Requirements and Effects of Palmitoylation of Rat PLD1*

Zhi Xie, Wan-Ting Ho, and John H. ExtonDagger

From the Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295

Received for publication, October 16, 2000, and in revised form, November 6, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs (HXKX4D), denoted HKD, located in the N- and C-terminal halves, which are required for phospholipase D activity. The two halves of rPLD1 can associate in vivo, and the association is essential for catalytic activity and Ser/Thr phosphorylation of the enzyme. In this study, we found that this association is also required for palmitoylation of rPLD1, which occurs on cysteines 240 and 241. In addition, palmitoylation of rPLD1 requires the N-terminal sequence but not the conserved C-terminal sequence, since rPLD1 that lacks the first 168 amino acids is not palmitoylated in vivo, while the inactive C-terminal deletion mutant is. Palmitoylation of rPLD1 is not necessary for catalytic activity, since N-terminal truncation mutants lacking the first 168 or 319 amino acids exhibit high basal activity although they cannot be stimulated by protein kinase C (PKC). The lack of response to PKC is not due to the lack of palmitoylation, since mutation of both Cys240 and Cys241 to alanine in full-length rPLD1 abolishes palmitoylation, but the mutant still retains basal activity and responds to PKC. Palmitoylation-deficient rPLD1 can associate with crude membranes; however, the association is weakened. Wild type rPLD1 remains membrane-associated when extracted with 1 M NaCl or Na2CO3 (pH 11), while rPLD1 mutants that lack palmitoylation are partially released. In addition, we found that palmitoylation-deficient mutants are much less modified by Ser/Thr phosphorylation compared with wild type rPLD1. Characterization of the other cysteine mutations of rPLD1 showed that mutation of cysteine 310 or 612 to alanine increased basal phospholipase D activity 2- and 4-fold, respectively. In summary, palmitoylation of rPLD1 requires interdomain association and the presence of the N-terminal 168 amino acids. Mutations of cysteines 240 and 241 to alanine abolish the extensive Ser/Thr phosphorylation of the enzyme and weaken its association with membranes.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phospholipase D (PLD)1 is a ubiquitous enzyme found in bacteria, fungi, plants, and mammals (1). It hydrolyzes phosphatidylcholine (PC) to phosphatidic acid and choline. Phosphatidic acid is generally recognized as the signaling product of PLD and functions as an effector in different physiological processes. It can also be converted to diacylglycerol or to lysophosphatidic acid. Diacylglycerol is a well characterized activator for protein kinase C (PKC), while lysophosphatidic acid is a major extracellular signal that acts on specific cell surface receptors. PLD also catalyzes a phosphatidyl transfer reaction using primary alcohols as nucleophilic acceptors to produce phosphatidylalcohols. This reaction is used as a specific measure of PLD activity.

PLD activity has been found to be highly regulated (2). Different factors, including protein-tyrosine kinases, PKC, heterotrimeric and small G proteins, and intracellular Ca2+ regulate PLD activity directly or indirectly (2-4). Based on the wide involvement of PLD in signaling pathways and the actions of its products, multiple functions of PLD have been proposed (2), which include signal transduction, secretion, membrane trafficking, cytoskeleton reorganization, and apoptosis (5).

Two isoforms of mammalian PLD (PLD1 and PLD2) have been cloned. These isoforms share about 50% amino acid similarity but exhibit quite different regulatory properties. PLD1 has a low basal activity and responds strongly to PKC and to members of the Rho and ARF families of small G proteins (6-9). The PKC interaction domain has been mapped to the N-terminal part of the molecule (10-12), while the Rho interaction domain has been localized to the C-terminal part of the enzyme (13). PLD2, on the other hand, exhibits a high basal activity (14-16), and some reports have shown that it is regulated by calcium, PKC, ARF, the epidermal growth factor receptor, and alpha -actinin (16-20).

PLD belongs to a superfamily defined by the motif, HXKX4D, denoted "HKD" (22-24). The enzymes within the family exhibit diverse functions and include phospholipid synthases, poxvirus envelope proteins, a Yersinia murine toxin, and the Nuc endonuclease. Despite the distinct substrate specificities of the superfamily members, the consensus HKD motif appears to be essential for their enzymatic activity. PLD contains two copies of the HKD motif located in the N- and C-terminal halves of the molecule, respectively. Mutation of either HKD motif inactivates human PLD1 and mouse PLD2 (12, 25). Biochemical and structural studies of the Nuc endonuclease and Yersinia toxin suggest that the histidine residue in the conserved motif is directly involved in the catalytic reaction by forming a phosphoenzyme intermediate (26-28).

