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Originally published In Press as doi:10.1074/jbc.M412409200 on March 23, 2005

J. Biol. Chem., Vol. 280, Issue 22, 21577-21587, June 3, 2005
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Oxysterols Inhibit Phosphatidylcholine Synthesis via ERK Docking and Phosphorylation of CTP:Phosphocholine Cytidylyltransferase*

Marianna Agassandian{ddagger}§, Jiming Zhou{ddagger}§, Linda A. Tephly{ddagger}, Alan J. Ryan{ddagger}, A. Brent Carter{ddagger}, and Rama K. Mallampalli{ddagger}¶||**

From the Departments of {ddagger}Internal Medicine and ||Biochemistry and the Department of Veterans Affairs, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242

Received for publication, November 2, 2004 , and in revised form, March 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Surfactant deficiency contributes to acute lung injury and may result from the elaboration of bioactive lipids such as oxysterols. We observed that the oxysterol 22-hydroxycholesterol (22-HC) in combination with its obligate partner, 9-cis-retinoic acid (9-cis-RA), decreased surfactant phosphatidylcholine (PtdCho) synthesis by increasing phosphorylation of the regulatory enzyme CTP:phosphocholine cytidylyltransferase-{alpha} (CCT{alpha}). Phosphorylation of CCT{alpha} decreased its activity. 22-HC/9-cis-RA inhibition of PtdCho synthesis was blocked by PD98059 or dominant-negative ERK (p42 kinase). Overexpression of constitutively active MEK1, the kinase upstream of p42 kinase, increased CCT{alpha} phosphorylation. Expression of truncated CCT{alpha} mutants lacking proline-directed sites within the C-terminal phosphorylation domain partially blocked oxysterol-mediated inhibition of PtdCho synthesis. Mutagenesis of Ser315 within CCT{alpha} was both required and sufficient to confer significant resistance to 22-HC/9-cis-RA inhibition of PtdCho synthesis. A novel putative ERK-docking domain N-terminal to this phosphoacceptor site was mapped within the CCT{alpha} membrane-binding domain (residues 287–300). The results are the first demonstration of a physiologically relevant phosphorylation site and docking domain within CCT{alpha} that serve as targets for ERKs, resulting in inhibition of surfactant synthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pulmonary surfactant is an essential mixture containing primarily disaturated phosphatidylcholine (DSPtdCho)1 and key proteins that provide stability to alveoli by lowering surface tension. Surfactant is synthesized within lung type II alveolar epithelia and packaged within lamellar bodies, an intracellular storage form of surfactant, prior to secretion into the alveolar lumen. Deficiency of surfactant DSPtdCho contributes to the pathogenesis of acute lung injury, a disorder characterized by leakage of serum proteins into the alveolus, resulting in severe respiratory compromise (1). Recent studies in our laboratory (2) and by others (3) suggest that cholesterol-enriched low density lipoproteins are important serum components that accumulate in the alveolus. Once oxidized, these modified lipoproteins have the ability to impair DSPtdCho synthesis within alveolar type II cells (2, 3).

Oxysterols are oxygenated derivatives of cholesterol and important constituents of oxidized low density lipoproteins that are detected in association with surfactant during acute lung injury (4). Oxysterols are present in human lung and exert potent biological effects by controlling expression of diverse genes via binding to liver X receptors (LXRs), members of the nuclear receptor superfamily (5, 6). As a prerequisite for nuclear transduction of oxysterol signaling, LXRs form heterodimers with their obligate partner, the 9-cis-retinoic acid (9-cis-RA) receptor (RXR), which binds to LXR/RXR response elements within target genes, thereby regulating transcription (5). However, not all regulation by oxysterols and 9-cis-RA is transcriptional, as rapid activation of target proteins (e.g. kinases) has been demonstrated within minutes by these agonists via non-genomic mechanisms (7, 8). One recognized target for oxysterols is the ATP-binding cassette transporter family of transmembrane proteins. The ABCA1 gene is involved in the efflux of cellular phospholipid and cholesterol from the plasma membrane; our recent study (9) shows that oxysterols deplete surfactant PtdCho in lung epithelia in part by ABCA1-driven basolateral phospholipid export. However, the effects of oxysterols on PtdCho synthesis have not been investigated.

The synthesis of surfactant PtdCho is tightly regulated by the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT; CTP:choline-phosphate cytidylyltransferase, EC 2.7.7.15 [EC] ), which catalyzes the conversion of choline phosphate to CDP-choline utilizing CTP (10). CCT has been purified to homogeneity, and cDNAs from several species have been identified and cloned (10). The primary structure of CCT (termed CCT{alpha}) in mammals consists of four functional domains, including an N-terminal nuclear localization signal, a catalytic core, an {alpha}-helical lipid-binding domain, and a C-terminal phosphorylation domain (10). CCT{alpha} is localized to the nuclear membrane, but in alveolar epithelia, it is cytoplasmic, largely associated with plasma and endoplasmic reticulum membranes (11). In addition to CCT{alpha}, two additional splice variants encoded by a different gene (CCT{beta}1 and CCT{beta}2) have been identified in human (12). These isoforms differ in the extent of their C-terminal phosphorylation, but share some similar regulatory features.

CCT activity in cells is regulated extensively by exogenous lipids, as both activating and inhibitory lipids have been identified (10). CCT{alpha} is also a phosphoenzyme, although the precise physiological role of CCT{alpha} phosphorylation in vivo is unknown. The degree by which lipids alter CCT activity is influenced by its phosphorylation status, and phosphorylation of CCT{alpha} reduces its activity in vitro (13, 14). CCT{alpha} phosphorylation is restricted to 16 serines located within the C terminus. Seven of these serines are followed by prolines, suggestive of a role for proline-directed kinases, such as p34cdc2 and mitogen-activated protein kinase (MAPK), in CCT{alpha} regulation (15). Accordingly, CCT{alpha} is an in vitro substrate for MAPK, and ras-transfected cells exhibit increased CCT{alpha} phosphorylation in vivo (16–18). However, mutagenesis of consensus proline-directed phosphorylation sites or expression of a truncated CCT{alpha} mutant lacking the phosphorylation domain results in an enzyme that is phenotypically similar to wild-type CCT{alpha}, thus calling into question the physiological role of proline-directed kinases and their link with PtdCho synthesis (19, 20).

There are mounting data suggesting not only that proline-directed kinases target specific motifs ((Ser/Thr)-Pro) within substrates, but that the efficiency and specificity of phosphorylation of the acceptor site are regulated by binding of MAPKs to scaffolding complexes and distinct docking sites (21). Docking domains are present within all members of the MAPK cascade, including c-Jun N-terminal kinase (JNK), p38 kinase, and extracellular signal-regulated kinase (ERK), such as p42/44 kinase. Many MAPKs use a negatively charged conserved docking domain that interacts with motifs within target substrates that contain basic or hydrophobic residues; these sequences are usually located upstream of the kinase phosphorylation site (22).

In this study, we investigated the pathophysiological role of CCT{alpha} phosphorylation within the context of oxysterol exposure in alveolar epithelia. We observed that LXR/RXR agonists greatly diminished cellular PtdCho levels by inhibiting its biosynthesis via site-specific phosphorylation of CCT{alpha} catalyzed by ERK. In the process of investigating MAPK phosphorylation of CCT{alpha}, we identified a distinct docking domain for ERK residing within the membrane-binding domain of the enzyme.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—22-Hydroxycholesterol (22-HC) was purchased from Steraloids (Newport, RI). 9-cis-RA were obtained from Sigma. The murine lung epithelial (MLE) cell line MLE-12 was obtained from American Type Culture Collection (Manassas, VA). Hite's medium was from the University of Iowa Tissue Culture and Hybridoma Facility. Radiochemicals were purchased from PerkinElmer Life Sciences. Immunoblotting membranes were obtained from Millipore Corp. (Bedford, MA). Rabbit anti-CCT{alpha} polyclonal antiserum raised against a synthetic peptide was generated by Covance Research Products Inc. (Richmond, CA), and anti-phosphoserine antibody was from Zymed Laboratories Inc. (San Francisco, CA). Anti-p42 kinase and anti-p42/44 antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-protein kinase C antibody and the rabbit IgG TrueBlot set were from eBioscience, Inc. (San Diego, CA), and the Cdc2 (cell division control-2) kinase was purchased from Cell Signaling Technology (Beverly, MA). The p44 kinase, glutathione S-transferase (GST)-agarose, and N-terminally GST-tagged p38 protein were from Upstate Biotechnologies, Inc. (Lake Placid, NY). The ECL Plus Western blotting detection system was purchased from Amersham Biosciences. The dominant-negative p42 kinase and constitutively active MEK1 plasmids were a kind gift from Dr. Roger Davis (University of Massachusetts Medical Center) (23), and the phosphatase inhibitor mixture and PD98059 were obtained from Calbiochem. The CCT{alpha}314 C-terminal phosphorylation deletion mutant (pCMV5-CCT{alpha}314) was a kind gift from Dr. Claudia Kent (University of Michigan) (19). The TNT reticulocyte assay system was from Promega (Madison, WI). ERK-GST-agarose was obtained from Stressgen Biotech Corp. (Victoria, British Columbia, Canada). The Advantage cDNA polymerase and in vivo transactivator kinase assay (MAPK) kits containing the pTRE-luc and pTET-Elk plasmids were obtained from Clontech (Palo Alto, CA). The Geneclean II kit was obtained from BIO 101, Inc. (Vista, CA). The QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). The pCR4-TOPO plasmids and Escherichia coli TOP10 competent cells were obtained from Invitrogen, and FuGENE 6 transfection reagent was purchased from Roche Diagnostics. All DNA sequencing was performed by the University of Iowa DNA Core Facility.

