PhosphorylationofPhosphatidatePhosphataseRegulatesIts ... Files/Choi-JBC-2011-1486-98.pdf · the anemia and inflammatory disorders associated with the Majeed syndrome (15–18). PAP

Phosphorylation of Phosphatidate Phosphatase Regulates ItsMembrane Association and Physiological Functions inSaccharomyces cerevisiaeIDENTIFICATION OF SER602, THR723, AND SER744 AS THE SITES PHOSPHORYLATED BYCDC28 (CDK1)-ENCODED CYCLIN-DEPENDENT KINASE*

Received for publication, June 17, 2010, and in revised form, November 15, 2010 Published, JBC Papers in Press, November 16, 2010, DOI 10.1074/jbc.M110.155598

Hyeon-Son Choi‡, Wen-Min Su‡, Jeanelle M. Morgan‡, Gil-Soo Han‡, Zhi Xu‡, Eleftherios Karanasios§,Symeon Siniossoglou§, and George M. Carman‡1

From the ‡Department of Food Science and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, New Jersey08901 and the §Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/Medical Research CouncilBuilding, Hills Road, Cambridge CB2 0XY, United Kingdom

The Saccharomyces cerevisiae PAH1-encoded phosphatidatephosphatase (PAP) catalyzes the penultimate step in the synthesisof triacylglycerol and plays a role in the transcriptional regulationof phospholipid synthesis genes. PAP is phosphorylated atmulti-ple Ser and Thr residues and is dephosphorylated for in vivo func-tion by the Nem1p-Spo7p protein phosphatase complex localizedin the nuclear/endoplasmic reticulummembrane. In this work,we characterized seven previously identified phosphorylationsites of PAP that are within the Ser/Thr-Promotif.When ex-pressed on a low copy plasmid, wild type PAP could not comple-ment the pah1� mutant in the absence of the Nem1p-Spo7pcomplex. However, phosphorylation-deficient PAP (PAP-7A)containing alanine substitutions for the seven phosphorylationsites bypassed the requirement of the phosphatase complex andcomplemented the pah1� nem1� mutant phenotypes, such astemperature sensitivity, nuclear/endoplasmic reticulummem-brane expansion, decreased triacylglycerol synthesis, and dere-pression of INO1 expression. Subcellular fractionation coupledwith immunoblot analysis showed that PAP-7Awas highly en-riched in themembrane fraction. In fluorescence spectroscopyanalysis, the PAP-7A showed tighter association with phospho-lipid vesicles than wild type PAP. Using site-directedmutagenesisof PAP, we identified Ser602, Thr723, and Ser744, which belong tothe seven phosphorylation sites, as the sites phosphorylated bytheCDC28 (CDK1)-encoded cyclin-dependent kinase. Comparedwith the dephosphorylationmimic of the seven phosphorylationsites, alanine substitution for Ser602, Thr723, and/or Ser744 had apartial effect on circumventing the requirement for the Nem1p-Spo7p complex.

In the yeast Saccharomyces cerevisiae, the PAH1-encodedphosphatidate phosphatase (PAP)2,3 catalyzes the dephosphor-

ylation of PA, yielding DAG and Pi (1, 2). This reaction isdependent on Mg2� ions and is based on a DXDX(T/V) cata-lytic motif within a haloacid dehalogenase-like domain in theenzyme (2–4). PAP is associated with the cytosolic and mem-brane fractions of the cell, and the association with the mem-brane is peripheral in nature (2). Chromatin immunoprecipi-tation analysis indicates that PAP is also localized in thenucleus (5). The DAG generated in the PAP reaction is usedfor the synthesis of TAG (2) and for the synthesis of phos-phatidylethanolamine and phosphatidylcholine via the CDP-ethanolamine and CDP-choline branches, respectively, of theKennedy pathway (4, 6). The enzyme also plays a major rolein controlling the cellular concentration of its substrate PA(2), the precursor of phospholipids that are synthesized viathe CDP-DAG pathway (6–8). In addition, the substrate PAplays a signaling role in the transcriptional regulation of phos-pholipid synthesis genes (9). In fact, mutants defective inPAH1-encoded PAP activity exhibit a �90% reduction inTAG content, a derepression of phospholipid synthesis genes,and an expansion of the nuclear/ER membrane (3, 5). Thus,the regulation of PAP activity governs the synthesis of TAG,the pathways by which phospholipids are synthesized, PAsignaling, and the growth of the nuclear/ER membrane (6).The importance of PAP in lipid metabolism and cell physi-

ology is further emphasized by the fact that the overexpres-sion of Lpin1-encoded PAP (also known as lipin 1) in miceleads to obesity and insulin sensitivity, whereas loss of lipin 1prevents normal adipose tissue development, resulting in lip-odystrophy and insulin resistance (10, 11). Moreover, micelacking PAP activity exhibit peripheral neuropathy (12–14)caused by degradation of myelin through the MEK/ERK sig-naling pathway that is activated by elevated levels of PA (14).

* This work was supported, in whole or in part, by National Institutes ofHealth Grant GM-50679 (to G. M. C.). This work was also supported byMedical Research Council Grant G0701446 (to S. S.).

1 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); E-mail: [email protected].

2 The abbreviations used are: PAP, phosphatidate phosphatase; PA, phos-phatidate; DAG, diacylglycerol; TAG, triacylglycerol; CDK, cyclin-depen-

dent kinase; 7A, alanine mutations of Ser110, Ser114, Ser168, Ser602, Thr723,Ser744, and Ser748; ER, endoplasmic reticulum.

3 PAP is also referred to as Pah1p, protein product of PAH1. PAP is distin-guished in catalytic and physiological functions from the S. cerevisiaeDPP1- and LPP1-encoded lipid phosphate phosphatase enzymes thatdephosphorylate a broad spectrum of substrates (e.g. PA, lyso-PA, DAGpyrophosphate, sphingoid base phosphates, and isoprenoid phos-phates) by a distinct catalytic mechanism that does not require divalentcations (4, 67, 68).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 2, pp. 1486 –1498, January 14, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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In humans, mutations in LPIN1-encoded PAP are associatedwith metabolic syndrome, type 2 diabetes, and recurrentacute myoglobinuria in children, whereas mutations inLPIN2-encoded PAP (also known as lipin 2) are the basis forthe anemia and inflammatory disorders associated with theMajeed syndrome (15–18).PAP is subject to the covalent modification of phosphoryla-

tion (19, 20). The yeast enzyme has been identified in pro-teome-wide in vitro phosphorylation analyses to be a targetfor multiple protein kinases, including those encoded byCDC28 (CDK1) (21), PHO85 (22, 23), and DBF2 (24). Massspectrometry analysis of purified PAP, in combination withimmunoblot analysis using anti-MPM2 antibodies that recog-nize phosphorylated serine and threonine residues in a Ser/Thr-Pro motif, has identified 16 sites of phosphorylation,seven of which (Ser110, Ser114, Ser168, Ser602, Thr723, Ser744,and Ser748) are contained within the minimal Ser/Thr-Promotif that is a target for protein kinases regulated during thecell cycle (25). Moreover, data indicate that PAP is phosphor-ylated by CDC28 (CDK1)-encoded CDK in a cell cycle-depen-dent manner (5). The nine remaining sites are putative targetsfor protein kinases, such as protein kinases A and C, caseinkinases I and II, and MAPK, indicating that the regulation ofPAP by phosphorylation is complex.The simultaneous mutation of the seven sites within the

Ser/Thr-Pro motif to a nonphosphorylatable alanine residue(also known as 7A mutations) results in a 1.8-fold increase inPAP activity (25). In addition, the overexpression of thePAP-7A mutant enzyme causes inositol auxotrophy by allevi-ating the PA-mediated inhibition of Opi1p repressor activityon INO1, the gene that encodes inositol-3-phosphate syn-thase (9, 25). Moreover, cells that lack the Nem1p-Spo7p pro-tein phosphatase complex, which is responsible for the de-phosphorylation of PAP, show phenotypes characteristic ofcells lacking PAP activity (5, 26). These observations supportthe conclusion that phosphorylation of the seven sites nega-tively regulates PAP function in vivo (25). Due to the im-portance of these phosphorylations in controlling PAP func-tion, the major aims of this work were 1) to furthercharacterize the physiological consequences of the seven sitesof phosphorylation and 2) to establish that CDC28 (CDK1)-encoded CDK is in fact the relevant protein kinase responsi-ble for these phosphorylations. We showed that lack of phos-phorylation at the seven sites caused a great increase in theamount of PAP associated with membranes and that this cor-related with a significant increase in TAG synthesis in cells

lacking the Nem1p-Spo7p protein phosphatase complex. Wealso showed that among the seven sites of phosphorylation,only Ser602, Thr723, and Ser744 were targets of CDC28(CDK1)-encoded CDK. Moreover, mutations of these sites,individually and in combination, partially mimicked the phys-iological consequences of the 7A mutations.