Our studies on the rat PLD1 (rPLD1) isoform showed that the enzyme could be split into two halves and that PLD activity could be restored when the two fragments were coexpressed in COS7 cells. Coimmunoprecipitation experiments showed that the N- and C-terminal fragments could physically associate (11), and it was proposed that the association brought the two HKD domains together to form a catalytic center. Further studies showed that conserved amino acids in the HKD domains were important for the interdomain association and that the association was essential for the catalysis of the enzyme (29). In addition, we found that rPLD1 can be modified by Ser/Thr phosphorylation, and this modification is also required for the association of the N- and C-terminal halves of rPLD1. We also found that the phosphorylated rPLD1 localizes exclusively in the membrane fraction (29). In the present study, we found that the interdomain association was also required for the palmitoylation of rPLD1. We investigated the role of this lipid modification and of cysteine amino acids in the catalysis and properties of rPLD1.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Materials-- 4beta -Phorbol 12-myristate 13-acetate, phosphatidylinositol 4,5-bisphosphate, bovine serum albumin, Triton X-100, and microcystin were from Sigma. Phosphatidylethanolamine, PC, and phosphatidylbutanol standard were from Avanti Polar Lipids Corp. Dipalmitoyl[2-palmitoyl-9,10-3H]PC, dipalmitoyl[choline-methyl-3H]PC, [3H]myristic acid, and [3H]palmitic acid were from PerkinElmer Life Sciences. Protein A-agarose beads, Dulbecco's modified Eagle's medium, penicillin, streptomycin, and fetal bovine serum were from Life Technologies, Inc. The transfection reagent FuGENE6 and the protease inhibitor mixture were from Roche Molecular Biochemicals. COS7 cells were from the American Type Culture Collection. SDS-polyacrylamide gels were from Novex. The PcDNA3 vectors and the monoclonal antibodies against the V5 and Xpress epitope tags were from Invitrogen. Anti-mouse antibodies conjugated with horseradish peroxidase were from Vector Laboratories. The AmplifyTM and Hyperfilm-MP were from Amersham Pharmacia Biotech.

Plasmid Construction-- The N-terminal Xpress-tagged full-length or truncated rPLD1 or the N- or C-terminal fragments of rPLD1 with coding regions corresponding to amino acids 1-584 and 585-1036, respectively, were created by polymerase chain reaction amplification and subcloned (11). The deletion or site-directed mutations of rPLD1 were generated as described in the QuickChangeTM Site-Directed Mutagenesis instruction manual from Stratagene. All of the constructs were sequenced to verify the coding regions of rPLD1. The oligonucleotide pairs from 5' to 3' for site-directed mutagenesis were as follows: rPLD1(C79A), GATCTACCTCTCTGGCGCTCCTGTAAAAGCAC and GTGCTTTTACAGGAGCGCCAGAGAGGTAGATC; rPLD1(C240A), GATACCAGGTGTGAATGCCTGTGGCCATGGAAGA and TCTTCCATGGCCACAGGCATTCACACCTGGTATC; rPLD1(C241A), CCAGGTGTGAATTGCGCTGGCCATGGAAGAGCC and GGCTCTTCCATGGCCAGCGCAATTCACACCTGG; rPLD1(C240A,C241A), GATACCAGGTGTGAATGCCGCTGGCCATGGAAGAGC and GCTCTTCCATGGCCAGCGGCATTCACACCTGGTATC; rPLD1(C310A), GACACTGATTTTAAAAGCTAACAGCTACAGACA and TGCATGTCTGTAGCTGTTAGCTTTTAAAATCAGTGTC; rPLD1(C403A), CGCTGGAGGCTGGACGCCATCCTCAAACGGAA and TTCCGTTTGAGGATGGCGTCCAGCCTCCAGCG; rPLD1(C612A), CATGGGAAGGATTACGCCAACTTTGTCTTCAAG and CTTGAAGACAAAGTTGGCGTAATCCTTCCCATG.

Cell Culture and Transfection-- COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum in humidified 10% CO2. Six-well plates were seeded with 2 × 105 COS7 cells/well, and 10-cm dishes were seeded with 6 × 105 cells and transfected with FuGENE6 according to the manufacturer's instructions.

In Vivo PLD Assay-- After a 6-h transfection with FuGENE6, COS7 cells in six-well plates were serum-starved (0.5% fetal bovine serum in Dulbecco's modified Eagle's medium) in the presence of 1 µCi/ml [3H]myristic acid. After overnight starvation, the cells were washed with phosphate-buffered saline (PBS) and incubated in serum-free medium supplemented with 0.3% bovine serum albumin for 50 min. PLD activity was then assayed as described (30). Briefly, cells were incubated in 0.3% 1-butanol for 25 min. Cells were then washed with ice-cold PBS and stopped with methanol. Lipids were extracted, and the phosphatidylbutanol product was resolved by thin layer chromatography. Bands comigrating with a phosphatidylbutanol standard were quantitated by scintillation counting.