Cell Culture—MLE cells were maintained in Hite's medium with 2% fetal bovine serum at 37 °C in an atmosphere containing 5% CO2. After reaching 70% confluence, the cells were harvested using 0.25% trypsin with 0.1% EDTA and plated onto either 12-well or 60-mm tissue culture dishes. After incubation overnight, the medium was replaced with serum-free Hite's medium alone (control medium) or in combination with various amounts of 22-HC (5–30 µM) with or without 9-cis-RA (1 µM) for up to 48 h. In some experiments, cells were exposed to PD98059 (10 µM) for 1 h prior to addition of 22-HC and 9-cis-RA. Cells lysates were prepared by brief sonication in 150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol, 0.025% sodium azide, and 1 mM phenylmethylsulfonyl fluoride (pH 7.4) at 4 °C.

PtdCho and DSPtdCho Analysis—Cells cultured in Hite's medium alone or containing agonists for up to 48 h were pulsed with 2 µCi of [methyl-3H]choline chloride during the final 2–4 h of incubation. Total cellular lipids were extracted from equal amounts of cellular protein using the method of Bligh and Dyer (24). Lipids were resolved by thin layer chromatography, and PtdCho or DSPtdCho was quantitated by scintillation counting as described (25). PtdCho mass was assayed by measuring lipid phosphorus content (26).

Enzyme Activities—CCT activity assays were performed in cell lysates as described without inclusion of lipid activator in the reaction mixture (26).

Immunoblot Analysis—Immunoblotting was performed as described (2). In some experiments, CCT{alpha} was immunoprecipitated from equal amounts of cell lysate prior to SDS-PAGE (26). Immunoreactive proteins were probed using the ECL Western blotting detection system. The dilution factor for anti-CCT{alpha}, anti-phosphorylated p42/44 MAPK, and anti-Cdc2 kinase antibodies was 1:1000, and that for anti-protein kinase C antibody was 1:100. To control for loading, blots were also probed with rabbit polyclonal antibody to {beta}-actin or total p42/44 kinase at 1:1000 dilution. In separate experiments, lysates from cells transiently transfected with pCMV5-CCT{alpha}-His plasmids were harvested using M-PER mammalian protein extraction reagent. CCT{alpha}-His proteins were purified using the B-PER 6xHis spin purification kit (Pierce) following the manufacturer's instructions. The levels of phosphoserine, phosphorylated p42/44 MAPK, protein kinase C, Cdc2 kinase, and CCT{alpha} were then determined by probing membranes with antibody at 1:1000 dilution, and those of protein kinase C at 1:100 dilution.

ERK Kinase Assay—To assess ERK kinase activity in vivo, cells were cotransfected with the pTET-Elk and pTRE-luc plasmids following the manufacturer's instructions in the presence or absence of a constitutively active MEK1 plasmid as described previously (27, 28). The pTET-Elk plasmid expresses Elk-1 (Ets-like transcription factor-1), an ERK-dependent transactivator that, if phosphorylated, activates the luciferase reporter gene contained in pTRE-luc. After 24 h, cells were stimulated with or without 22-HC (25 µM) and 9-cis-RA (1 µM) for up to 24 h, and luciferase activity, which was normalized to protein, was measured (27).

Immunoprecipitation of CCT{alpha} and p42/44 Kinase—Immunoprecipitation was performed with the rabbit IgG TrueBlot set according to the manufacturer's instructions. Cell lysates (250 µl) were first precleared with 25 µl of anti-rabbit IgG beads for 1 h on ice. Subsequently, 2.5 µg of primary antibody to CCT{alpha} or p42/44 kinase, normal rabbit IgG, or preimmune serum was added to precleared lysates and incubated on ice for an additional 1 h. Samples were then incubated overnight at 4 °C with 25 µl of anti-rabbit IgG beads. The next day, the beads were washed three times with TrueBlot lysis buffer, and immunoprecipitates were released from the beads by 5 min of boiling in Laemmli buffer prior to separation by SDS-PAGE. Immunoblotting was performed with anti-CCT{alpha} or anti-p42/44 primary antibody as described above. A rabbit IgG TrueBlot at 1:1000 dilution was used as a secondary antibody.

Construction of Putative CCT{alpha} Docking Domain Mutants—The cDNA encoding the open reading frame (~1100 bp) of rat CCT{alpha} was generated by PCR as described previously and ligated into a pCMV5 expression vector (pCMV5-CCT{alpha}) (2). C-terminally histidine-tagged full-length CCT{alpha} (pCMV5-CCT{alpha}-His) was generated by PCR using pCMV5-CCT{alpha} as a template as we described previously (2). An N-terminally truncated CCT{alpha} mutant termed CCTN40, which lacks the first 40 amino acid residues, was generated as follows. pCMV5-CCT{alpha} was used as a template for PCR using the sense primer 5'-aga tct atg tta cgg cag cca gct cc-3' and the antisense primer 5'-tct aga tta gtc ctc ttc atc ctc gct g-3' in a two-step PCR amplification using Advantage cDNA polymerase under the following reaction conditions: 94 °C for 2 min and 18 cycles at 94 °C for 30 s and 68 °C for 3 min. An ~1000-bp fragment was purified using the Geneclean II kit and cloned into pCR4-TOPO, and plasmid minipreparations were verified by DNA sequencing.

A CCT{alpha} variant termed CCTm158, in which 158LAEHR was mutated to 158ASEHA, was generated using the QuikChange site-directed mutagenesis kit. The oligonucleotides used were 5'-cgc ccg agt tcg cga gcg agc acg cga ttg att tcg-3' (sense) and 5'-cga aat caa tcg cgt gct cgc tcg cga act cgg gcg-3' (antisense), with pCMV5-CCT{alpha} plasmid DNA used as a template (2). The PCR conditions were as follows: 95 °C for 30 s and 18 cycles at 95 °C for 30 s, 55 °C for 60 s, and 68 °C for 6 min.

The CCT{alpha} internal deletion mutant termed CCT289, which lacks the membrane-binding domain (residues 240–290), has been described previously (2). A related CCT{alpha} variant termed CCTm289, in which 13KRRK was mutated to 13KWWK, was generated by site-directed mutagenesis with oligonucleotides 5'-gct aaa gtc aat tca agg aag tgg tgg aaa gag gta cct ggc cct-3' (sense) and 5'-agg gcc agg tac ctc ttt cca cca ctt cct tga att gac ttt agc-3' (antisense) using pCMV5-CCT289 as a template. The PCR conditions were as follows: 95 °C for 30 s and 18 cycles at 95 °C for 30 s, 55 °C for 60 s, and 68 °C for 5 min.

A third CCT{alpha} internal deletion mutant termed CCTd21, which lacks residues 231–251, was constructed following the same PCR protocol used for generation of full-length CCT{alpha} with pCMV5-CCT{alpha} as a template and sense primer 5'-gga gct caa agt gaa aga tgt gga gg-3' and antisense primer 5'-tcc ccg ggt cta gat tag tcc tct tca tcc tcg c-3', resulting in a 351-bp PCR product that was cloned into pCR4-TOPO for transformation into E. coli TOP10 competent cells. Plasmids were double-digested with SacI and XbaI, and pCMV5-CCT{alpha} was digested with BglII and SacI. The 351- and 687-bp fragments were purified and ligated into pCMV5 (previously digested with BglII and XbaI) using T4 ligase overnight at 15 °C and then transferred into TOP10 competent cells. The 1038-bp insert was directionally cloned into pCMV5 as confirmed by partial DNA sequencing and restriction enzyme digestion.