EXPERIMENTAL PROCEDURES

Materials—All chemicals were reagent grade or better.Growth medium supplies were obtained from Difco. NewEngland Biolabs was the source of modifying enzymes, re-combinant Vent DNA polymerase, restriction endonucleases,and recombinant human CDK1-cyclin B complex. The DNAgel extraction kit, plasmid DNA purification kit, and nickel-nitrilotriacetic acid-agarose resin were purchased from Qia-gen. Sigma-Aldrich was the source of aprotinin, benzamidine,bovine serum albumin, leupeptin, pepstatin, phenylmethylsul-fonyl fluoride, phosphoamino acids, L-1-tosylamido-2-phenyl-ethyl chloromethyl ketone-trypsin, the CDK peptide sub-strate, and Triton X-100. PCR primers were preparedcommercially by Genosys Biotechnologies. Tobacco etch vi-rus protease was purchased from Invitrogen. TheQuikChange site-directed mutagenesis kit was purchasedfrom Stratagene. Carrier DNA for yeast transformation wasfrom Clontech. GE Healthcare supplied IgG-Sepharose, poly-vinylidene difluoride paper, and the enhanced chemifluores-cence Western blotting detection kit. DNA size ladders, elec-trophoresis reagents, immunochemical reagents, molecularmass protein standards, and protein assay reagents were fromBio-Rad. Lipids were from Avanti Polar Lipids, and thin layerchromatography plates (cellulose and silica gel 60) were fromEM Science. Scintillation counting supplies and acrylamidesolutions were from National Diagnostics, and radiochemicalswere PerkinElmer Life Sciences. Alkaline phosphatase-conju-gated goat anti-rabbit IgG antibodies were from Thermo Sci-entific. Mouse anti-phosphoglycerate kinase antibodies andalkaline phosphatase-conjugated goat anti-mouse IgG anti-bodies were from Invitrogen and Pierce, respectively.Strains and Growth Conditions—The strains used in this

work are listed in Table 1. The pah1� nem1� mutant wasconstructed by transforming a pah1�::TRP1 fragment (5) intoa nem1�::HIS3mutant (26). The nem1�::HIS3 spo7�::HIS3HEH2-CHERRY::TRP1mutant was constructed by integrationof plasmid YIplac204-HEH2-CHERRY at TRP1 in thenem1�::HIS3 spo7�::HIS3mutant (26). The homologous re-combinations were verified by PCR analysis. Plasmids were

TABLE 1Strains used in this work

Strain Relevant characteristics Source/Reference

E. coliDH5� F� �80dlacZ�M15 �(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk� mk

�)phoA supE44 l�thi-1 gyrA96 relA1

Ref. 29

BL21(DE3)pLysS F� ompT hsdSB (rB�mB�) gal dcm (DE3) pLysS Novagen

S. cerevisiaeRS453 MATa ade2-1 his3-11,15 leu2–3,112 trp1-1 ura3-52 Ref. 69SS1026 pah1�::TRP1 derivative of RS453 Ref. 5SS1132 pah1�::TRP1 nem1�::HIS3 derivative of RS453 This studySS1010 nem1�::HIS3 spo7�::HIS3 derivative of RS453 Ref. 26SS1594 HEH2-CHERRY::TRP1 derivative of RS453 Ref. 33SS1571 HEH2-CHERRY::TRP1 derivative of SS1010 This study

Phosphorylation of Phosphatidate Phosphatase

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propagated in Escherichia coli strain DH5�. E. coli cells weregrown at 37 °C in 1% tryptone, 0.5% yeast extract, 1% NaCl,pH 7 (LB medium). Ampicillin (100 �g/ml) was added to se-lect for cells carrying plasmids. PAP was expressed in E. coliBL21(DE3)pLysS cells bearing wild type and mutant PAH1derivatives of plasmid pET-15b as described by Han et al.(27). Yeast cultures were generally grown in standardsynthetic complete medium. Appropriate amino acids wereomitted from the synthetic growth medium to select for cellscarrying specific plasmids. Cells were also cultured in inositol-lacking synthetic medium (28) containing 2% glucose or 2%galactose. Inositol (75 �M) and choline (1 mM) were added tothis growth medium where indicated. Cell numbers in liquidcultures were determined spectrophotometrically at an ab-sorbance of 600 nm. The growth medium was supplementedwith agar (2% for yeast or 1.5% for E. coli) for growth onplates. For the heterologous expression of wild type and mu-tant forms of PAH1-encoded PAP enzymes, E. coliBL21(DE3)pLysS cells bearing pET-15b-based plasmids weregrown to A600 � 0.5 at room temperature in 500 ml of LBmedium containing ampicillin (100 �g/ml) and chloramphen-icol (34 �g/ml). Cultures were incubated for 1 h with 1 mM

isopropyl-�-D-thiogalactoside to induce the expression ofHis6-tagged PAP enzymes.DNAManipulations, PCR Amplification of DNA, Site-di-

rected Mutagenesis, and DNA Sequencing—Standard proto-cols were used to isolate genomic and plasmid DNA, to digestand ligate DNA, and to amplify DNA by PCR (29, 30). DNA

sequencing was performed by GENEWIZ, Inc. (South Plain-field, NJ) using Applied Biosystems BigDye version 3.1. Thereactions were then run on an Applied Biosystems 3730xlDNA Analyzer. Site-specific mutations were generated withthe QuikChange site-directed mutagenesis kit using appropri-ate primers and plasmid templates, and mutant constructswere confirmed by DNA sequencing. Transformation ofE. coli (29) and yeast (31, 32) with plasmids was performed asdescribed previously.Construction of Plasmids—Plasmids used in this study are

listed in Table 2. The plasmid pGH313 directs the overex-pression of His6-tagged yeast PAH1-encoded PAP in E. coli(2). pGH315 was constructed by insertion of the PAH1 DNAfragment (2) into the low copy plasmid pRS415 and was usedfor the expression of PAP in yeast. The high copy plasmidYEplac181-GAL1/10-PAH1 directs the overexpression of PAPin yeast (25). Plasmids containing the PAH1S110A, PAH1S114A,PAH1S168A, PAH1S602A, PAH1T723A, PAH1S744A, andPAH1S748A were constructed by site-directed mutagenesis usingplasmids pGH313, pGH315, and YEplac181-GAL1/10-PAH1 astemplates where indicated in Table 2. PAH1S602A/T723A was con-structed with the primers for the PAH1S723A mutation usingplasmids pJM106, pHC201, and YEplac181-GAL1/10-PAH1-S602A as the templates where indicated in Table 2.PAH1S602A/T723A/S744A was constructed with the primers forthe PAH1S744A mutation using plasmids pHC203 andYEplac181-GAL1/10-PAH1-S602A/T723A as the templateswhere indicated in Table 2. Plasmid pHC204 was constructed