Subcellular Fractionation-- COS7 cells in 100-mm plates were harvested after transfection with wild type or mutant rPLD1 constructs and starved overnight as described above. For each rPLD1 construct, we transfected six 100-mm plates of COS7 cells, and the cells were combined and washed and harvested with ice-cold PBS buffer. They were then centrifuged and resuspended in 750 µl of ice-cold lysis buffer (25 mM Hepes, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture containing leupeptin, aprotinin, and pefabloc). The cells were passed through a 27-gauge needle seven times, and the cell lysate was centrifuged at 500 × g for 10 min to remove unbroken cells and nuclei. The supernatant was then centrifuged at 120,000 × g for 45 min at 4 °C to separate the cytosolic and crude membrane fractions. The particulate fraction was washed twice with the lysis buffer and resuspended in 450 µl of this buffer by passing through a 27-gauge needle until the pellet was resuspended. The suspension (150 µg of protein) was then split into three parts and supplemented with 100 µl of lysis buffer containing the appropriate concentration of either NaCl, Na2CO3 (pH 11), or Triton X-100 to make the final concentration of these reagents in the buffer 1 M NaCl, 0.1 M Na2CO3 (pH 11), or 1% Triton X-100, respectively. The mixtures were further incubated at 4 °C for 30 min and then centrifuged at 120,000 × g for 45 min at 4 °C to separate the supernatant and pellet fraction. The pellet fraction was rinsed with 750 µl of hypotonic lysis buffer two times and then resuspended with 250 µl of lysis buffer. Equal volumes of the cytosolic and membrane fractions were analyzed by Western blotting. The intensity of the bands corresponding to rPLD1 in the cytosol or membrane fractions was quantitated by densitometry. The amounts of the indicated rPLD1 constructs in the cytosol or membrane fractions were calculated as percentage of the total enzyme in both.

Western Blotting-- Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% gels and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp.). The blots were then blocked with 5% nonfat milk and incubated with appropriate primary antibodies (1 mg/ml) (1:5000) followed by incubation with horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were detected using enhanced chemiluminescence.

Immunoprecipitation-- COS7 cells cultured on 6-well plates were transfected and starved as described above. The cells (3 µg of protein) were washed twice with ice-cold PBS and then resuspended in 300 µl of immunoprecipitation buffer containing 25 mM Hepes, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 50 mM KC1, 0.5% Triton X-100, 10 mM NaF, 10 mM Na4P2O7, 1.2 mM Na3VO4, 1 µM microcystin, and two tablets of protease mixture. The cell suspension was then passed through a 27-gauge needle five times, and the resulting cell lysate was centrifuged at 15,000 rpm in an Eppendorf microcentrifuge for 10 min at 4 °C to pellet the unbroken cells. The supernatant was then precleared by mixing it with 1 µg of affinity-purified mouse IgG and 20 µl of a 1:1 slurry of protein A beads for 1 h at 4 °C. The mixture was then centrifuged, and the resulting supernatant was incubated with 2 µl of Xpress mouse antibody (1 mg/ml) and 20 µl of protein A beads overnight. The immunoprecipitates were washed four times with the lysis buffer and then resuspended in SDS sample buffer and analyzed by Western blotting.

Palmitoylation Assay-- After 24 h of transfection, COS7 cells cultured on six-well plate's were preincubated for 1 h in serum-free medium supplemented with 0.3% bovine serum albumin and then incubated with 0.2 mCi/ml [3H]palmitic acid for 4 h in the preincubation medium. [3H]palmitic acid was dried under N2 and resuspended in ethanol, and the final volume of ethanol was 1% of that of the incubation medium. After the radiolabeling, cells were washed twice with cold PBS buffer and then scraped with the immunoprecipitation buffer and immunoprecipitated followed by washes as described above. The resulting immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting.

For fluorography, protein samples were separated by SDS-PAGE and then incubated in fixing solution containing isopropyl alcohol/H2O/acetic acid (25:65:10) for 30 min. The gel was then treated with AmplifyTM for 30 min, dried under vacuum, and exposed to Hyperfilm-MP at -80 °C. For hydroxylamine treatment, duplicate gels were treated with either 1 M Tris-HCl (pH 7.0) or 1 M hydroxylamine (pH 7.0) overnight at room temperature and then subjected to fluorography as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Palmitoylation of rPLD1-- Our study of the palmitoylation of rPLD1 was initiated when we tried to examine the role of the conserved amino acids at the C-terminal end in PLD catalysis. Mutagenesis studies showed that an intact C-terminal end and the hydrophobicity of the last four amino acids, EVWT, were essential for the catalytic activity of the enzyme (31). We investigated whether this hydrophobicity was required for a potential palmitoylation of rPLD1, since human PLD1 had been shown to be palmitoylated, and lack of this lipid modification was reported to destroy the catalytic activity of the enzyme (32, 33). We first transfected COS7 cells with either wild type rPLD1 or an inactive C-terminal deletion mutant rPLD1-(1-1032) and radiolabeled the cells with [3H]palmitic acid. All of the rPLD1 constructs used in the studies were tagged with Xpress epitope if not specified. The exogenously transfected rPLD1 constructs were then immunoprecipitated by anti-Xpress antibodies, and the immunoprecipitates were analyzed by Western blotting (Fig. 1A) and fluorography (Fig. 1B) for the expression and the palmitoylation of the constructs, respectively. As shown in Fig. 1B, tritium incorporation was observed in a 120-kDa band that corresponded to the size of both wild type and the C-terminal deleted rPLD1. The reduced tritium incorporation observed in the rPLD1-(1-1032) is attributable to the reduced level of this mutant in the immunoprecipitates (Fig. 1A). Thus, both wild type rPLD1 and the C-terminal deletion mutant of the enzyme were palmitoylated in COS7 cells. The incorporation of tritium was analyzed further by treating gels containing tritium-labeled rPLD1 constructs with M Tris-HCl (pH 7.0) or 1 M hydroxylamine (pH 7.0). Only the hydroxylamine treatment released the tritium incorporated into wild type rPLD1 and its C-terminal deletion mutant, confirming that the palmitoylation of the enzyme is through a thioester linkage (data not shown).


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Fig. 1.   Palmitoylation of rPLD1. Wild type or C-terminally truncated rPLD1 was transfected into COS7 cells as indicated in the figure. After 24 h of expression, COS7 cells were labeled with [3H]palmitic acid for 4 h and harvested and immunoprecipitated by anti-Xpress antibodies. The immunoprecipitates were then analyzed either by SDS-PAGE followed by Western blotting with anti-Xpress antibodies (A) or by fluorography (exposed at -80 °C for 30 days) (B) as described under "Experimental Procedures."

Palmitoylation of rPLD1 Requires the First 168 Amino Acids-- Alanine mutagenesis of cysteine residues of rPLD1 showed that the palmitoylation of rPLD1 occurs on Cys240 and Cys241. As shown in Fig. 2, wild type PLD1 was expressed (Fig. 2A, lane 1) and palmitoylated (Fig. 2B, lane 1) in COS7 cells labeled with [3H]palmitic acid. However, no tritium incorporation was observed in the double cysteine mutant, rPLD1(C240A,C241A) (Fig. 2B, lane 2), although the constructs were expressed and immunoprecipitated from COS7 cells (Fig. 2A, lane 2). Mutation of the other cysteine residues did not affect the palmitoylation of rPLD1 (see Fig. 4 or data not shown). The finding that rPLD1 was palmitoylated on Cys240 and Cys241 was consistent with studies of human PLD1 (32).


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Fig. 2.   Requirement of the first 168 amino acids for palmitoylation of rPLD1. Wild type or site-directed or deletion mutants of rPLD1 were transfected into COS7 cells as indicated in the figure. After 24 h of expression, COS7 cells were labeled with [3H]palmitic acid for 4 h and harvested and immunoprecipitated by anti-Xpress antibodies. A, the immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting with anti-Xpress antibodies. A*, to obtain a comparable ECL signal so that single and double bands of different constructs could be visualized, the same samples in A were loaded at different amounts and reanalyzed by SDS-PAGE followed by Western blotting with anti-Xpress antibodies. B, after the immunoprecipitates were analyzed by SDS-PAGE, the gel was first incubated with 1 M Tris-HCl (pH 7.0) overnight and then analyzed by fluorography (exposed at -80 °C for 40 days). C, after the immunoprecipitates were analyzed by SDS-PAGE, the gel was incubated with 1 M hydroxylamine (pH 7.0) overnight and then analyzed by fluorography as described under "Experimental Procedures."

Characterization of the palmitoylation-deficient human PLD1 had shown that the lipid modification was required for efficient action of the enzyme, since mutation of both of the palmitoylation sites abolished most of the catalytic activity of the enzyme (32). This was surprising, since our previous studies of N-terminal deletion mutants of rPLD1 showed that the basal catalytic activity of rPLD1-(320-1036) was not decreased but was enhanced (10, 11). A possible explanation for this discrepancy was that alternative cysteines were used for palmitoylation in this N-terminal deletion mutation. We therefore investigated the palmitoylation of this mutant along with that of two other rPLD1 mutants that lacked either the first 50 or the first 168 amino acids. COS7 cells transiently transfected with the N-terminal deletion mutants were labeled with [3H]palmitic acid, and the expressed rPLD1 constructs were immunoprecipitated with anti-Xpress antibodies and analyzed by Western blotting (Fig. 2A) and fluorography (Fig. 2, B and C). As shown in Fig. 2B, rPLD1-(51-1036) was palmitoylated in COS7 cells, but no apparent tritium incorporation was observed into rPLD1-(320-1036), which lacks Cys240 and Cys241. Surprisingly, rPLD1-(169-1036), which still retains Cys240 and Cys241, also lacked detectable tritium incorporation. This indicates that palmitoylation of rPLD1 requires the presence of the N-terminal 168 amino acids. More importantly, the fact that the N-terminal deletion mutants, rPLD1-(169-1036) and rPLD1-(320-1036), that lacked palmitoylation still exhibited high basal activity (10, 11) indicates that palmitoylation of rPLD1 is not necessary for its catalytic activity. The same protein samples shown in Fig. 2A were also reanalyzed in Fig. 2A*, with the difference that different amounts were loaded so that a comparable ECL signal of the protein samples could be obtained by Western blotting.

Palmitoylation of rPLD1 Requires Interdomain Association between the N- and C-terminal Fragments-- Our previous studies showed that rPLD1 could be split into two halves and that the N- and C-terminal halves associated in vivo. This interdomain association was important for the catalytic activity of the enzyme and the Ser/Thr phosphorylation of the rPLD1 (11, 29). We therefore investigated the role of the interdomain association on the palmitoylation of rPLD1. COS7 cells were cotransfected with wild type or mutated N- and C-terminal halves of rPLD1 as indicated in Fig. 3. The N-terminal fragments were tagged with V5 epitope, while the C-terminal fragment was tagged with Xpress epitope. After 24 h of expression followed by 4 h of labeling with [3H]palmitic acid, COS7 cells were harvested and immunoprecipitated with both anti-Xpress and anti-V5 antibodies. The immunoprecipitates were analyzed by Western blotting (Fig. 3A) with both anti-V5 and anti-Xpress antibodies to probe the expression of the N- and C-terminal halves of rPLD1. The palmitoylation was analyzed by fluorography to detect tritium incorporation (Fig. 3B). As shown in Fig. 3B (lane 4), tritium incorporation was detected in the ~66-kDa band, which corresponds to the size of the N-terminal half of rPLD1 when COS7 cells were cotransfected with the wild type N- and C-terminal halves. Mutation of Ile470 in the N-terminal half or of Ile870 in the C-terminal half of rPLD1 has been shown to disrupt the interdomain association (29). The interdomain interaction was almost abolished when the Ser873 in the C-terminal half was mutated to alanine (Fig. 3, C and D). When COS7 cells were transfected with the N- and C-terminal fragments carrying these mutations, no apparent tritium incorporation was detected in the N-terminal fragment of rPLD1 (Fig. 3B). Thus, the interdomain interaction between the N- and C-terminal halves of rPLD1 appears to be required for the palmitoylation of the enzyme. In addition, we found that mutation of both Cys240 and Cys241 in the N-terminal half of rPLD1 also diminished the tritium incorporation (Fig. 3B), which is consistent with the finding with full-length rPLD1 (Fig. 2). However, single mutation of Cys240 or Cys241 did not abolish the palmitoylation (data not shown), suggesting that palmitoyltransferase can act on either residue.


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Fig. 3.   Requirement of interdomain association for palmitoylation of rPLD1. Wild type or the indicated mutant N- and C-terminal halves of rPLD1 were cotransfected into COS7 cells. The N-terminal half of rPLD1 was tagged with a V5 epitope, while the C-terminal half was tagged with an Xpress epitope. After 24 h of expression, COS7 cells were labeled with [3H]palmitic acid for 4 h and harvested and immunoprecipitated by anti-Xpress and anti-V5 antibodies. A, the immunoprecipitates were then analyzed by SDS-PAGE followed by Western blotting with anti-V5 and anti-Xpress antibodies. B, the immunoprecipitates were analyzed by SDS-PAGE and then analyzed by fluorography (exposed at -80 °C for 60 days). The C-terminally V5-tagged N-terminal fragment of rPLD1-(1-584) was coexpressed either with beta -galactosidase tagged with Xpress epitope at the N terminus (LacZ) (lane 1), with the N-terminally Xpress-tagged C-terminal fragment of rPLD1 (lane 2), or with the C-terminal fragment of rPLD1 carrying an S873A mutation (lane 3) in COS7 cells. C, the cell lysate was analyzed by SDS-PAGE and Western blotted with both anti-V5 and anti-Xpress antibodies. D, the immunoprecipitates by monoclonal anti-Xpress antibodies were analyzed by SDS-PAGE followed by Western blotting with anti-V5 antibodies. A*, a darker exposure of part of the Western blot shown in A, so that the modification of the wild type N-terminal fragment could be visualized. NT, C-terminally V5-tagged N-terminal fragment of rPLD1-(1-584). CT, N-terminally Xpress-tagged C-terminal fragment of rPLD1-(585-1036).

Cysteines 240 and 241 Are Required for Extensive Ser/Thr Phosphorylation of rPLD1-- The preceding results (Fig. 3) showed that the interdomain association between the N- and C-terminal fragments is important for palmitoylation. We have previously shown that this association is also required for Ser/Thr phosphorylation modification of the enzyme (29), and the phosphorylation has been reported to play roles in locating the enzymes and responding to PMA activation (34). We therefore wondered whether these two types of posttranslational modification of rPLD1 were interrelated. As shown in the Western blot analysis in Figs. 2A* and 5B, we found that rPLD1(C240A,C241A) that lacked palmitoylation also lacked extensive Ser/Thr phosphorylation, as demonstrated by the disappearance of the bands with slower electrophoretic mobility as compared with wild type rPLD1 or those with single mutations. In addition, bands with slower electrophoretic mobility were also not observed in the N-terminal deletion mutants, rPLD1-(169-1036) and rPLD1-(320-1036), which both lacked palmitoylation (Fig. 2A*). However, rPLD1-(51-1036) that still retained palmitoylation retained extensive Ser/Thr phosphorylation (Fig. 2A*). Furthermore, when wild type and indicated mutant rPLD1 constructs (Fig. 4A) were analyzed by Western blotting with anti-Thr(P) antibodies, we found similar results. A 120-kDa band corresponding to the size of full-length rPLD1 was detected by anti-Thr(P) antibodies in wild type or single Cys-mutated rPLD1 molecules that still retained palmitoylation. However, no apparent band was detected by the antibodies when the palmitoylation-deficient mutant, rPLD1(C240A,C241A), was analyzed. In summary, these results suggested that palmitoylation of rPLD1 is required for extensive Ser/Thr phosphorylation of the enzyme. On the other hand, we found that [3H]palmitate was incorporated into rPLD1 molecules with either slow or fast electrophoretic mobility (i.e. both upper and lower bands) when analyzed by Tritium Screen (Fig. 4, B and C). Thus, extensive Ser/Thr phosphorylation appeared not to be required for palmitoylation of rPLD1.


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Fig. 4.   Palmitoylation of rPLD1 is required for extensive Ser/Thr phosphorylation of the enzyme. Wild type rPLD1 or the indicated cysteine mutants were transfected into COS7 cells. After 24 h of expression, the cells were harvested and immunoprecipitated with anti-Xpress antibodies. The immunoprecipitates were then analyzed by SDS-PAGE followed by Western blotting with antiphospho-Thr antibodies (A) or anti-Xpress antibodies (C). D, after Western blotting and enhanced chemiluminescence detection, the blot from C was rinsed with PBS, air-dried, and exposed to Tritium Screen at room temperature for 10 days and scanned by the StormTM 860 detection system from Molecular Dynamics, Inc. (Sunnyvale, CA).

Palmitoylation of rPLD1 Is Not Required for Its Response to PKC in COS7 Cells-- We noticed that rPLD1-(169-1036) and rPLD1-(320-1036), which lacked the palmitoylation and extensive Ser/Thr phosphorylation, did not respond to PMA stimulation, which activates PKC in COS7 cells. rPLD1-(51-1036), on the other hand, which retained both of the posttranslational modifications, responded to PMA stimulation. The lack of the response to PKC of the N-terminal truncation mutants could be due to the lack of N-terminal amino acids or the lack the posttranslational modifications. We therefore examined whether full-length mutant rPLD1(C240A,C241A) that lacks these posttranslational modifications could respond to PMA stimulation in COS7 cells. The cells were transfected with wild type or the indicated cysteine mutants (Fig. 5), and in vivo PLD assays were carried out by measuring the transphosphatidylation activity as described under "Experimental Procedures." As shown in Fig. 5A, although the basal activity of rPLD1(C240A,C241A)was decreased by about 60% compared with wild type rPLD1, it still retained a significant response to PMA stimulation. The single Cys mutant, rPLD1(C241A), retained wild type activity, while the basal activity and PMA response of of rPLD1(C240A) were reduced by about 30%. The expression levels of these wild type and mutant rPLD1 constructs in COS7 cells were comparable (Fig. 5B). Thus, palmitoylation (Fig. 2) and extensive Ser/Thr phosphorylation of rPLD1 (Fig. 5B) are not absolutely required for PKC stimulation of the enzyme. The finding that extensive Ser/Thr phosphorylation is not required for the rPLD1 response to PKC in vivo is consistent with our studies in vitro (29).


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Fig. 5.   Palmitoylation of rPLD1 is not required for its activation by PKC. Wild type rPLD1 or the indicated cysteine mutants were transfected into COS7 cells, and the PLD activity (A) was measured in the absence or presence of 100 nM PMA treatment for 20 min as described under "Experimental Procedures." The PLD activity is represented by the percentage of the phosphatidylbutanol synthesized versus the total tritium incorporated into the lipid of the COS7 cells. The results are representative of three experiments performed in triplicate. Mean values ± S.E. are shown. The gray and black bars represent the PLD activity in the absence and presence of PMA treatment, respectively. The corresponding cell lysate was analyzed by Western blotting (B) with anti-Xpress antibodies to examine the expression level of wild type or mutant rPLD1 constructs in COS7 cells.

Palmitoylation of rPLD1 Affects the Association Affinity of the Enzyme with Membranes-- Palmitoylation has been shown to play a role in membrane association of proteins (35). Here we utilized biochemical fractionation to examine the effect of the palmitoylation of rPLD1 on its membrane association. As described under the "Experimental Procedures," membrane fractions of COS7 cells expressing either wild type or mutant rPLD1 were first isolated under the hypotonic conditions, where no salt or detergent was added to the extraction buffer. The resulting membrane fractions were resuspended and incubated with the extraction buffer containing 1 M NaCl, 0.1 M Na2CO3 (pH 11), or 1% Triton X-100 at 4 °C for 30 min and then centrifuged to separate the membrane and supernatant fractions. As shown in Fig. 6, wild type rPLD1 remained membrane-associated when extracted with hypotonic buffer, 1 M NaCl, or 0.1 M Na2CO3 (pH 11). About 10% of wild type rPLD1 was released into supernatant when the membrane fractions were further extracted with 1% Triton X-100. rPLD1 mutants that carried a single cysteine mutation at either position 240 or 241 also remained associated with the membrane fraction when extracted with hypotonic buffer (Fig. 6A, lanes 7 and 10) and were minimally released when the membranes were extracted with either 1 M NaCl, 0.1 M Na2CO3 (pH 11) or 1% Triton X-100 (Fig. 6, A-C, lanes 8 and 11). However, when palmitoylation-deficient rPLD1(C240A,C241A) was analyzed, we found that ~10% of the protein was present in the cytosolic fraction (Fig. 6A, lane 4). When the membrane fractions were further extracted, we found an additional 20-30% of rPLD1(C240A,C241A) was released by 1 M NaCl or 1% of Triton X-100 (Fig. 6, A and C, lane 5). However, when the membrane fraction was further extracted with 0.1 M Na2CO3 (pH 11), an additional 60% was released (Fig. 6B, lane 5). Na2CO3 (pH 11), at a concentration of 0.1 M, primarily acts to denature protein structure without disrupting the organization of the bilayer and, thus, often dissociates peripheral membrane proteins from membranes (36). These results indicate that although the palmitoylation-deficient rPLD1 can associate with membranes, its association is weakened compared with wild type enzyme.


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Fig. 6.   Membrane association of palmitoylation-deficient rPLD1. Wild type rPLD1 or the indicated cysteine mutants were transfected into COS7 cells. After 24 h of expression, the cells were harvested, fractionated by hypotonic buffer, and ultracentrifuged to separate the cytosol and crude membrane fractions. The crude membrane fractions were then further extracted by incubation with either 1 M NaCl (A), 0.1 M Na2CO3 (B), or 1% Triton X-100 (C) for 30 min at 4 °C and ultracentrifuged to separate the supernatant and pellet fractions. Equal volumes of the cytosol and membrane fractions were analyzed by Western blotting with anti-Xpress antibodies as described under "Experimental Procedures." The intensity of the bands corresponding to rPLD1 in the cytosol or membrane fractions was quantitated by densitometry. The amounts of the indicated rPLD1 constructs in the cytosol or membrane fractions were calculated as percentage of the total enzyme in both. C1, the cytosol fraction obtained from the first fractionation using the hypotonic buffer; C2, the supernatant fraction obtained from the additional extraction with indicated salt or detergent. M, the resulting membrane fraction after the two consecutive fractionations with hypotonic buffer and the indicated salt or detergent.

Alanine Mutation of Cysteine 310 or 612 Dramatically Elevates Basal rPLD1 Activity in Vivo-- During our investigation of the role of palmitoylation of rPLD1, we also generated multiple Cys mutations that did not affect palmitoylation of the enzyme. We transfected these mutants into COS7 cells and examined the effect of the mutations on the basal PLD activity and the response to PMA stimulation in vivo. We found that mutation of Cys247 or Cys403 to Ala did not affect the basal activity of rPLD1 or its response to PMA. Mutation of Cys79 to Ala increased the basal PLD activity ~40%. When Cys310 or Cys612 was mutated to Ala, the basal PLD activity was increased ~2- and 4-fold, respectively (Fig. 7A). These two mutants still responded to PMA stimulation, but the -fold increase was lower compared with wild type rPLD1 (Fig. 7B). The high basal activity of these mutants was not due to their higher expression level in COS7 cells (Fig. 7C). These data suggest that Cys310 and Cys612 play roles in regulating the catalytic activity of rPLD1, but the detailed mechanisms need further investigation.


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Fig. 7.   Effect of alanine mutation of Cys310 and Cys612 of rPLD1 on Its Catalytic Activity. Wild type rPLD1 or indicated cysteine mutants were transfected into COS7 cells, and the PLD activity was measured in the absence or presence of 100 nM PMA as described under "Experimental Procedures." The PLD activity is represented by the percentage of the phosphatidylbutanol synthesized versus the total tritium incorporated into the lipid of the COS7 cells. The results are representative of three experiments performed in triplicate. Mean values ± S.E. are shown. A, the PLD activity in the absence of stimulation. B, the PLD activity in the absence (gray bars) or presence (black bars) of PMA treatment for 20 min. C, the corresponding cell lysate was analyzed by Western blotting with anti-Xpress antibodies to examine the expression level of wild type or mutant rPLD1 constructs in COS7 cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our previous studies of rPLD1 showed that the N- and C-terminal halves of the enzyme could associate in vivo to restore PLD activity when cotransfected (11). The interdomain association between the N- and C-terminal halves of rPLD1 is also required for Ser/Thr phosphorylation of the enzyme (29). In this study, we found that rPLD1 can be modified by palmitoylation, and this lipid acylation also requires the association of the N- and C-terminal halves of rPLD1.2 A previous study of the palmitoylation of human PLD1 showed that mutation of Ser911 to alanine abolished the lipid modification of the enzyme (33); however, the mechanism was not elucidated. Here, we found that mutation of the corresponding Ser873 in rPLD1 to alanine disrupted the interdomain association between the N- and C-terminal halves (Fig. 3D), which is the likely reason for the diminished palmitoylation of this mutant enzyme (Fig. 3A). Examination of the palmitoylation of the N- and C-terminal halves showed that the lipid modification occurred on the N-terminal half. We mutated all of the Cys residues in the N-terminal half except Cys55, since this residue is not conserved between human and rat PLD1. Studies of all the Cys mutants in the N-terminal half of rPLD1 showed that only mutation of both Cys240 and Cys241 abolished the palmitoylation of rPLD1. Thus, Cys240 and Cys241 are the probable sites for the palmitoylation of rPLD1 (Fig. 2 and 4).

An important finding in the present study is that palmitoylation of rPLD1 is not necessary for its catalytic activity. This is because the N-terminal deletion mutants, rPLD1-(169-1036) and rPLD1-(320-1036), that lack the palmitoylation modification (Fig. 2) exhibited higher basal activity compared with wild type enzyme (10, 11). These results suggested that palmitoylation is not required for catalytic activity of rPLD1. However, the palmitoylation-deficient mutant, rPLD1(C240A,C241A), showed a reduced basal catalytic activity compared with wild type enzyme (Fig. 5), which is in agreement with the in vivo finding of Sugars et al. (32). One explanation for this difference may be the presence of the N-terminal sequence in the full-length palmitoylation-deficient mutant. The N-terminal 168 amino acids have been shown to be suppressive to the basal activity but are required for the PKC activation of rPLD1 (10, 11). It is possible that in the full-length enzyme, palmitoylation of rPLD1 may relieve part of the suppressive effect mediated by the N-terminal sequence in vivo, and this effect is not seen when the inhibitory N-terminal fragment of rPLD1 is deleted. Proof of this hypothesis will require detailed studies on how the N-terminal sequence regulates the activity of PLD1. In addition, the finding that the palmitoylation-deficient full-length human PLD1 had wild type activity when assayed in vitro (32) again suggests that this lipid modification is not essential for the catalysis of PLD1 but may play some regulatory role in vivo. It is also possible that lack of Cys residues instead of lack of palmitoylation is partly responsible for the changes in the properties of the PLD mutant, such as the dramatically reduced basal activity of the double Cys mutant. The fact that rPLD1-(320-1036), which lacks both Cys240 and Cys241, still exhibits high basal activity (Refs. 10 and 11) suggests that lack of the Cys residues may not be solely responsible for the changes in PLD activity, although the possibility still exists that lack of Cys240 and Cys241 may have different effects depending on the presence or absence of the N-terminal amino acid. A related question is whether or not palmitoylation of rPLD1 requires an active enzyme. In Fig. 1, we showed that a C-terminal deletion mutant of rPLD1 that lacks the last four amino acids, EVWT, is palmitoylated. However, it is completely inactive when assayed in vivo or in vitro for transphosphatidylation or choline release activity (31).

It is interesting to find that rPLD1 can be modified by both Ser/Thr phosphorylation and palmitoylation and that these two post-translational modifications are interrelated (Ref. 29 and this study). Both of the modifications require the interdomain association between the N- and C-terminal halves of rPLD1 and occur in the N-terminal half of the enzyme (Fig. 3 and Ref. 11). