cAMP-dependent protein kinase catalytic subunit (10 pmol/ min) and [y-32P]ATP for the indicated time intervals. The cAMPdependent protein kinase catalytic subunit was omitted from the control (C) sample. Following incubation, the sample was subjected to SDS/polyacrylamide gel electrophoresis, electrophoretic transfer to nitrocellulose paper, autoradiography, and immunoblotting. A portion of an autoradiogram and the corresponding immunoblot are shown. The positions of the PtdSer synthase M, 23,000 and M, 30,000 subunits are indicated.

phorylated itself (21). The presence of this inhibitor during the incubation of pure PtdSer synthase with the catalytic subunit reversed the inhibitory effect of the protein kinase on PtdSer synthase activity (Fig. 6) and the phosphorylation of the PtdSer synthase Mr 23,000 (Fig. 7). The stoichiometry of the phosphorylation reaction raised the suggestion that there was only one phosphorylation site per PtdSer synthase molecule. To further investigate this possibility, 32P-labeled PtdSer synthase was treated with either chymotrypsin or trypsin and the resulting peptide fragments were separated by isoelectric focusing. Treatment with these proteases resulted in different 32P-labeled peptide fragments (Fig. 8). In both cases, only one labeled fragment was observed (Fig. 8). That these labeled fragments represented a single phosphorylated peptide was confirmed by SDS/polyacrylamide gel electrophoresis in the second dimension (data not shown). Phospho amino acid analysis of the 32P-labeled enzyme indicated that the single phosphorylation site on the PtdSer synthase molecule was at a serine residue (data not shown).

DISCUSSION PtdSer synthase plays a major role in the regulation of phospholipid biosynthesis in S. cerevisiae (2, 3, 5). In this report we provide evidence that PtdSer synthase activity is regulated by cAMP-dependent protein kinase. The phosphorylated form of the PtdSer synthase Mr 23,000 subunit was immunoprecipitated from exponential-phase wild-type cells,

0

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.

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whereas the dephosphorylated form of the enzyme was immunoprecipitated from stationary-phase wild-type cells. It is known that cAMP-dependent protein kinase is active during cell cycle initiation and vegetative growth and is inactivated during the stationary (or G1) phase of growth (41, 43, 44). Furthermore, PtdSer synthase activity was reduced in the bcyl mutant (which has high cAMP-dependent protein kinase activity), whereas PtdSer synthase activity was elevated in the cyrl mutant (which has low cAMP-dependent protein kinase activity). The reduced PtdSer synthase activity in the bcyI mutant correlated with elevated levels of immunoprecipitated phosphorylated form of the PtdSer synthase Mr 23,000 subunit. The elevated PtdSer synthase activity in the cyri mutant correlated with reduced levels of immunoprecipitated phosphorylated form of the enzyme. The results of the phosphorylation experiments in vivo suggested that PtdSer synthase would be a substrate for cAMP-dependent protein kinase in vitro. Indeed, the purified PtdSer synthase Mr 23,000 subunit was phosphorylated by the cAMP-dependent protein kinase catalytic subunit. Phosphorylation of the enzyme resulted in a 60-70% reduction in PtdSer synthase activity. The specific cAMP-dependent protein kinase inhibitor prevented the phosphorylation of PtdSer synthase and the inhibition of its activity by the catalytic subunit. Initial attempts to activate PtdSer synthase by dephosphorylation of the phosphorylated form of the enzyme with alkaline phosphatase were unsuccessful. PtdSer synthase activity was inhibited by zinc, the cofactor required for alkaline phosphatase activity.

c o

0

___

m

FIG. 7. Effect of protein kinase inhibitor on the phosphorylation of the PtdSer synthase M, 23,000 subunit by cAMP-dependent protein kinase. Pure PtdSer synthase (5 Aug) was incubated under phosphorylation conditions with cAMP-dependent protein kinase catalytic subunit (10 pmol/min) and [y-32P]ATP with the indicated concentrations of the synthetic protein kinase inhibitor. Following incubation, the sample was subjected to SDS/polyacrylamide gel electrophoresis, electrophoretic transfer to nitrocellulose paper, autoradiography, and immunoblotting. A portion of an autoradiogram and the corresponding immunoblot are shown. The positions of the PtdSer synthase Mr 23,000 and Mr 30,000 subunits are indicated.

0

~= u

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7965

Proc. Natl. Acad. Sci. USA 85 (1988)


E

vue 4)

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1

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0.

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FIG. 6. Effect of protein kinase inhibitor on the inhibition of PtdSer synthase activity by cAMP-dependent protein kinase. Pure PtdSer synthase (0.05 nmol/min) was preincubated under phosphorylation conditions with cAMP-dependent protein kinase catalytic subunit (10 pmol/min) with the indicated concentrations of the synthetic protein kinase inhibitor. Following incubation, PtdSer synthase activity was measured.

no0

IA

20 10 Fraction

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30

FIG. 8. Peptide fragment analysis of phosphorylated PtdSer synthase. Pure PtdSer synthase was phosphorylated by cAMPdependent protein kinase and treated with trypsin (o) or chymotrypsin (e), and the resulting peptide fragments were analyzed by isoelectrofocusing.

7966


The in vitro experiments with pure PtdSer synthase and the catalytic subunit indicated that not more than 1 mol of phosphate was incorporated per mol of PtdSer synthase Mr 23,000 subunit. The analysis of peptides derived from protease-treated phosphorylated PtdSer synthase showed only one labeled peptide. Taken together, these results raised the suggestion that there is only one phosphorylation site per PtdSer synthase molecule. Phospho amino acid analysis of the 32P-labeled enzyme indicated that this site was at a serine residue. It is likely, therefore, that PtdSer synthase is phosphorylated at ger, which is contained within the se46 quence Arg-Arg-Ala-Ser-Ser of the CHOI gene product (13, 14) and corresponds to a cAMP-dependent protein kinase recognition sequence (35). Sequencing of proteolytic fragments from the labeled protein will be necessary to confirm this. The PtdSer synthase Mr 30,000 subunit, which appears to be an inactive precursor ofthe Mr 23,000 subunit (13), was not phosphorylated either in vivo or in vitro by cAMP-dependent protein kinase under the conditions of our experiments. The processing of the Mr 30,000 precursor appears to result in an activation of the enzyme and, at the same time, a rendering of the active enzyme susceptible to regulation by phosphorylation. The mechanism of this effect is not known. Future studies must be directed toward gaining an understanding of the ways in which PtdSer synthase regulation by cAMP-dependent protein kinase may interact with other regulatory mechanisms, such as modulation of PtdSer synthase activity by inositol (9) and phospholipids (19). We are grateful to Kunihiro Matsumoto for providing us the bcyl and cyri mutant strains. This work was supported by Public Health Service Grant GM-28140 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund. This is New Jersey Agricultural Experiment Station Publ. D-10531-3-88. 1. Henry, S. A. (1982) in The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, eds. Strathern, J. N., Jones, E. W. & Broach, J. R. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 101-158. 2. Letts, V. A., Klig, L. S., Bae-Lee, M., Carman, G. M. & Henry, S. A. (1983) Proc. Natl. Acad. Sci. USA 80,7279-7283. 3. Letts, V. A. & Henry, S. A. (1985) J. Bacteriol. 163, 560-567. 4. Atkinson, K. D., Jensen, B.,' Kolat, A. I., Storm, E. M., Henry, S. A. & Fogel, S. (1980) J. Bacteriol. 141, 558-564. 5. Poole, M. A., Homann, M. J., Bae-Lee, M. & Carman, G. M. (1986) J'. Bacteriol. 168, 668-672. 6. Kanfer, J. N. & Kennedy, E. P. (1964) J. Biol. Chem. 239,

1720-1726.

7. Bae-Lee, M. & Carman, G. M. (1984) J. Biol. Chem. 259, 10857-10862. 8. Raetz, C. R. H., Carman, G. M., Dowhan, W., Jiang, R.-T., Waszkuc, W., Loffredo, W. & Tsai, M.-D. (1987) Biochemistry 26, 4022-4027. 9. Kelley,' M. J., Bailis, A. M., Henry, S. A. & Carman, G. M. (1988) J. Biol. Chem., in press. 10. Cobon, G. S., Crowfoot, P. D. & Linnane, A. W. (1974) Biochem. J. 144, 265-275.

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