TABLE 2Plasmids used in this work

Plasmid Relevant characteristics Source/Reference

pET-15b E. coli expression vector with N-terminal His6 tag fusion NovagenpGH313 PAH1 derivative of pET-15b Ref. 2pHC208 PAH1S110A derivative of pGH313 This studypHC209 PAH1S114A derivative of pGH313 This studypHC210 PAH1S168A derivative of pGH313 This studypJM106 PAH1S602A derivative of pGH313 This studypWS15 PAH1T723A derivative of pGH313 This studypHC211 PAH1S744A derivative of pGH313 This studypHC212 PAH1S748A derivative of pGH313 This studypWS16 PAH1S602A/T723A derivative of pGH313 This studypRS415 Low copy E. coli/yeast shuttle vector with URA3 Ref. 70pGH315 PAH1 derivative of pRS415 This studypHC201 PAH1S602A derivative of pGH315 This studypHC202 PAH1T723A derivative of pGH315 This studypHC213 PAH1S744A derivative of pGH315 This studypHC203 PAH1S602A/T723A derivative of pGH315 This studypHC214 PAH1S602A/T723A/S744A derivative of pGH315 This studyYCplac111 Low copy number E. coli/yeast shuttle vector with LEU2 Ref. 71YCplac111-CDC28-PtA Protein A-tagged CDC28 derivative of YCplac111 This studyYCplac111-PAH1-7Aa PAH1S110A/S114A/S168A/S602A/T723A/S744A/S748A derivative of YCplac111 Ref. 25YCplac111-PAH1-GFP PAH1-GFP derivative of YCplac111 Ref. 33YCplac111-PAH1–7A-GFP PAH1S110A/S114A/S168A/S602A/T723A/S744A/S748A derivative of YCplac111-PAH1-GFP Ref. 33YCplac111-PAH1-S602A-GFP PAH1S602A derivative of YCplac111-PAH1-GFP This studyYCplac111-PAH1-T723A-GFP PAH1T723A derivative of YCplac111-PAH1-GFP This studyYCplac111-PAH1-S744A-GFP PAH1S744A derivative of YCplac111-PAH1-GFP This studyYCplac111-PAH1-S602A/T723A-GFP PAH1S602A/T723A derivative of YCplac111-PAH1-GFP This studyYCplac111-PAH1-S602A/T723A/S744A-GFP PAH1S602A/T723A/S744A derivative of YCplac111-PAH1-GFP This studypHC204 PAH1S110A/S114A/S168A/S602A/T723A/S744A/S748A derivative of pGH315 This studyYEplac181 High copy number E. coli/yeast shuttle vector with LEU2 Ref. 71YEplac181-GAL1/10-PAH1 PAH1 under control of the GAL1/10 promoter in YEplac181 Ref. 25YEplac181-GAL1/10-PAH1-S602A PAH1S602A derivative of YEplac181-GAL1/10-PAH1 This studyYEplac181-GAL1/10-PAH1-T723A PAH1T723A derivative of YEplac181-GAL1/10-PAH1 This studyYEplac181-GAL1/10-PAH1-S744A PAH1S744A derivative of YEplac181-GAL1/10-PAH1 This studyYEplac181-GAL1/10-PAH1-S602A/T723A PAH1S602A/T723A derivative of YEplac181-GAL1/10-PAH1 This studyYEplac181-GAL1/10-PAH1-S602A/T723A/S744A PAH1S602A/T723A/S744A derivative of YEplac181-GAL1/10-PAH1 This studyYEplac181-GAL1/10-PAH1-7Aa PAH1S110A/S114A/S168A/S602A/T723A/S744A/S748A derivative of YEplac181-GAL1/10-PAH1 Ref. 25YIplac204-HEH2-CHERRY HEH2-CHERRY under control of the NOP1 promoter into integrative/TRP1 vector Ref. 33

aPAH1-7A, which was originally called PAH1-7P (25), encodes PAP, where seven phosphorylation sites, each within the Ser/Thr-Pro motif, are changed to alanine.



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by replacing the wild type PAH1 DNA of pGH315 with thePAH1S110A/S114A/S168A/S602A/T723A/S744A/S748A fragment ofYCplac111-PAH1-7A (25). PAH1-7A, which was originallycalled PAH1-7P (25), refers to a PAH1 allele in which theseven sites were mutated to alanine. The YCplac111-PAH1-GFP and YCplac111-PAH1–7A-GFP constructs express thewild type and the S110A/S114A/S168A/S602A/T723A/S744A/S748A septuple phosphorylation-deficient mutant ofPAH1, respectively (33). The YCplac111-PAH1-S602A-GFP,YCplac111-PAH1-T723A-GFP, YCplac111-PAH1-S744A-GFP, YCplac111-PAH1-S602A/T723A-GFP, and YCplac111-PAH1-S602A/T723A/S744A-GFP were constructed by sub-cloning the DNA fragments of the individual mutations orcombinations of them from the YCplac111-PAH1–7A-GFPinto the YCplac111-PAH1-GFP. The CDC28-PtA fusion wasconstructed by inserting the tobacco etch virus cleavage site-Protein A fragment to the C terminus of CDC28 at a uniqueBamHI site prior to the stop codon. Expression was driven bythe CDC28 promoter and terminator. The fusion gene wascloned into the low copy plasmid YCplac111. All plasmidswere sequenced to confirm the mutation in the PAH1 codingregion.Preparation of Cell Extracts and Subcellular Fractions and

Purification of Enzymes—All steps were performed at 4 °C.Cell extracts were prepared by disruption of yeast cells withglass beads (0.5-mm diameter) using a BioSpec ProductsMini-BeadBeater-16 (34). The cell disruption buffer con-tained 50 mM Tris-HCl (pH 7.5), 0.3 M sucrose, 10 mM 2-mer-captoethanol, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM

benzamidine, 5 �g/ml aprotinin, 5 �g/ml leupeptin, and 5�g/ml pepstatin. The cytosolic (supernatant) and total mem-brane (pellet) fractions were prepared by centrifugation at100,000 � g for 1 h (34). The membrane pellets were sus-pended in the disruption buffer to the same volume of thecytosolic fraction. Protein concentration was estimated bythe method of Bradford (35) using bovine serum albumin asthe standard. His6-tagged wild type and mutant PAP enzymesexpressed in E. coli were purified by affinity chromatographyusing nickel-nitrilotriacetic acid-agarose as described by Hanet al. (2). Protein A-tagged wild type and 7A mutant forms ofPAP expressed in S. cerevisiae were purified by affinity chro-matography using IgG-Sepharose as described previously(25). The CDK-cyclin B complex was purified from S. cerevi-siae that expressed protein A-tagged CDK by affinity chroma-tography using IgG-Sepharose (26). Elution of the untaggedPAP enzymes and CDK from IgG-Sepharose columns wasachieved by treatment with tobacco etch virus protease (36).SDS-PAGE analyses showed that the purified preparations ofthe wild type and mutant PAP enzymes and CDK-cyclin Bcomplex were nearly homogeneous.Phosphorylation Reactions—Phosphorylation reactions

were routinely performed in triplicate for 5–10 min at 30 °Cin a total volume of 20 �l. The standard reaction mixture con-tained 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 2 mM dithio-threitol, 20 �M [�-32P]ATP (2,400 cpm/pmol), 50 �g/ml PAP,and yeast (1 �g) or recombinant human (20 ng) CDK. At theend of the phosphorylation reactions, samples were treatedwith 4� Laemmli sample buffer (37), subjected to SDS-PAGE,

and transferred to polyvinylidene difluoride membranes.Phosphorimaging was used to visualize phosphorylated en-zyme, and the extent of phosphorylation was quantified withImageQuant software. For some experiments, the CDK reac-tions were terminated by spotting the reaction mixtures ontoP81 phosphocellulose paper. The papers were washed threetimes with 75 mM phosphoric acid and then subjected to scin-tillation counting. A unit of CDK activity was defined as theamount of enzyme that catalyzed the formation of 1 nmol ofphosphorylated product/min.SDS-PAGE and Immunoblot Analysis—SDS-PAGE (37) and

immunoblotting (38) with polyvinylidene difluoride mem-brane were performed by standard protocols. Anti-PAP anti-bodies were prepared in rabbits against the C-terminal por-tion (residues 778–794) of the protein at BioSynthesis, Inc.Rabbit anti-PAP antibodies, rabbit anti-phosphatidylserinesynthase antibodies (39), and mouse anti-phosphoglyceratekinase antibodies were used at a concentration of 2 �g/ml.Alkaline phosphatase-conjugated goat anti-rabbit IgG anti-bodies and goat anti-mouse IgG antibodies were used at adilution of 1:5,000. Immune complexes were detected usingthe enhanced chemifluorescence Western blotting detectionkit. Fluorimaging was used to acquire images from immuno-blots, and the relative densities of the images were analyzedusing ImageQuant software. Signals were in the linear rangeof detectability.Phosphoamino Acid and Phosphopeptide Mapping

Analyses—For phosphoamino acid analysis, 32P-labeled PAPon polyvinylidene difluoride membrane was subjected to acidhydrolysis with 6 N HCl, followed by two-dimensional electro-phoresis on cellulose thin-layer chromatography plates (40,41). Phosphorimaging was used to visualize 32P-labeled phos-phoamino acids, whereas ninhydrin spraying was used to vi-sualize standard phosphoamino acids (41). For phosphopep-tide mapping analysis, 32P-labeled PAP on polyvinylidenedifluoride membrane was subjected to proteolytic digestionwith L-1-tosylamido-2-phenylethyl chloromethyl ketone-tryp-sin, followed by electrophoresis and TLC using cellulose thin-layer chromatography plates (42). Radioactive phosphopep-tides were visualized by phosphorimaging analysis.Preparation of 32P-labeled PA and Measurement of PAP

Activity—[32P]PA was synthesized enzymatically from DAGand [�-32P]ATP with E. coli DAG kinase, and the radioactiveproduct was purified by thin-layer chromatography (34). PAPactivity was measured by following the release of water-solu-ble 32Pi from chloroform-soluble [32P]PA (10,000 cpm/nmol)(34). The reaction mixture contained 50 mM Tris-HCl buffer(pH 7.5), 1 mM MgCl2, 0.2 mM PA, 2 mM Triton X-100, andenzyme protein in a total volume of 0.1 ml. All enzyme assayswere conducted in triplicate at 30 °C. The average S.D. valueof the assays was �5%. The reactions were linear with timeand protein concentration. A unit of PAP activity was definedas the amount of enzyme that catalyzed the formation of 1nmol of product/min.Labeling and Analysis of Lipids—Steady-state labeling of

lipids with [2-14C]acetate was performed as described pre-viously (43). Lipids were extracted from labeled cells by themethod of Bligh and Dyer (44). Lipids were analyzed by one-



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dimensional thin-layer chromatography on silica gel platesusing the solvent system hexane/ethyl ether/acetic acid (40:10:1) (45). The identity of the labeled TAG and total phospho-lipids on thin-layer chromatography plates was confirmed bycomparison with standards after exposure to iodine vapor.Radiolabeled lipids were visualized by phosphorimaging anal-ysis, and their relative quantities were analyzed using Image-Quant software.Preparation of Unilamellar Phospholipid Vesicles—Unila-

mellar phospholipid vesicles were prepared with dioleoyl-phosphatidylcholine and dioleoyl-PA at a molar ratio of 10:1by the lipid extrusion method of MacDonald et al. (46). Chlo-roform was evaporated from phospholipids under nitrogen toform a thin film. The phospholipids were then resuspended in20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and1 mM EDTA. After five cycles of freezing and thawing, thephospholipid suspension was extruded 11 times through apolycarbonate filter (100-nm diameter).Light Microscopy—The expression of the HEH2-CHERRY

fusion gene in wild type and the nem1� spo7� mutant wasused to visualize nuclear morphology. Images were acquiredon an epifluorescence microscope consisting of an invertedmicroscope (Zeiss Axiovert 200M), a camera (HamamatsuOrca ER cooled CCD type), and a 100� plan-apochromatic1.4 numerical aperture objective lens (Carl Zeiss Ltd.). Themicroscope was controlled by the Improvision OpenLab soft-ware (version 5). mCherry was recorded with Carl Zeiss filterset 00 (488000-0000-000) (excitation BP 530–585 nm, emis-sion LP 615). The brightness and contrast of the images wereadjusted using Adobe Photoshop software.Fluorescence Measurements—Fluorescence measurements

were carried out in a FluoroMax-3 fluorimeter (HORIBA Jo-bin Yvon Inc.) at room temperature in 200 �l of 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 105 nM PAP,and the indicated concentrations of phospholipid vesicles.The excitation wavelength was 280 nm, and the emissionspectra were collected from 300–450 nm after a 10-min incu-bation period.Analyses of Data—Kinetic data were analyzed according to

the Michaelis-Menten and Hill equations using the EnzymeKinetics module of SigmaPlot software. Dissociation con-stants for the interaction of PAP with phospholipid vesicleswere determined by the method of Lear and DeGrado (47).Statistical analyses were performed with SigmaPlot software.The p values of �0.05 were taken as a significant difference.

RESULTS

Phosphorylation of PAP by CDK—Data indicate that CDC28(CDK1)-encoded CDK might be the protein kinase responsi-ble for the phosphorylation of the seven sites within the Ser/Thr-Pro motif of PAP previously shown to be phosphorylatedin vivo (5, 25). To examine the phosphorylation of PAP byCDK in vitro, we utilized a preparation of PAP that was heter-ologously expressed in E. coli (2). In this manner, our phos-phorylation studies were conducted with a pristine substratethat was free from the endogenous phosphorylations that oc-cur when PAP is expressed in S. cerevisiae (25). Purified PAPwas incubated with yeast CDK in the presence of [�-32P]ATP,

and its phosphorylation was monitored by following the in-corporation of the radioactive �-phosphate into the enzyme.Phosphorimaging analysis of reaction products resolved bySDS-PAGE showed that purified PAP was a substrate foryeast CDK (Fig. 1A). In addition, PAP was also phosphory-lated by recombinant human CDK1 (CDC2)-encoded proteinkinase (Fig. 1A), which is functionally homologous to yeastCDC28 (CDK1)-encoded protein kinase (48, 49). Phos-phopeptide mapping analysis showed similar patterns, indi-cating that the yeast and human CDK enzymes phosphory-lated PAP on the same sites (Fig. 1B). Phosphoamino acidanalysis of the 32P-labeled protein derived from phosphoryla-tion by human CDK showed that PAP was phosphorylated atboth serine and threonine residues (Fig. 1C). Due to the factthat human CDK had a 10-fold higher specific activity (as de-termined with the CDK peptide substrate PKTPKKAKKL)and was more stable to storage when compared with the yeastCDK preparation, we utilized human CDK for the remainderof this work.Using PAP as substrate, CDK activity was dependent on the

time of the reaction (Fig. 2A) and the concentrations of thekinase (Fig. 2B), ATP (Fig. 2C), and PAP (Fig. 2D). The depen-dences of CDK activity on ATP and PAP followed saturationkinetics and positive cooperative kinetics, respectively. Analy-ses of the data according to the Michaelis-Menten and Hillequations showed that the Km values for ATP and PAP were

FIGURE 1. PAP is phosphorylated by yeast and human CDK enzymes.A, purified recombinant PAP (50 �g/ml) was phosphorylated with yeast (1�g) or human (20 ng) CDK and 20 �M [�-32P]ATP (2,400 cpm/pmol) for 10min, followed by SDS-PAGE, transfer to polyvinylidene difluoride mem-brane, and phosphorimaging analysis. B, polyvinylidene difluoride mem-branes containing 32P-labeled PAP that were phosphorylated with yeast orhuman CDK were treated with L-1-tosylamido-2-phenylethyl chloromethylketone-trypsin. The resulting peptides were separated on cellulose thinlayer plates by electrophoresis (from left to right) in the first dimension andby chromatography (from bottom to top) in the second dimension. The ma-jor (labeled 1 and 2) and minor (labeled 3) phosphopeptides were presentin the maps of PAP phosphorylated by both CDK enzymes. The radioactivespots labeled 3 in the map for the yeast CDK phosphorylation were observedwhen the image was overexposed (not shown). C, polyvinylidene difluoridemembrane containing 32P-labeled PAP phosphorylated with human CDK washydrolyzed with 6 N HCl for 90 min at 110 °C, and the hydrolysate was sepa-rated by two-dimensional electrophoresis. The positions of phosphorylatedPAP and the standard phosphoamino acids phosphoserine (P-Ser), phospho-threonine (P-Thr), and phosphotyrosine (P-Tyr) (dotted lines) are indicated. Thedata shown are representative of three independent experiments.



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5.8 and 0.21 �M, respectively, and that the Hill number forPAP was 2. These data indicated that PAP was a bona fidesubstrate for CDK.We examined the stoichiometry of the phosphorylation of

PAP by CDK. The enzyme was incubated with [�-32P]ATP(2,500 cpm/pmol) and recombinant human CDK for 60 min.Following the incubation, samples were subjected to SDS-PAGE followed by transfer to polyvinylidene difluoride mem-brane. The amount of phosphate incorporated into PAP wasdetermined by ImageQuant analysis. At the point of maxi-mum phosphate incorporation, CDK catalyzed the incorpora-tion of 0.8 mol of phosphate/mol of PAP. This stoichiometrywas low given the results of the peptide mapping experimentsthat indicated multiple sites of phosphorylation (Fig. 1B). Anexplanation for this result might be that the phosphorylationof one site inhibited the phosphorylation of another site (50)(see below).To examine the effect of CDK on PAP activity, the en-

zyme was preincubated with a constant amount of CDKand unlabeled ATP for various times. Following the phos-phorylations, the reaction mixtures were diluted 10-foldand used for the measurement of PAP activity. The time-dependent increase in PAP phosphorylation had little ef-fect on its enzyme activity (Fig. 3). Increasing the amounts

of CDK in this experiment did not affect the extent of PAPphosphorylation or PAP activity. These results indicatedthat PAP phosphorylation by CDK had essentially no effecton its enzyme activity.Identification of Ser602, Thr723, and Ser744 as the CDK Phos-

phorylation Sites in PAP—The seven sites with the Ser/Thr-Pro motif previously shown to be phosphorylated in vivo (25)are putative CDK sites (51), which are located at the N- andC-terminal portions of the protein (Fig. 4A). Individual Ser/Thr to alanine mutations of the seven sites were constructedby site-specific mutagenesis. Each of the phosphorylation sitemutant enzymes was expressed and purified from E. coli and

FIGURE 2. Characterization of CDK activity using PAP as a substrate.Human CDK activity was measured by following the incorporation of the�-phosphate of [�-32P]ATP (2,400 cpm/pmol) into purified recombinant PAPunder standard phosphorylation conditions except for the variation in time(A), CDK concentration (B), ATP concentration (C), and PAP concentration(D). The CDK reactions were terminated by spotting the mixtures onto P81phosphocellulose paper. The papers containing the phosphorylated PAPenzyme were washed three times with 75 mM phosphoric acid and thensubjected to scintillation counting. The values reported were the average ofthree experiments � S.D. (error bars). Some error bars are contained withinthe symbols.

FIGURE 3. Effect of CDK on PAP activity. Purified recombinant PAP wasphosphorylated with human CDK (20 ng) and 20 �M ATP for the indicatedtimes. Following the phosphorylation reactions, samples were diluted 10-fold, and PAP activity was measured by following the dephosphorylation of32P-labeled PA. The CDK enzyme was omitted from the control reactions.The values reported were the average of three experiments � S.D. (errorbars). Some error bars are contained within the symbols.

FIGURE 4. Phosphopeptide mapping analysis of PAP mutants. A, the dia-gram shows the positions of the NLIP domain, the haloacid dehalogenase(HAD)-like domain, and the serine (S) and threonine (T) residues within aSer/Thr-Pro motif that were previously identified as sites of phosphorylation(25). B, WT and the indicated phosphorylation site PAP mutant enzymeswere expressed and purified from E. coli. The recombinant PAP enzymeswere phosphorylated with human CDK (20 ng) and 20 �M [�-32P]ATP (2,400cpm/pmol) for 10 min. After phosphorylation, the samples were subjectedto SDS-PAGE and transferred to polyvinylidene difluoride membrane. The32P-labeled proteins were digested with L-1-tosylamido-2-phenylethyl chlo-romethyl ketone-trypsin. The resulting peptides were separated on cellulosethin layer plates by electrophoresis (from left to right) in the first dimension andby chromatography (from bottom to top) in the second dimension. The radioac-tive spots labeled 3 were observed when the images were overexposed (notshown). The positions of the phosphopeptides (labeled 1, 2, and 3) that wereabsent in the S602A, T723A, S744A, and S602A/T723A mutants (indicated bydotted lines) but were present in the wild type enzyme are indicated. The dataare representative of three independent experiments.



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used as a substrate for CDK. Of the seven PAP mutants, onlyS602A, T723A, and S744A showed defects in phosphorylationby CDK. As discussed above, the phosphopeptide map of wildtype PAP contained two major phosphopeptides (labeled 1and 2) and one minor phosphopeptide (labeled 3) (Figs. 1Band 4B). Phosphopeptides 1, 2, and 3 were missing in theS602A, T723A, and S744A mutant enzymes, respectively (Fig.4B). These results indicated that Ser602, Thr723, and Ser744were contained in phosphopeptides 1, 2, and 3, respectively.The phosphopeptides that were attributed to Ser744 weremore evident in the phosphopeptide map of the S602A/T723A double mutant, which lacked phosphopeptides 1 and 2(Fig. 4B). The extents of phosphorylation at Ser602, Thr723,and Ser744 differed, with Thr723 being the most phosphory-lated and Ser744 being the least phosphorylated. The S602Aand T723A mutations reduced the extent of phosphorylationby 50 and 70%, respectively, whereas the S744A mutationcaused only a small decrease in the extent of phosphorylation(Fig. 5). In addition, the phosphopeptide map of the wildtype enzyme showed that Thr723 was more heavily labeledwhen compared with Ser602 and that Ser744 was hardly la-beled when compared with the other two sites (Figs. 1Band 4B). These observations indicated that the phosphory-lations at Ser602 and Thr723 might inhibit the phosphoryla-tion at Ser744 and may provide an explanation for a stoichi-

ometry of less than the theoretical value of 3 (see above).The phosphopeptide maps of the S110A, S114A, S168A,and S748A mutant enzymes were indistinguishable fromthe map of wild type PAP (data not shown), indicating thatSer110, Ser114, Ser168, and Ser748 were not sites of phos-phorylation for CDK. Individually, these sites were not ex-amined further in this work.Expression of CDK Phosphorylation Site PAP Mutants in

S. cerevisiae—Ser/Thr to alanine mutations were constructedfor the CDK phosphorylation sites in PAP to examine thephysiological effects of phosphorylation in pah1� and pah1�nem1� mutants. The rationale for using the pah1� nem1�double mutant, which lacks the NEM1-encoded protein phos-phatase catalytic subunit (5), was to assess the dependence ofPAP function on the Nem1p-Spo7p complex. In addition, thisafforded examination of the phosphorylation site mutationsin a background that favored the phosphorylation of the othernon-mutated phosphorylation sites in the PAP protein. Anti-bodies directed against the C-terminal portion of PAP recog-nized the wild type and mutant forms of the enzyme whenexpressed in pah1� (Fig. 6A) and pah1� nem1� (Fig. 6B)cells. ImageQuant analysis of the immunoblots shown in Fig.6 indicated that there were no major differences in the rela-tive amounts of the S602A, T723A, and S744A mutant en-zymes when compared with the wild type control. However,

FIGURE 5. Effects of the S602A, T723A, and S744A mutations on thetime-dependent phosphorylation of PAP. A, WT and the S602A, T723A,and S744A mutant PAP enzymes were expressed and purified from E. coli.The recombinant PAP enzymes (50 �g/ml) were phosphorylated with hu-man CDK (20 ng) and 20 �M [�-32P]ATP (2,400 cpm/pmol) for the indicatedtimes. After the phosphorylation reactions, the samples were separated bySDS-PAGE; the polyacrylamide gel was dried and then subjected to phos-phorimaging analysis. B, the data shown in A were quantified with Image-Quant software. The extent of phosphorylation of the wild type and mutantenzymes at each time point was determined relative to the extent of phos-phorylation of the wild type enzyme at 8 min. The maximum amount ofphosphorylation for wild type PAP was set at 100%. The data are represen-tative of two independent experiments.

FIGURE 6. Expression of the phosphorylation-deficient PAP mutant en-zymes in S. cerevisiae. Cell extracts were prepared from pah1� (A) andpah1� nem1� (B) cells expressing the indicated WT and PAH1 mutant alleleson low copy plasmids. Samples (40 �g of protein) were subjected to immu-noblot analysis using anti-PAP and anti-phosphoglycerate kinase (PGK;loading control) antibodies. C, the relative amounts of PAP/phosphoglycer-ate kinase proteins from the cells were determined by ImageQuant analysisof the data. Representative immunoblots are shown in A and B, whereas thequantitation data shown in C are the average of three independent experi-ments � S.D. (error bars). The positions of the PAP and phosphoglyceratekinase proteins are indicated. 3A, S602A/T723A/S744A triple mutant.



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the relative amount of the 7A (50%), S602A/T723A (37%), andS602A/T723A/S744A (30%) mutant enzymes was reducedwhen compared with the wild type control (Fig. 6). Why thephosphorylation state of PAP appeared to govern its relativeabundance in the cell was unclear, but it may be related to anunknown mechanism that controls protein stability. As de-scribed previously (5, 25), the phosphorylation state of PAPaffected its electrophoretic mobility; the phosphorylatedforms migrated more slowly upon SDS-PAGE (Fig. 6, A and B,compare wild type and 7A). The change in electrophoreticmobility can be attributed to the phosphorylation of Thr723(25) (Fig. 6, A and B).Phosphorylation-deficient PAP Complements the Tempera-

ture-sensitive Phenotype of the pah1� nem1� Mutant—Mu-tants defective in PAP activity exhibit changes in cell physiol-ogy that are reflected in growth inhibition at elevatedtemperature (2, 3, 5). We examined whether phosphorylation-deficient PAP enzymes expressed via low copy plasmidscould complement the temperature-sensitive phenotype ofpah1� and pah1� nem1� mutant cells. Wild type PAH1 com-plemented the temperature sensitivity of the pah1� mutant(Fig. 7A). However, it did not complement this phenotype inthe pah1� nem1� mutant (Fig. 7B), indicating the require-ment of the Nem1p-Spo7p protein phosphatase complex forPAP function in vivo. In contrast to wild type PAH1, thephosphorylation-deficient 7A allele and, to a lesser extent, theCDK phosphorylation site mutant alleles complemented (e.g.bypassed) the temperature sensitivity of the pah1� nem1�mutant (Fig. 7B).

Phosphorylation-deficient PAP Complements the Nucle-ar/ER Membrane Expansion Phenotype of the nem1� spo7�Mutant—Cells lacking a functional Nem1p-Spo7p proteinphosphatase complex (e.g. nem1�, spo7�, and nem1� spo7�mutants) exhibit an abnormally expanded structure of thenuclear/ER membrane (26) (Fig. 8A). This phenotype is alsoexhibited by mutants defective in PAP activity (3, 5). Wequestioned whether low copy expression of wild type andphosphorylation-deficient PAH1 alleles could complementthe defect of nuclear morphology exhibited by nem1� spo7�mutant cells. Wild type PAH1 did not complement the phe-notype (Fig. 8B). However, normal nuclear morphology wasobserved when the mutant was transformed with the 7A allele(Fig. 8B), indicating that the lack of phosphorylation of theseven sites bypassed the requirement of the Nem1p-Spo7pprotein phosphatase complex. On the other hand, the individ-ual S602A, T723A, and S744A CDK phosphorylation site mu-tations and the double and triple combinations of these muta-tions did not complement the nem1� spo7� mutantphenotype (Fig. 8B).Overexpression of Phosphorylation-deficient PAP Inhibits

Growth on Media Lacking Inositol—The INO1-encoded inosi-tol-3-phosphate synthase catalyzes the committed step for thesynthesis of inositol in S. cerevisiae (8, 52). Cells that expressINO1 are prototrophic for inositol, whereas cells with re-pressed INO1 expression are auxotrophic for inositol (8, 52).The repression of INO1 is mediated by the Opi1p repressor,whose function is controlled by its interaction with PA at thenuclear/ER membrane (9, 53). Conditions that cause a de-crease in PA concentration lead to the dissociation of Opi1pfrom the membrane and its translocation into the nucleus (9,

FIGURE 7. Phosphorylation-deficient PAP complements the tempera-ture-sensitive phenotype of the pah1� nem1� mutant. Serial dilutions(10-fold) of pah1� (A) and pah1� nem1� (B) cells transformed with lowcopy pRS415-based plasmids bearing the indicated WT and phosphor-ylation site mutant forms of PAH1 were spotted onto glucose-containingagar plates. The plates were incubated at the indicated temperatures for 3days. 3A, S602A/T723A/S744A triple mutant.

FIGURE 8. Phosphorylation-deficient PAP-7A complements the nucle-ar/ER membrane expansion phenotype of the nem1� spo7� mutant.The nuclear morphology of wild type and nem1� spo7� mutant cells ex-pressing the HEH2-CHERRY fusion (to label the nucleus) was visualized byfluorescence microscopy. A, wild type and nem1� spo7� cells expressingthe empty vector. B, nem1� spo7� cells expressing low copy YCplac111-based plasmids bearing the indicated WT and phosphorylation site mutantalleles of PAH1. White bar, 5 �m. 3A, S602A/T723A/S744A triple mutant.



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53). In the nucleus, Opi1p represses the expression of INO1by binding to the transcriptional activator Ino2p that is asso-ciated with the INO1 promoter (9, 53). Previous studies haveshown that PAP activity plays a role in controlling PA contentand the transcriptional regulation of INO1 as well as otherphospholipid synthesis genes (3, 5, 25). For example, the lossof PAH1 causes an increase in PA content and the derepres-sion of INO1 and other phospholipid synthesis genes (3, 5),whereas the massive overexpression (driven by the GAL1/10promoter) of PAH1-7A causes a decrease in PA content andthe repression of INO1, rendering cells auxotrophic for inosi-tol (25) (Fig. 9). The decrease in PA would also decrease thesynthesis of phospholipids via the CDP-DAG pathway. Inosi-tol supplementation could only partially complement the ino-sitol auxotrophy caused by the overexpression of the 7A mu-tant, but supplementation of inositol plus choline fullycomplemented this growth defect (Fig. 9). The enhanced ef-fect of choline might be attributed to the consumption of thePAP-mediated production of DAG for phospholipid synthesisvia the Kennedy pathway as well as to the alleviation of anytoxicity that might be caused by the accumulation of DAG.The S602A/T723A/S744A allele partially mimicked thegrowth properties of the 7A mutant, whereas the individualCDK phosphorylation site mutations did not affect thegrowth on media lacking inositol (Fig. 9).Effects of the Phosphorylation-deficient PAP on the Amounts

of TAG and Phospholipids—Because PAP catalyzes the penul-timate step in the synthesis of TAG, we examined the effectsof the 7A and CDK phosphorylation site mutations on TAGcontent. Because the effects of PAH1-encoded PAP activity onTAG content are most pronounced in stationary phase cells(2, 3), our experiments were performed at this phase ofgrowth. The phosphorylation-deficient PAP mutant enzymeswere expressed in pah1� and pah1� nem1� cells. Cells werelabeled to steady state with [2-14C]acetate followed by theextraction and analysis of lipids. In pah1� cells expressingwild type PAH1, TAG accounted for 22% of the total 14C-la-beled lipids (Fig. 10A). The TAG content was not significantlyaffected by the 7A mutations or the CDK phosphorylation sitemutations (Fig. 10A). In contrast, when wild type PAH1 wasexpressed in pah1� nem1� cells, TAG accounted for only4.2% of the total lipids (Fig. 10B), indicating that the dephos-

phorylation of PAP by the Nem1p-Spo7p protein phosphatasecomplex is required for PAP function in the synthesis of TAG.The decrease in TAG content in these cells was accompaniedby an increase in total phospholipids; pah1� nem1� cells ex-pressing PAH1 had 65% phospholipids (Fig. 10B), whereaspah1� cells expressing PAH1 had 36% phospholipids(Fig. 10A).In pah1� nem1� cells, the expression of the 7A mutant

allele caused a 5-fold increase in the amount of TAG whencompared with the wild type PAH1 allele (Fig. 10B), and theamount of TAG (22%) in pah1� nem1� cells expressing the7A mutant allele reached to the level of TAG found in pah1�cells expressing the wild type allele (Fig. 10A). In addition, theexpression of the 7A mutant allele in pah1� nem1� cellscaused a decrease (from 65 to 44%) in total phospholipidswhen compared with the wild type allele (Fig. 10B). Whenexpressed in pah1� nem1� cells, the CDK phosphorylationsite mutant alleles alone, and in combinations, also causedincreases (1.6–2.9-fold) in the amounts of TAG but not to thesame extent as the 7A allele (Fig. 10B). These data indicatedthat the phosphorylations of PAP at the seven sites had a ma-jor effect on TAG synthesis and that the phosphorylations atthe three CDK sites played a partial role in this regulation.

FIGURE 9. Overexpression of phosphorylation-deficient PAP mutantsreduces growth on medium lacking inositol. Serial dilutions (10-fold) ofwild type cells transformed with the indicated WT and phosphorylation sitemutant forms of PAH1 on galactose (GAL1/10)-inducible high copy YE-plac181-based plasmids were spotted on glucose- or galactose-containingagar plates with or without 75 �M inositol and 1 mM choline where indi-cated. The plates were incubated for 3 days at 30 °C. 3A, S602A/T723A/S744A triple mutant.

FIGURE 10. Effects of the phosphorylation-deficient PAP mutants on thecontents of TAG and phospholipids. pah1� (A) and pah1� nem1� (B) cellsexpressing the indicated WT and mutant alleles of PAH1 on low copypRS415-based plasmids were grown to the stationary phase in 5 ml of syn-thetic complete medium containing [2-14C]acetate (1 �Ci/ml). Lipids wereextracted and separated by one-dimensional TLC, and the phosphorimageswere subjected to ImageQuant analysis. The percentages shown for TAGand phospholipids were normalized to the total 14C-labeled chloroform-soluble fraction. Each data point represents the average of three experi-ments � S.D. (error bars). 3A, S602A/T723A/S744A triple mutant.



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Effects of the Phosphorylation Site Mutations on the Local-ization of PAP—The effects of the 7A, S602A/T723A, andS602A/T723A/S744A mutations on the localization of PAPwere examined by immunoblot analysis of cell fractions de-rived from pah1� and pah1� nem1� mutant cells (Fig. 11, Aand B). In both genetic backgrounds, most of the wild typePAP enzyme was associated with the cytosolic fraction (82–88%) (Fig. 11C). The 7A mutations had a dramatic effect onthe association of PAP with membranes (Fig. 11, A and B). Incontrast to the wild type enzyme, most of the 7A mutant en-zyme was associated with the membranes of both pah1�(73%) and pah1� nem1� (76%) mutant cells (Fig. 11). In other

words, the 7A mutation caused a 10-fold increase in theassociation of PAP with the membrane. The combinations ofthe CDK phosphorylation mutations also affected the associa-tion of PAP with membranes but not to the same extent asthe 7A mutations. The S602A/T723A and S602A/T723A/S744A mutations caused an increase in membrane associationby 2–3.6-fold and by 3.4–4-fold, respectively (Fig. 11C). Theindividual CDK phosphorylation site mutations did not have asignificant effect on the membrane association of PAP (datanot shown).Effects of the Phosphorylation Site Mutations on the Interac-

tion of PAP with Phospholipid Vesicles—To gain further in-sight into the effect of phosphorylation on PAP associationwith membranes, we used a phospholipid vesicle binding as-say based on fluorescence emission. For this study, we usedthe wild type and 7A mutant enzymes that were expressed inS. cerevisiae and were phosphorylated in vivo (25). The inter-action of PAP with PA-containing phospholipid vesicles re-sults in an increase in fluorescence emission intensity of tryp-tophan residues as well as a shift from 350 to 343 nm in thewavelength of the maximum emission.4 The increase in fluo-rescence is probably a result of a change from a hydrophilic toa more hydrophobic environment around the tryptophan res-idues (54). For wild type PAP, there was a dose-dependentincrease in fluorescence by the addition of phospholipid vesi-cles (Fig. 12A). The 7A mutations caused a significant in-crease in the fluorescence of PAP (Fig. 12A), indicating anincrease in phospholipid vesicle interaction. The dissociationconstant (Kd) for the phospholipid vesicles of the PAP-7Aenzyme was 4.4-fold lower when compared with the constantdetermined for the wild type PAP enzyme (Fig. 12B).

DISCUSSION

The PAH1-encoded PAP, the enzyme that catalyzes thepenultimate step in the synthesis of TAG and that plays animportant role in the transcriptional regulation of phospho-lipid synthesis genes, is subject to multiple site phosphoryla-tions (2, 3, 5, 25). In fact, a bioinformatics analysis indicates

4 Z. Xu and G. M. Carman, unpublished data.

FIGURE 11. Effects of the phosphorylation-deficient mutations on thelocalization of PAP. The indicated WT and phosphorylation-deficient mu-tant forms of PAP were expressed from a low copy pRS415-based plasmidin pah1� (A) and pah1� nem1� (B) mutant cells. The cells that containedempty vector (V) are also indicated. Extracts (E) prepared from exponentialphase cells were fractionated into the cytosolic (C) and membrane (M) frac-tions by centrifugation. The membrane fraction was resuspended in thesame volume as the cytosolic fraction, and equal volumes of the fractionswere subjected to immunoblot analysis using anti-PAP, anti-phosphoglyc-erate kinase (PGK; cytosol marker), and anti-phosphatidylserine synthase(PSS; ER marker) antibodies. C, the relative amounts of cytosol- and mem-brane-associated PAP were determined for the wild type and mutant formsof the enzyme by ImageQuant analysis of the data. Representative immu-noblots are shown in A and B, whereas the quantitation data shown in C arethe average of three independent experiments � S.D. (error bars). The posi-tions of the PAP, phosphoglycerate kinase, and phosphatidylserine syn-thase (the upper and lower bands are the phosphorylated and dephosphor-ylated forms of the enzyme, respectively (39)) proteins are indicated. 3A,S602A/T723A/S744A triple mutant.

FIGURE 12. Effect of the 7A mutations on the interaction of PAP withphospholipid vesicles. A, the indicated WT and phosphorylation-deficient(7A) PAP enzymes that had been expressed and purified from S. cerevisiaewere incubated with the indicated concentrations of phospholipid vesicles.Following a 10-min incubation, the increase in PAP fluorescence was mea-sured. The values reported were the average of three experiments � S.E.(error bars). Some error bars are contained within the symbols. B, dissocia-tion constants (Kd) were determined from the data shown in A.



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that PAP may be one of the most heavily phosphorylated pro-teins in S. cerevisiae, with �90 putative target sites for severalprotein kinases (51). In this work, we extended studies on thePAP-7A mutant enzyme to shed light on the physiologicalconsequences of phosphorylation of PAP at the seven sitespreviously shown to be phosphorylated in vivo (25) and estab-lished the target sites and contribution of CDC28 (CDK1)-encoded CDK to this regulation.That PAP is a target for CDC28 (CDK1)-encoded CDK

phosphorylation in vivo is supported by the observations thatits electrophoretic mobility increases in a temperature-sensi-tive cdc28-4mutant defective in CDK activity and in a cyclinclb3� clb4� mutant but decreases in cells at the mitotic phaseof the cell cycle (5). Moreover, the slower migrating PAP pro-tein is recognized by the anti-MPM2 antibody that is specificfor cell cycle-regulated phosphoepitopes having the minimalSer/Thr-Pro motif (5, 55), and the seven sites of phosphoryla-tion previously identified in PAP have this motif (25). Thestudies performed with unphosphorylated enzyme prepara-tions isolated from E. coli demonstrated that PAP was a bonafide substrate of CDK with targets on serine and threonineresidues. The phosphorylation of PAP was dependent on timeand the concentration of the kinase, and the Km values forPAP and ATP were in the low micromolar range, indicatingthat PAP was a good substrate for CDK. Biochemical and mo-lecular approaches were used to identify Ser602, Thr723, andSer744 as the targets of CDK phosphorylation, and these resi-dues were among the seven sites previously shown to be phos-phorylated in vivo (25).

Although we did not identify all of the protein kinases re-sponsible for the phosphorylation of the seven sites, this workadvanced the understanding of how the phosphorylation ofthese sites regulated PAP function. Insights into this regula-tion were obtained through the analysis of phosphorylation-deficient mutants expressed in pah1� nem1� cells. The lipidcomposition analysis as well as the complementation analyseswith respect to phenotypes related to temperature sensitivityand the anomalous nuclear/ER membrane expansion substan-tiated the importance of the Nem1p-Spo7p protein phospha-tase complex in PAP function. Indeed, the wild type PAH1gene did not complement physiological defects (e.g. reducedTAG content) caused by the pah1� mutation unless a func-tional Nem1p-Spo7p protein phosphatase complex was pres-ent. The requirement of dephosphorylation was shown fur-ther by the ability of PAP-7A to complement the pah1�phenotypes in the absence of functional Nem1p-Spo7p com-plex. Taken together, these data indicated that dephosphory-lation at the seven sites was sufficient to confer the physiolog-ical functions of PAP. That CDK phosphorylation at Ser602,Thr723, and Ser744 contributed to this regulation was primar-ily supported by the enzyme localization and lipid composi-tion data. Indeed, the S602A/T723A and S602A/T723A/S744A mutants partially mimicked the physiologicalconsequences of the 7A mutations.Based on the specific activities of purified wild type and 7A

mutant enzymes (25), the phosphorylation of all seven siteswould cause only a 1.8-fold decrease in PAP activity. This is arelatively small effect on the in vitro catalytic activity when

compared with the large effects that the 7A mutations causedin vivo. Moreover, the CDK phosphorylation of the E. coli-expressed PAP had little effect on in vitro enzyme activity.Thus, the effects of phosphorylation on PAP functions in vivoshould be mediated by an additional mechanism. The associa-tion of PAP with the membrane where its substrate PA re-sides is essential to its function in vivo. In the absence of theNem1p-Spo7p complex, wild type PAP was enriched inthe cytosol, where it was physiologically inactive, whereas thephosphorylation-deficient PAP-7A mutant enzyme was en-riched in the membrane and was physiologically active. Therequirement of the Nem1p-Spo7p protein phosphatase com-plex, which is located in the nuclear/ER membrane (5, 26),indicates that PAP is recruited to the membrane for its physi-ological function. Indeed, the Nem1p-Spo7p-dependentmembrane localization of PAP has been shown in the pres-ence of elevated levels of PA (33). Moreover, dephosphory-lated PAP anchors onto the nuclear/ER membrane via a shortN-terminal amphipathic helix, allowing for the production ofDAG for TAG synthesis (33). In the absence of the Nem1p-Spo7p complex, PAP-7A associates with the membrane for invivo function (33). The phospholipid vesicle binding assays ofthe wild type and 7A mutant enzymes that were purified fromyeast supported this conclusion. Thus, phosphorylation is amechanism to control PAP function in vivo by regulating itsassociation with the membrane.5

Under normal physiological conditions (i.e. presence of theNem1p-Spo7p complex), the level of wild type PAP detectedon the membrane was very low. In fact, microscopic analysisof live S. cerevisiae cells expressing PAP-green fluorescentprotein shows a cytoplasmic localization without a detectablefluorescence signal associated with the nuclear/ER membraneunless PA levels are overexpressed (33). Yet we know thatPAP is physiologically active with respect to lipid metabolismthroughout cell growth (2). Purified PAP has a relatively highcatalytic efficiency when compared with other enzymes ofphospholipid metabolism (1). The lethal phenotype of cellsthat overexpress the Nem1p-Spo7p complex (5) indicates thatan excess of PAP function is detrimental to cell physiology.We speculate that under normal physiological conditions, theamount of PAP associated with membranes must be small tocontrol its physiological activity (e.g. function), and this regu-lation is mediated by the amount of the Nem1p-Spo7p com-plex on the membrane. In support of this hypothesis, a globalanalysis of protein expression indicates that the expressionlevel of Nem1p is 10-fold lower when compared with PAP(56).Like yeast PAP, the mammalian enzyme (e.g. lipin 1) is also

localized to the cytosol, ER, and nucleus (16, 57, 58). Covalentmodifications of phosphorylation (59–61) and sumoylation(62) as well as interaction with 14-3-3 proteins (63) governlipin 1 localization. Interestingly, lipin 1 has been identified asone of the most heavily phosphorylated proteins in rat adipo-

5 Measurement of phosphorylated PAP activity in vitro using the substratePA in a Triton X-100 (detergent) micellar assay is not constrained by theabsence or presence of the Nem1p-Spo7p protein phosphatase complexin a membrane phospholipid bilayer.



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cytes following treatment with insulin (59). This phosphoryla-tion is dependent on the mTOR signaling pathway (59) andoccurs at 19 sites (60). Phosphorylation also regulates lipin 1and 2 in human cells (61). As in S. cerevisiae (5), the humanlipins are phosphorylated during the mitotic phase of cellgrowth at sites within a Ser/Thr-Pro motif, and their PAPactivities are inhibited by the mitotic phosphorylation (61).Similarly, phosphorylated forms of mammalian lipins are en-riched in the cytosolic fraction, whereas the dephosphorylatedforms are enriched in the membrane fraction (60, 61). How-ever, unlike the yeast PAP, the protein kinases responsible forthe phosphorylations of mammalian lipins have yet to beidentified.The only other enzyme of lipid metabolism in S. cerevisiae

known to be phosphorylated by CDC28 (CDK1)-encodedCDK is the TGL4-encoded TAG lipase (64). This particularlipase is important for the resumption of logarithmic growthfrom the stationary phase because it supplies precursors (e.g.DAG and fatty acids) for lipid synthesis (65, 66). The phos-phorylation of this TAG lipase at Thr675 and Ser890 occurs ina cell cycle-dependent manner but at an earlier point (G1/Stransition) in the cell cycle when compared with the phos-phorylation of PAP (G2/M transition) (5, 64). Thus, the phos-phorylation of PAP (synthesis) and TAG lipase (degradation)controls the homeostasis of TAG during cell cycle progres-sion (66). However, we now know that CDC28 (CDK1)-en-coded CDK is not the only cyclin-dependent kinase that phos-phorylates and regulates PAP function during the cell cycle.The identification of the protein kinase(s) responsible forphosphorylating the remaining four sites among the sevensites that contain the Ser/Thr-Pro motif will permit studies todelineate the sequence of phosphorylations that occur duringgrowth for the PAP-mediated regulation of lipid metabolism.

Acknowledgments—We thank Judith Storch for helpful commentsabout the fluorescence experiments and acknowledge Richard Lude-scher for use of the fluorimeter.

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