A series of C-terminally truncated CCT{alpha} mutants were generated as follows. pCMV5-CCT{alpha} was used as a template for PCR using sense primer 5'-gga tcc ata tgg atg cac aga gtt cag c-3' and antisense primers 5'-acg cgt tta tga ctt ttc ctc cac atc-3' for CCT260, 5'-acg cgt tta ctc ctc cca ctt ctg gat gag-3' for CCT280, 5'-acg cgt tta aat gaa ctc tcg gga ctt c-3'for CCT286, and 5'-acg cgt tta ctt cag cgc tcc ttc tgg acc-3' for CCT300. The PCR products were purified using the Geneclean II kit and cloned into pCR4-TOPO, and plasmid minipreparations were verified by DNA sequencing. These CCT mutants were truncated at the indicated amino acid.

Construction of CCT{alpha} Phosphorylation Mutants—A truncated mutant termed CCT{alpha}328, which lacks the distal portion of the phosphorylation domain of CCT{alpha} (residues 329–367), was generated as follows. pCMV5-CCT{alpha} was used as a template for PCR using sense primer 5'-aga tct atg gat gca cag agt tca g-3' and antisense primer 5'-tct aga tta gcg ctc atg agt agg gct gc-3' to generate an ~990-bp fragment. The PCR product was purified using the Geneclean II kit and cloned into pCR4-TOPO, and plasmid minipreparations were verified by DNA sequencing. This clone was then digested with BgIII and XbaI, purified using the Geneclean II kit, and ligated into a pCMV5 expression vector previously digested with the same restriction enzymes.

A phosphorylation variant termed CCT{alpha}328SA, which is similar to CCT{alpha}328 but with Ser321-Ser322-Ser323 mutated to Ala321-Ala322-Ala323, was constructed by site-directed mutagenesis. The oligonucleotides used were 5'-ccc aag cag agt ccc gca gca gca cct act cat gag cgc-3' and 5'-gcg ctc atg agt agg tgc tgc tgc ggg act ctg ctt ggg-3', with CCT{alpha}328 plasmid DNA used as a template. The PCR conditions were as follows: 95 °C for 30 s and 18 cycles at 95 °C for 30 s, 55 °C for 60 s, and 68 °C for 6 min.

Another truncated phosphorylation variant termed CCT{alpha}328SAquad, in which Ser315-Ser321-Ser322-Ser323 was mutated to Ala315-Ala321-Ala322-Ala323, was generated similarly using oligonucleotides 5'-gat gct gca ggc cat cgc tcc caa gca gag tcc-3' and 5'-gga ctc tgc ttg gga gcg atg gcc tgc agc atc-3', with CCT{alpha}328SA plasmid DNA used as a template. The PCR conditions were identical to those used for construction of CCT{alpha}328SA.

A full-length mutant termed CCT{alpha}315, in which Ala was substituted only at Ser315, was constructed using oligonucleotides 5'-ccc aag cag agt ccc gca gca gca cct act cat gag cgc-3' and 5'-gcg ctc atg agt agg tgc tgc tgc ggg act ctg ctt ggg-3', with pCMV5-CCT{alpha} plasmid DNA used as a template (2). The PCR conditions were identical to those used for construction of CCT{alpha}328SAquad. A second full-length CCT{alpha} phosphorylation mutant termed CCT{alpha}quad, in which Ser315-Ser321-Ser322-Ser323 was changed to Ala315-Ala321-Ala322-Ala323, was generated as follows. pCMV5-CCT{alpha}328SAquad and pCMV5-CCT{alpha} were digested with BglII/XbaI, generating ~1- and ~1.1-kb inserts, respectively. These fragments were purified and digested with BspHI. An ~970-bp fragment from CCT{alpha}328SAquad and an ~200-bp fragment from CCT{alpha} were purified and ligated into pCMV5 predigested with BglII/XbaI by T4 ligase and transferred into TOP10 cells. All CCT{alpha} constructs mentioned above were verified by DNA sequencing.

Preparation of p38-GST-agarose—p38-GST-agarose was prepared from N-terminally GST-tagged full-length recombinant p38 protein and GST-agarose. Proteins (5 µg of p38-GST and 25 µl of GST-agarose beads) were incubated in binding buffer containing 20 mM Tris-HCl, 150 mM NaCl, and 5 mM imidazole (pH 7.5) at 4 °C for 2 h according to the manufacturer's recommendations. The beads were subsequently washed three times with 20 mM Tris-HCl, 150 mM NaCl, and 50 mM imidazole (pH 7.5) and suspended in phosphate-buffered saline containing 50% glycerol and 0.05% sodium azide. Immunoblotting of preparations using anti-p38 antibody confirmed that the kinase was effectively conjugated to GST-agarose.

In Vitro CCT{alpha} Transcription and Translation and ERK Pull-down Assays—For in vitro synthesis of CCT{alpha} mutants, cDNA constructs cloned into pCR4-TOPO4 (2 µg/reaction) were added directly to the rabbit reticulocyte lysate (TNT coupled reticulocyte lysate system) and incubated with T7 RNA polymerase in a 50-µl reaction containing [35S]methionine (20 µCi/reaction) for 90 min at 30 °C following the manufacturer's instructions. Aliquots (10 µl) of in vitro translation products were boiled for 5 min in 2x Laemmli loading buffer and stored at -80 °C; alternatively, aliquots (40 µl) were directly rotated with ERK-GST-agarose, p38-GST-agarose, or GST-agarose alone at 4 °C overnight. After 18 h, the agarose beads were washed three times with ice-cold phosphate-buffered saline, and proteins were eluted from the beads after resuspension in 4x Laemmli buffer and boiling for 5 min. Proteins eluted from GST and products of in vitro translation (10-µl aliquots) were resolved by SDS-PAGE; the gels were dried; and autoradiography was performed.

Transfection Analysis—For expression of CCT{alpha} plasmids, the MEK1 plasmid, or the dominant-negative p42 kinase plasmid, cells were transfected for 4 h (4 µg/60-mm dish) with test plasmids. Immediately after transfections, cells were transferred to serum-free medium containing agonists for various times before pulsing the cells with [methyl-3H]choline chloride as described above for PtdCho determination.

CCT{alpha} Phosphorylation in Vivo and in Vitro—Purified His-tagged CCT{alpha}, CCT{alpha}315, and CCT{alpha}quad were dephosphorylated using alkaline phosphatase (5 units) in 50 mM Tris-HCl and 100 µM EDTA (pH 8.5) (16). After 1 h of incubation at 30 °C, CCT{alpha} proteins were purified again using the B-PER 6xHis spin kit. In vitro phosphorylation was catalyzed using 10 µg of p42 (or p44) kinase in MAPK buffer (25 mM MOPS, 6 mM MgCl2, 25 mM {beta}-glycerol phosphate, 100 µM ATP, and 2 mM dithiothreitol) in the presence of phosphatase inhibitor mixture (1:100) with 10 µCi of [{gamma}-32P]ATP (29). After 1 h of incubation at 30 °C, reactions were terminated with 4x Laemmli protein loading buffer, and products were heated at 5 min at 100 °C. Samples were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by CCT{alpha} immunoblotting and autoradiography.

For in vivo 32P labeling, MLE cells were exposed to medium with or without agonists as described above. After 20 h, cells were washed twice with phosphate-free medium and incubated in the same medium with 750 µCi of [32P]orthophosphate for 24 h (13). Cells were then harvested in radioimmune precipitation assay buffer containing 10 mM Na2HPO4, 100 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 mM NaF, 10 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 1% (v/v) Triton X-100 and precleared for 2 h at 4°C using Sepharose CL-4B, protein A, and preimmune rabbit serum. Cleared supernatants (500 µl) were incubated overnight at 4 °C with 1 µg of rabbit anti-CCT antibody, which was previously bound to protein A. The following morning, the immunoprecipitates were washed with lysis buffer (50 mM HEPES, 150 mM NaCl, 0.5 mM EGTA, 50 mM NaF, 10 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 1% (v/v) Triton X-100), and the pellets were placed in SDS protein sample buffer and heated to 95 °C for 5 min. Soluble proteins were separated by 10% SDS-PAGE, and the gels were dried and subjected to autoradiography as described above (2). Alternatively, to determine CCT{alpha} phosphorylation, cells were transfected with His-tagged CCT{alpha} constructs with or without the constitutively active MEK1 plasmid as described above. After 24 h, cells were harvested, purified, and processed for phosphoserine immunoblotting.

Statistical Analysis—Statistical analysis was performed by one-way analysis of variance or Student's t test (30). Data are presented as means ± S.E.



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  		FIG. 1.
Oxysterol/9-cis-RA inhibition of PtdCho synthesis and CCT activity in lung epithelia. A, MLE cells were incubated in serum-free medium alone (Control) or with 22-HC (25 µM) and 9-cis-RA (1 µM)(22HC/RA) for various times. Cells were pulsed with 2 µCi of [3H]choline during the final 2 h of incubation, and radioactivity in cellular [3H]PtdCho was determined following cellular lipid extraction, separation by TLC, and quantification by scintillation counting. Inset,[3H]choline incorporation into DSPtdCho at 24 h was also determined as described above. Values are means ± S.E. from three independent experiments. B and C, cells were exposed to various amounts of 22-HC (B) or different oxysterols (24-hydroxycholesterol (24-OH), 25-hydroxycholesterol (25-OH), or 7-{beta}-hydroxycholesterol (7-{beta}diol)) at 25 µM (C) for 24 h, and [3H]choline incorporation into PtdCho was determined. Both B and C were performed with inclusion of 9-cis-RA (1 µM) in the medium. D–F, MLE cells were cultured with 22-HC (25 µM) and 9-cis-RA (1 µM) in combination (D) or individually (E and F) for 6, 24, or 48 h. CCT activity was assayed in the absence of exogenous lipid activator in the assay mixture. *, p < 0.05 for 22-HC and 9-cis-RA versus the control; +, p < 0.001 for 22-HC/9-cis-RA versus the control or 25-hydroxycholesterol group versus all other groups. Values are means ± S.E. from three independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Oxysterol/9-cis-RA Inhibition of PtdCho Synthesis—Incubation of MLE cells with 22-HC and 9-cis-RA significantly decreased incorporation of [methyl-3H]choline into PtdCho and DSPtdCho by nearly 60 and 76%, respectively, after 24 h of exposure (Fig. 1A and inset). However, significant inhibitory effects on [methyl-3H]choline incorporation into PtdCho were also achieved with 22-HC and 9-cis-RA as early as 6 h after exposure, with maximal effects seen by 48 h (Fig. 1A). Increasing concentrations of oxysterol in combination with fixed amounts of 9-cis-RA (1 µM) produced a dose-dependent decrease in choline incorporation into PtdCho (Fig. 1B). Comparative analysis with other oxysterols revealed that 25-hydroxycholesterol also produced similar inhibitory effects on PtdCho synthesis (Fig. 1C). LXR/RXR agonists decreased PtdCho synthesis in cells by significantly altering the activities of one of the enzymes within the CDP-choline pathway. There was no significant effect of 22-HC and 9-cis-RA on the activity of choline kinase, the first committed enzyme of the pathway, or on the activity of choline phosphotransferase, the terminal enzyme involved in PtdCho synthesis (data not shown). However, after 6, 24, or 48 h of exposure, 22-HC/9-cis-RA inhibited CCT activity by ~30–50% relative to the control (Fig. 1D). Only modest inhibitory effects of 22-HC and 9-cis-RA on CCT activity were observed when these agents were used individually (Fig. 1, E and F). These effects of 22-HC and 9-cis-RA on PtdCho synthesis resulted in a net decrease in the mass of PtdCho by 30% within MLE cells (139 ± 16 (control) versus 96 ± 5 (22-HC and 9-cis-RA) nmol of lipid phosphorus, n = 12; p < 0.05).

Oxysterols and 9-cis-RA Increase CCT{alpha} Phosphorylation—To investigate the mechanisms whereby 22-HC and 9-cis-RA reduce CCT activity, we assayed CCT{alpha} protein mass. Overall, 22-HC and 9-cis-RA did not alter enzyme levels relative to control levels (Fig. 2A). We next examined whether these agents might increase the levels of CCT{alpha} phosphorylation, thereby reducing enzyme activity and PtdCho synthesis. First, to examine the phosphorylation status of endogenous CCT{alpha}, cells were stimulated with agonists and labeled with [32P]orthophosphate. Cells were then harvested, and CCT{alpha} was immunoprecipitated and processed for autoradiography (Fig. 2B). 22-HC and 9-cis-RA clearly increased the intensity of a major product at ~42 kDa compared with the control (Fig. 2B). Next, cells were transfected with plasmids encoding His-tagged full-length CCT{alpha} and exposed to agonists; the transfectants were purified to remove endogenous CCT{alpha} and lysates were processed for immunoblotting using anti-phosphoserine antibody (Fig. 2C, upper panel). As a control, we also probed for total levels of overexpressed CCT{alpha} protein (Fig. 2C, lower panel). After correction for the quantity of the total enzyme, 22-HC and 9-cis-RA increased the levels of CCT{alpha} phosphorylation by nearly 2- and 3-fold at 6 and 24 h of analysis, respectively (Fig. 2D). In separate experiments, endogenous CCT{alpha} was immunoprecipitated and probed with anti-phosphoserine antibody after stimulation of cells with 22-HC and 9-cis-RA (Fig. 2D, inset). Three bands exhibiting varying mobilities (~42 kDa) on autoradiograms were detected; these CCT{alpha} phosphorylation bands increased in intensity after 22-HC and 9-cis-RA treatment. Thus, 22-HC and 9-cis-RA increased phosphorylation of both endogenous and overexpressed CCT{alpha}.

Oxysterols and 9-cis-RA Increase CCT{alpha} Phosphorylation via MAPK—We used two complementary approaches to assess p42/44 activation by 22-HC and 9-cis-RA. As shown by immunoblotting, 22-HC and 9-cis-RA exerted a biphasic temporal pattern of activation of p42/44 kinase in MLE cells as described previously (31–34). First, the agonists increased the levels of phosphorylated p42/44 MAPK in cells within 15 min, and kinase activation was detected for up to 6 h (Fig. 3A). In addition, a late phase pattern of p42/44 MAPK activation was seen from 18 to 21 h after the stimulus, with activity waning by 24 h (Fig. 3A). Second, we measured p42/44 kinase activity in vivo using a luciferase reporter plasmid driven by Elk (Fig. 3B). In this assay, when Elk is phosphorylated by p42/44 kinase via constitutively active MEK1, phosphorylated Elk binds to a tetracycline response element within the luciferase promoter to induce reporter expression. Cells were transiently cotransfected with the luciferase reporter plasmid, the Elk plasmid, and either an empty vector or constitutively active MEK1. Cells exposed to 22-HC and 9-cis-RA exhibited an ~2-fold increase in reporter gene expression compared with the control. In addition, expression of constitutively active MEK1 also produced an ~30% increase in p42/44 kinase activation in response to agonist stimulation (n = 4; p < 0.001) (Fig. 3B). Additional experiments examining the kinetics of p42/44 kinase activation using this system demonstrated that luciferase activity was increased by 22-HC and 9-cis-RA uniformly from 6 to 24 h (Fig. 3C). These latter experiments strongly link 22-HC and 9-cis-RA inhibition of CCT activity with p42/44 kinase activation (Fig. 3A), as the luciferase reporter method is a functional readout that assesses activation of the transcription factor Elk, an important downstream physiological target for p42/44 kinase.



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  		FIG. 2.
Oxysterols and 9-cis-RA increase CCT{alpha} phosphorylation. A, MLE cells were exposed to medium with (+) or without (-) 22-HC (25 µM) in combination with 9-cis-RA (1 µM) for various times, and cells were harvested for CCT{alpha} immunoblotting. B, cells were exposed to medium with (+) or without (-) 22-HC (25 µM) with 9-cis-RA (1 µM) as described above for 24 h; cells were labeled with [32P]orthophosphate (750 µCi/dish); and CCT{alpha} was immunoprecipitated. The immunoprecipitates were subjected to SDS-PAGE; the gels were dried; and autoradiography was performed to detect [32P]CCT{alpha}. C, MLE cells were transiently transfected with a His-tagged full-length pCMV5-CCT{alpha} plasmid and cultured with (+) or without (-) 22-HC (25 µM) in combination with 9-cis-RA (1 µM) for various times. Lysates were harvested, and fusion proteins were purified on a His-tagged column. Purified proteins were separated by SDS-PAGE as described above, and the levels of phosphorylated CCT{alpha} (upper panel) were determined by probing immunoblot membranes with rabbit anti-phosphoserine polyclonal antibody or for the levels of total CCT{alpha} (lower panel). D, densitometric analysis of autoradiograms shows the amounts of immunoreactive phosphorylated CCT{alpha} versus the control using arbitrary densitometric values after correction for the levels of CCT{alpha} loading. 22HC/RA, 22-HC/9-cis-RA. Inset, cells were cultured with (+) or without (-) 22-HC (25 µM) in combination with 9-cis-RA (1 µM) for 24 h. CCT{alpha} was immunoprecipitated using a polyclonal antibody to the enzyme and resolved by SDS-PAGE prior to immunoblotting with anti-phosphoserine antibody. Arrowheads show multiple CCT{alpha} phosphorylated forms. The results in A are from four independent experiments, and those in B are from a representative experiment. The results in C are from four experiments, and those in D (inset) are from two experiments. *, p < 0.05 for 22-HC and 9 cis-RA versus the control.

 
To determine whether the effects of 22-HC and 9-cis-RA on PtdCho synthesis are ERK-dependent, cells were pretreated with an inhibitor of MEK1, the upstream kinase of p42/44 kinase, or transfected with a dominant-negative plasmid encoding p42 kinase. Both pretreatment with the MEK1 kinase inhibitor PD98059 and expression of dominant-negative p42 kinase significantly blocked 22-HC- and 9-cis-RA-induced inhibition of [methyl-3H]choline incorporation into PtdCho (Fig. 3, D and E). In these experiments, 22-HC and 9-cis-RA decreased CCT activity from 1.57 ± 0.04 (control) to 1.12 ± 0.01 nmol/min/mg of protein; PD98059 alone did not alter CCT activity (1.50 ± 0.02 nmol/min/mg of protein), but in combination with 22-HC and 9-cis-RA, PD98059 significantly blocked agonist-induced inhibition of enzyme activity (1.38 ± 0.02 nmol/min/mg of protein for 22-HC and 9-cis-RA versus all other groups; p < 0.05). Finally, to further investigate p42/44 kinase and CCT{alpha} interaction, cells were transfected with pCMV5-CCT{alpha}-His in the presence or absence of 22-HC and 9-cis-RA; CCT{alpha} was purified using a His-tagged column and proteins were resolved by SDS-PAGE prior to immunoblotting for phosphorylated or total p42/44 kinase (Fig. 3F). The results show that phosphorylated (active) p42/44 kinase was variably associated with CCT{alpha} protein and that this association increased at 3, 6, 21, and 24 h after 22-HC and 9-cis-RA exposure (Fig. 3F). In contrast, nonspecific binding to the His-tagged column was not observed with either endogenous CCT{alpha} protein or p42/44 kinase in cell lysates (Fig. 3G, lanes 1 and 2) or after recombinant p44 kinase was applied to the column (data not shown). To further assess the specificity of kinase binding to our His-CCT columns, we probed for retention of protein kinase C or Cdc2 kinase on these columns. MLE cells were first transfected with pCMV5-CCT{alpha}-His (Fig. 3H, Lysates). Next, CCT{alpha} was purified from cells transfected with pCMV5-CCT{alpha}-His as described above. Unlike p42/44 kinase, we did not detect Cdc2 kinase or protein kinase C in association with CCT{alpha} protein in these experiments (Fig. 3H, Fraction). To determine whether endogenous CCT{alpha} was associated with total p42/44, we performed immunoprecipitation experiments after cells were cultured in the presence or absence of agonists (Fig. 3I). We first performed immunoprecipitation using anti-p42/44 kinase antibody or normal rabbit IgG, followed by immunoblotting with anti-CCT{alpha} antibody. As shown in Fig. 3I (left panel), CCT{alpha} was detected with immunoprecipitated p42/44 kinase in the presence or absence of oxysterol and 9-cis-RA exposure, whereas this association was not detectable using control antibody (rabbit IgG). Conversely, immunoprecipitation using anti-CCT{alpha} antibody followed by immunoblotting with antibody to total p42/44 kinase also revealed that the kinase was detected in association with CCT{alpha} (Fig. 3I, right panel). This association was not observed using preimmune serum as a negative control. Thus, endogenous CCT{alpha} and total p42/44 kinase were detected in close association, as were overexpressed CCT{alpha} and the activated kinase.



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  		FIG. 3.
Oxysterol/9-cis-RA inhibition of PtdCho synthesis is mediated by p42/44 kinase. A, MLE cells were cultured with (+) or without (-) 22-HC (25 µM) with 9-cis-RA (1 µM) for various times, and the levels of phosphorylated (upper panel) and total (lower panel) p42/44 kinase were assayed by immunoblotting. B, cells were transiently transfected with pTRE-luc and pTET-Elk and either an empty vector or the constitutively active pCMV-MEK1 expression vector. After 24 h, the cells were stimulated with ethanol (Control) or 22-HC with 9-cis-RA (22HC/RA) for 6 h. Luciferase activity, which was normalized to protein, is expressed as -fold increase from the control. C, using the same assay system described for B, cells were cultured with 22-HC and 9-cis-RA for up to 24 h, and luciferase activity measured. D, cells were cultured in 12-well plates with or without 22-HC and 9-cis-RA for 24 h and with PD98059 (10 µM) alone for 1 h and then exposed to medium for 24 h or pretreated with PD98059 (10 µM) for 1 h prior to agonist stimulation for 24 h. [3H]Choline incorporation into PtdCho was then determined. E, cells were cultured with or without 22-HC and 9-cis-RA in 60-mm dishes as described for D, transfected with the dominant-negative (DN) p42 kinase plasmid (4 µg) alone for 4 h, and then exposed to medium for 24 h or transfected with the dominant-negative p42 kinase plasmid prior to agonist stimulation. F, cells were transfected with the His-tagged full-length pCMV5-CCT{alpha} plasmid and cultured with (+) or without (-) 22-HC (25 µM) in combination with 9-cis-RA (1 µM) for 3, 6, 21, or 24 h. Lysates were harvested and purified on a His-tagged column and then probed with a polyclonal antibody to phosphorylated (upper panel) or total (lower panel) p42/44 kinase. G, to control for nonspecific kinase binding to the His-tagged column, untransfected MLE cell lysates alone (200 µg) were applied to the column, and protein was eluted in two fractions (fractions 1 and 2). We then used equal amounts of protein from the crude cell lysate (L lanes) and eluted fractions (lanes 1 and 2) for SDS-PAGE and immunoblotting for CCT{alpha} (left) and p42/44 kinase (right). H, in separate experiments, cells were transfected with the His-tagged full-length pCMV5-CCT{alpha} plasmid, and the lysate was harvested and used for immunoblotting (left) for CCT{alpha} (lane 1), p42/44 kinase (lane 2), Cdc2 kinase (lane 3), and protein kinase C (lane 4). The transfected lysate was also applied to a nickel column; the protein was eluted; and samples were used for immunoblotting (right) for analysis of protein binding: CCT{alpha} (lane 1), p42/44 kinase (lane 2), Cdc2 kinase (lane 3), and protein kinase C (lane 4). I, left panel, immunoprecipitation of ERK and immunoblotting for CCT{alpha}. Cells were cultured in the presence (+) or absence (-) of agonists for 24 h as described above. After treatment, cells were harvested in lysis buffer, and samples were processed using the rabbit IgG TrueBlot set. Lysates were precleared and incubated with either anti-p42/44 kinase antibody (first and second lanes) or normal rabbit IgG (third lane), followed by immunoblotting with anti-CCT{alpha} antibody. Thefourth lane represents CCT{alpha} in cell lysates. Right panel, immunoprecipitation of CCT{alpha} and immunoblotting for ERK. Lysates from cells cultured as described above were also immunoprecipitated using anti-CCT{alpha} antibody, followed by immunoblotting with an antibody to total p42/44 kinase (first and second lanes). As a negative control, lysates were first incubated with preimmune serum (P; third lane), followed by immunoblotting with antibody to total p42/44 kinase. The fourth lane represents the levels of total p42/44 in cell lysates. *, p < 0.05 versus the control; **, p < 0.01 versus the control; +, p < 0.001 versus the control. Values are means ± S.E. from three independent experiments for A–C, at least six experiments for D and E, four experiments for F, and one experiment for G–I.

 
Mapping of ERK-docking Sites within CCT{alpha}—MAPK phosphorylation of substrates involves docking interactions at motifs often very distant to phosphoacceptor sites that are essential for efficient phosphorylation. These motifs often harbor clusters of basic residues, FXFP motifs, or LXL motifs (35). Data base analysis of the CCT{alpha} sequence uncovered similar regions suggestive of potential p42/44 kinase-docking sites. Thus, we initially used a candidate approach by deleting or mutating these core regions within CCT{alpha} (Fig. 4A). Four mutants (CCTN40, CCT158, CCTm289, and CCTd21) in which candidate sites were removed or mutated were constructed, tested for kinase binding after in vitro translation (Fig. 4, left panels), and analyzed using an ERK pull-down assay (right panels). As shown in Fig. 4 (B, D, and F), each of these mutants was sufficiently translated. Deletion of 40 residues from the CCT{alpha} N terminus (CCTN40) or 21 residues (positions 231–251) internally (CCTd21) or mutations within potential docking sites (CCTm158) failed to alter ERK-CCT{alpha} binding (Fig. 4, C and E). Moreover, a CCT{alpha} double mutant (CCTm289) lacking the entire membrane-binding domain (residues 240–290) and harboring an N-terminal site mutation at Arg14-Arg15 resulted in total loss of kinase-CCT{alpha} binding (Fig. 4C). The results using a similar mutant (CCT289) devoid of this membrane-binding region (but lacking the N-terminal mutation) confirmed that an ERK-docking domain likely resides between residues 252 and 314 of CCT{alpha} (Fig. 4E). CCT289 is an internal deletion mutant that contains the entire phosphorylation domain (residues 315–367) of CCT{alpha}, and the absence of kinase binding to this construct indicates that this domain is not sufficient for ERK docking. Conversely, ERK pull-down analysis using a C-terminally truncated mutant (CCT314) lacking only the phosphorylation domain demonstrated ERK-CCT{alpha} binding similar to that observed with full-length CCT{alpha} (data not shown), indicating that the phosphorylation domain is not required for kinase docking.

We next tested several CCT{alpha} mutants that were progressively truncated within the membrane-binding domain (at the C terminus) to map an ERK-docking site (Fig. 4, F and G). Although full-length CCT{alpha} and a CCT mutant harboring the first 300 residues effectively bound ERK, binding was not observed with mutants containing <287 amino acids. Thus, the results indicate that a putative ERK-docking domain likely resides in a span of 14 residues localized within the membrane-binding domain of CCT{alpha} (residues 287–300) (Fig. 4, E and G). Finally, to assess binding specificity, newly synthesized full-length CCT{alpha} was incubated with ERK-GST-agarose, p38-GST-agarose, or GST-agarose alone. As shown in Fig. 4H, only ERK exhibited robust binding to CCT{alpha}.

Because the above data suggested that an ERK-docking domain is present within the membrane-binding region of CCT{alpha}, we further investigated whether these in vitro results could be recapitulated in vivo by transfecting lung epithelia with histidine-tagged full-length CCT{alpha} or CCT289. CCT was purified on a nickel column. Samples were processed for immunoblotting for phosphorylated p42/44 kinase (Fig. 4I, middle panel), and blots were probed again with anti-CCT{alpha} antibody (lower panel). Furthermore, to determine whether p42/44 kinase docking is necessary for efficient CCT{alpha} phosphorylation, similar transfectants were purified from cells and reacted in vitro with recombinant p44 kinase in the presence of [{gamma}-32P]ATP after dephosphorylation using alkaline phosphatase (Fig. 4I, upper panel). Compared with full-length CCT{alpha}, minimal {gamma}-32P labeling, if any, was detected with the internal deletion mutant, suggesting that CCT289 was less efficiently phosphorylated compared with wild-type CCT{alpha} (Fig. 4I, upper panel). As anticipated, significantly greater amounts of active kinase were bound to CCT in transfectants in which full-length CCT{alpha} was expressed compared with cells transfected with the mutant lacking the membrane-binding domain (Fig. 4I, middle panel). Thus, ERK docking with CCT{alpha} in the membrane-binding domain was observed in vivo and was required for optimal phosphorylation of the enzyme.

Oxysterol/9-cis-RA and MAPK Sensitivity Is Altered by CCT{alpha} C-terminal Phosphorylation Domain Mutants—To investigate physiologically relevant ERK phosphorylation sites within CCT{alpha} in response to 22-HC and 9-cis-RA, we expressed other truncated enzyme mutants lacking various portions of the C-terminal phosphorylation domain. In addition, we generated full-length and truncated CCT{alpha} mutants harboring point mutations at specific proline-directed sites (Fig. 5A). The sensitivity of cells to the inhibitory effects of 22-HC and 9-cis-RA on PtdCho synthesis varied after transfection with CCT{alpha}, CCT{alpha}314, CCT{alpha}328, CCT{alpha}328SA, and CCT{alpha}328SAquad plasmids. Cells transfected with full-length CCT{alpha} exhibited a 53% decrease in the rates of [methyl-3H]choline incorporation into PtdCho after 24 h of 22-HC and 9-cis-RA exposure, whereas similar treatment did not significantly alter synthetic rates in cells transfected with CCT{alpha}314 (Fig. 5B). In these experiments, 22-HC and 9-cis-RA treatment decreased CCT activity from 3.11 ± 0.1 to 1.76 ± 0.2 nmol/min/mg of protein after transfection of full-length CCT{alpha}, whereas the agonists did not significantly alter enzyme activity after expression of CCT{alpha}314 (1.92 ± 0.3 (control) to 1.71 ± 0.1 (22-HC and 9-cis-RA) nmol/min/mg of protein). Thus, deletion of the C-terminal phosphorylation domain of CCT{alpha} totally abolished the inhibitory effects of 22-HC and 9-cis-RA on PtdCho synthesis. When cells were transfected with CCT{alpha}328, treatment with 22-HC and 9-cis-RA led to a 41% decrease in [methyl-3H]choline incorporation into PtdCho compared with matched controls (CCT{alpha}328-transfected untreated cells). Thus, expression of a construct lacking 11 of 16 consensus serine phosphorylation sites within CCT{alpha} still led to significant agonist-induced inhibition of PtdCho synthesis. These results suggested that additional sites contained within the proximal C-terminal domain were targets for p42/44 kinase phosphorylation of CCT{alpha} in response to 22-HC and 9-cis-RA. To investigate this, cells were transfected with CCT{alpha}328SA, in which one of three proline-directed sites within CCT{alpha}328 (Ser323-Pro324) was modified to alanine, and subsequently tested for 22-HC and 9-cis-RA sensitivity. As shown in Fig. 5B, cells transfected with CCT{alpha}328SA still displayed a 42% decrease in the rates of [3H]choline incorporation into PtdCho after agonist stimulation compared with untreated control transfectants. We next expressed a CCT{alpha} mutant similar to CCT{alpha}328SA but also containing an alanine substitution at Ser315 (CCT{alpha}328SAquad). Cells transfected with CCT{alpha}328SAquad were resistant to treatment with 22-HC and 9-cis-RA, as the rates of PtdCho synthesis were comparable between treatment and control groups.



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  		FIG. 4.
Mapping of a CCT{alpha} docking domain for ERK. A, map illustrating the sequences of individual CCT{alpha} mutants using candidate or deletion mutagenesis of putative docking sites. Dashed lines represent deleted residues. Specific mutations within motifs are underlined. Constructs tested include wild-type CCT (CCT{alpha}), a variant harboring mutations at a candidate docking site (CCTm158), and an N-terminal deletion mutant (CCTN40) devoid of the nuclear localization signal (NLS). A double mutant lacking the membrane-binding region and containing mutations within a putative N-terminal docking site (CCTm289), an internal deletion mutant devoid of only the membrane-binding region (CCT289), and another internal deletion mutant lacking 21 residues within the catalytic membrane-binding hinge region (CCTd21) were also tested. Finally, a series of deletion mutants progressively truncated within the distal membrane-binding region (CCT260, CCT280, CCT286, and CCT300) were generated. B, D, and F, individual mutants were synthesized in vitro using rabbit reticulocyte lysate in a reaction containing [35S]methionine (20 µCi/reaction). C, E, and G, newly translated reaction products were mixed with ERK-GST-agarose, sedimented, and the pellets were analyzed for CCT by SDS-PAGE and autoradiography as described under "Experimental Procedures." Each panel is representative of at least four separate experiments. H, CCT{alpha} was synthesized in vitro using rabbit reticulocyte lysate as described above and then reacted with ERK-GST-agarose, p38-GST-agarose, or GST-agarose alone and processed subsequently for SDS-PAGE and autoradiography. I, cells were transfected with plasmids encoding His-tagged full-length CCT{alpha} or the membrane-binding deletion mutant CCT289. After 18 h, lysates were harvested and purified on a His-tagged column. In the upper panel, lysates from CCT{alpha} and CCT289 transfectants were dephosphorylated using alkaline phosphatase and then phosphorylated using recombinant p44 kinase (10 µg) in the presence of [{gamma}-32P]ATP. Reaction products were purified, resolved by SDS-PAGE, and processed for autoradiography to detect the levels of [{gamma}-32P]CCT{alpha}. In the middle and lower panels, lysates were processed for immunoblotting and probed with a polyclonal antibody to phosphorylated p42/44 kinase or to CCT{alpha}, respectively.

 



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  		FIG. 5.
Oxysterol/9-cis-RA sensitivity is altered by CCT{alpha} phosphorylation domain mutants. A, schematic illustrating the sequences of individual CCT{alpha} mutants. All constructs contain a nuclear localization signal (NLS), a catalytic core, and a membrane-binding domain, but vary in the C-terminal domain. Wild-type CCT (CCT{alpha}) and a mutant lacking the entire phosphorylation domain (CCT{alpha}314) were used as controls. Three truncated constructs each containing 328 residues (CCT{alpha}328, CCT{alpha}328SA, and CCT{alpha}328SAquad) differ only in the numbers of C-terminal proline-directed sites. Two full-length CCTs were tested: CCT{alpha}quad contains the entire C terminus but with modified residues identical to CCT{alpha}328SAquad, whereas CCT{alpha}315 harbors a point mutation at Ser315. B, cells were transfected with plasmids encoding full-length CCT{alpha} or one of four truncated mutants (CCT{alpha}328, CCT{alpha}328SA, CCT{alpha}328SAquad, and CCT{alpha}314). Cells were subsequently exposed to medium alone (Control) or in combination with 22-HC (25 µM) with 9-cis-RA (1 µM) (22HC/RA) for 24 h and pulsed with 2 µCi of [3H]choline to determine radioactivity in cellular [3H]PtdCho as described in the legend to Fig. 1. C, cells were transfected with plasmids encoding wild-type CCT{alpha} or one of two full-length mutants harboring mutations within the C terminus (CCT{alpha}quad and CCT{alpha}315). Cells were subsequently exposed to medium with or without 22-HC with 9-cis-RA and processed for PtdCho synthesis as described for B. Data in B and C are from at least three independent experiments. D, shown is the 32P labeling of CCT{alpha} mutants in vitro. Cells were transfected with His-tagged CCT{alpha} constructs, and CCT{alpha} was purified, dephosphorylated using alkaline phosphatase, and then phosphorylated using recombinant p42 kinase (10 µg) in the presence of [{gamma}-32P]ATP. Reaction products were purified, resolved by SDS-PAGE, and processed for autoradiography (upper panel) or CCT{alpha} immunoblotting (lower panel). The bar graph shows the densitometric ratio of CCT{alpha} phosphorylation mutants to total CCT{alpha} protein. E, shown is the in vivo phosphorylation of CCT{alpha} mutants. Cells were transfected with His-tagged CCT{alpha} constructs with (+) or without (-) the constitutively active MEK1 plasmid. After 24 h, cells were rinsed and harvested; His-tagged CCT{alpha} was purified; and samples processed for phosphoserine immunoblotting. F, cells transfected as described for E were also pulsed with 2 µCi of [3H]choline as described above, and radioactivity in cellular [3H]PtdCho was determined. *, p < 0.05 versus control plasmids as determined by analysis of variance.

 
When [3H] choline incorporation into PtdCho was expressed as disintegrations/min/mg of protein for these constructs, 22-HC and 9-cis-RA decreased labeling from 39,828 ± 5780 (control) to 19,063 ± 2258 dpm/mg of protein in wild-type CCT{alpha}, whereas expression of the CCT{alpha}328SAquad construct resulted in values that were comparable between the groups (34,102 ± 4665 dpm/mg (control) to 36,117 ± 5455 dpm/mg of protein). Thus, Ser315 of CCT{alpha} is a required phosphorylation site for 22-HC and 9-cis-RA to maximally inhibit PtdCho synthesis. To determine whether mutagenesis of Ser315 is sufficient to confer oxysterol resistance, cells were transfected with a full-length construct in which alanine was substituted only at Ser315 (CCT{alpha}315) (Fig. 5C). We also tested a full-length CCT{alpha} construct with mutations identical to those in CCT{alpha}328SAquad. Indeed, the rates of PtdCho synthesis were not significantly different between cells treated with 22-HC and 9-cis-RA versus the control after transfection with either CCT{alpha}315 or CCT{alpha}quad. Further analysis revealed that 22-HC and 9-cis-RA treatment decreased CCT activity from 4.05 ± 0.4 to 2.58 ± 0.3 nmol/min/mg of protein (p < 0.01) after transfection of full-length CCT{alpha}, whereas the agonists did not significantly alter enzyme activity after expression of CCT{alpha}315 (4.67 ± 0.3 (control) to 4.47 ± 0.3 (22-HC and 9-cis-RA) nmol/min/mg of protein). Collectively, these results suggest that mutagenesis of Ser315 is both necessary and sufficient to significantly overcome inhibition of PtdCho synthesis after LXR/RXR agonist treatment (Fig. 5C).

We next investigated whether the CCT{alpha} mutants exhibit different sensitivities to in vitro phosphorylation by ERK (Fig. 5D). Following cellular expression, CCT{alpha} mutants were purified, dephosphorylated in vitro with alkaline phosphatase, and phosphorylated with p42 kinase in the presence of [{gamma}-32P]ATP. Wild-type CCT{alpha} was substantially phosphorylated under these conditions, whereas CCT{alpha}315 and CCT{alpha}quad exhibited relatively low levels of {gamma}-32P labeling (Fig. 5D). To determine whether p44/42 kinase differentially increased phosphorylation of CCT{alpha} mutants in vivo, cells were cotransfected with His-tagged mutants and the MEK1 plasmid, and CCT{alpha} was purified and processed for phosphoserine analysis. Compared with control cells (not transfected with MEK1), cells cotransfected with MEK1 and the full-length CCT{alpha} plasmid displayed increased levels of enzyme phosphorylation (Fig. 5E, first and second lanes). In contrast, the levels of phosphorylation were unchanged in cells cotransfected with MEK1 and either CCT{alpha}315 or CCT{alpha}quad compared with matched controls not transfected with the kinase plasmid. These results demonstrate that CCT{alpha}315 is significantly less sensitive to MAPK-mediated phosphorylation both in vivo and in vitro. Finally, cells cotransfected in this manner were also analyzed for [3H]choline incorporation into PtdCho. Although cells cotransfected with MEK1 and the full-length CCT{alpha} plasmid exhibited a 38% reduction in PtdCho synthesis, MEK1 did not alter radiolabeling in the phospholipid in cells transfected with either CCT{alpha}315 or CCT{alpha}quad (Fig. 5F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
These results show that oxysterols in combination with their obligate partner, 9-cis-RA, significantly inhibit cellular PtdCho biosynthesis by ERK-dependent phosphorylation of the regulatory enzyme CCT{alpha}. ERK binds CCT{alpha} via a unique docking region that was mapped to an amphipathic helical region previously shown to interact with membranes (36, 37). This docking region is upstream of a CCT{alpha} phosphoacceptor site that was functionally characterized in vivo; indeed, by expressing CCT{alpha} variants harboring truncated C-terminal phosphorylation domains or mutations at proline-directed sites, we observed marked variations in the sensitivity of cells to LXR/RXR agonists. Specifically, we have shown that Ser315 is a critical site that is targeted by p42/44 kinase, as mutagenesis of this site was not only required but sufficient to substantially block the inhibitory effects of 22-HC/9-cis-RA on PtdCho synthesis. The significance of these results is that oxysterol activation of stress kinases could significantly reduce de novo synthesis of PtdCho and alveolar secretion of surfactant phospholipid, thereby accelerating pulmonary atelectasis.

Reversible phosphorylation of CCT{alpha} is the most likely mechanism whereby LXR/RXR agonists inhibit CCT activity and PtdCho synthesis. CCT{alpha} phosphorylation has been linked to membrane phospholipid synthesis with cell division (18, 38, 39), but to our knowledge, this study is the first showing the physiological relevance of specific phosphoacceptor sites. Our results mechanistically differ from the inhibitory effects of 25-HC on PtdCho synthesis that are regulated by cholesterol biosynthesis (40, 41). In our experiments, we used 22-HC, a more potent LXR ligand that, in combination with 9-cis-RA, did not reduce CCT{alpha} protein expression. Rather, the magnitude by which 22-HC and 9-cis-RA inhibited CCT{alpha} activity inversely correlated with the degree of enzyme phosphorylation (Figs. 1 and 2). Our data also indicate that the p42/44 MAPK pathway is the primary mediator for 22-HC and 9-cis-RA signaling, as (i) the kinetics of p42/44 kinase activation by 22-HC and 9-cis-RA coincided with CCT{alpha} docking, phosphorylation, and inhibition of enzyme activity; (ii) CCT{alpha} and p42/44 kinase were physically associated in vivo; (iii) p42/44 MAPK phosphorylated CCT{alpha}; and (iv) the effects of these agents on PtdCho synthesis were reversed by PD98059 and dominant-negative p42 kinase (Fig. 3). In particular, we observed a fairly tight correlation between p42/44 kinase activation and inhibition of CCT activity by 22-HC and 9-cis-RA. The biphasic activation of MAPKs (up to 6 h and at 18–21 h) in response to 22-HC and 9-cis-RA was temporally linked to inhibition of PtdCho synthesis at 6 and 24 h by these agents (Figs. 1 and 3A). Even though total cellular phosphorylated p42/44 kinase activity dissipated by 24 h, a distinct pool of active kinase was still bound to CCT{alpha} after 22-HC and 9-cis-RA exposure (Fig. 3F); this, together with the extended half-life of CCT{alpha} protein (26), provides a plausible explanation for the reduced levels of CCT activity and PtdCho synthesis observed 24 h following agonist treatment (Fig. 1). Furthermore, this pattern is highly consistent with the kinetics of ERKs, as they regulate a variety of physiological readouts such as platelet-derived growth factor-induced mitogenesis (31), cellular propagation of influenza (32), leukotactin-1 control of cell cycle progression (33), and effects of phorbol esters and nerve growth factors on cell survival (34, 42). Although the data favor 22-HC and 9-cis-RA activation of p42/44 kinase, concurrent activation of p38 kinase, JNK, or p34cdc2 is also possible, as these enzymes utilize similar minimal recognition motifs ((Ser/Thr)-Pro) for substrate phosphorylation (43, 44). On the other hand, because (Ser/Thr)-Pro sequences are fairly ubiquitous within substrates, the specificity and prevention of inappropriate cross-talk between related kinases are provided by assembly of scaffolding proteins and interaction with docking regions on target proteins (45).

We localized an ERK-docking region to the distal membrane-binding region of CCT{alpha}. This motif (GSFLEMFGPEGALK) is rich in non-polar residues that help form a putative amphipathic {alpha}-helix, helix-1, for membrane insertion and lipid activation (36). This interaction resembles that of p44 kinase docking with PDE4 cAMP phosphodiesterase, in which a docking site is also located on an exposed {alpha}-helix (46). The results of deletion analysis using truncated mutants of helix-1 showed that residues 287–300 are required for p42/44 kinase-CCT{alpha} binding and that this domain is also required for optimal phosphorylation of the enzyme. Our results do not rule out the possibility that the putative docking region within CCT{alpha} might involve a broader motif encompassing the entire {alpha}-helix or that other residues (e.g. residues 260–286) might optimize ERK-CCT{alpha} binding. This docking sequence is enriched with hydrophobic residues typical of many docking sites, but it has few basic residues and lacks LXL and FXF motifs commonly seen in some substrates (35, 47–49). However, many MAPK substrates either lack or display little similarity in their core sequences for docking, suggestive of yet unidentified binding motifs. Indeed, the sequence of the ERK-docking region within CCT{alpha} shows little similarity to other ERK targets, but resembles that of activating transcription factor-2, a p38/JNK substrate, suggesting the existence of a novel MAPK-docking motif (47–49). It is possible that this variation in the docking sequence for CCT{alpha} provides greater promiscuity for kinase binding or simply provides a recognition site whereby p42/44 kinase can influence CCT{alpha} membrane association. This scenario might be analogous to mutations within hydrophobic docking residues for MEK1 that alter the cellular distribution of p44 kinase (50), thioredoxin-1 docking with PTEN phosphatase inhibiting its catalytic activity and membrane association (51), or protein kinase C binding and phosphorylation of its adapter molecule triggering translocation of the complex to the cytoplasm (52). It is also possible that ERKs utilize other motifs within the CCT{alpha} sequence for binding. For example, sequence 13KRRK within the nuclear localization signal, 158LAEHR within the catalytic domain, and 242LQER in the membrane-binding region of CCT{alpha} share high similarity with known docking sites in ribosomal S6 kinase (53), p90rsk (54), and MAPK phosphatase (55), respectively. However, candidate mutagenesis or deletion of these regions failed to interrupt p42/44 binding to CCT{alpha}.

To identify the phosphorylation targets for ERK in response to 22-HC and 9-cis-RA, we expressed other enzyme mutants containing C-terminal phosphorylation domain truncations or point mutations at proline-directed sites within the CCT{alpha} C terminus. Indeed, the CCT{alpha} C-terminal phosphorylation region is a necessary signal to direct inhibition of PtdCho synthesis by LXR/RXR agonists, as expression of a mutant devoid of the entire phospho-terminal domain (CCT{alpha}314) significantly blocked the inhibitory effects of 22-HC and 9-cis-RA. In contrast, cells transfected with CCT{alpha}328, which lacks 11 of 16 C-terminal serines, largely retained sensitivity to these agonists. Thus, five C-terminal phosphorylation sites (Ser315, Ser319, Ser321, Ser322, and Ser323) within CCT{alpha}328 are potential kinase targets. Sequence analysis of CCT{alpha}328 revealed three Ser/Pro motifs recognized by p42/44 kinases at Ser315, Ser319, and Ser323, indicating that an oxysterol-sensitive region might be localized to residues within the N-terminal end of the phosphorylation domain. To examine this, two additional CCT{alpha} mutants were tested. Using CCT{alpha}328SA, we observed that mutagenesis of Ser at positions 321, 322, and 323 was insufficient to overcome the inhibitory effects of 22-HC and 9-cis-RA on PtdCho synthesis. Mutagenesis of Ser315 to alanine within the context of serial mutations at Ser321, Ser322, and Ser323 (CCT{alpha}328SAquad) dramatically abolished agonist-induced inhibition of phospholipid synthesis (Fig. 5). Moreover, expression of CCT{alpha}315, a construct with a point mutation at Ser315, restored the ability of the cells to synthesize PtdCho to near control levels after oxysterol treatment. Limited {gamma}-32P labeling was also detected by CCT{alpha}315 or CCT{alpha}quad using p42 kinase in vitro. Taken together, these data suggest that Ser315 is a mechanistically relevant site mediating p42/44 kinase-directed CCT{alpha} phosphorylation following 22-HC and 9-cis-RA exposure. Single site modification of such serine targets for p42/44 kinase leading to altered biological activity is well described for several proteins, including hormone-sensitive lipase, sterol regulatory element-binding protein, GTPase-activating protein, and the transcription factor GATA4 (56–59). Our demonstration that expression of CCT{alpha} mutants with modified ERK phosphorylation sites restores surfactant lipid synthesis to high levels raises intriguing possibilities for the use of gene transfer approaches employing "designer" surfactant biosynthetic enzymes in lung injury.


   FOOTNOTES
 
* This work was supported by Merit Review Awards (to A. B. C. and R. K. M.) from the Office of Research and Development, Department of Veterans Affairs, and by Grants RO1 HL055584, HL071040, HL080229, and HL068135 (to R. K. M.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

** To whom correspondence should be addressed: Dept. of Internal Medicine, Pulmonary and Critical Care Div., C-33K, GH, University of Iowa College of Medicine, 200 Hawkins Dr., Iowa City, IA 52242. Tel.: 319-356-1265; Fax: 319-353-6406; E-mail: rama-mallampalli{at}uiowa.edu.

1 The abbreviations used are: DSPtdCho, disaturated phosphatidylcholine; LXR, liver X receptor; 9-cis-RA, 9-cis-retinoic acid; RXR, 9-cis-retinoic acid receptor; PtdCho, phosphatidylcholine; CCT, CTP:phosphocholine cytidylyltransferase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; 22-HC, 22-hydroxycholesterol; MLE, murine lung epithelial; GST, glutathione S-transferase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MOPS, 4-morpholinepropanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Ronald Salome for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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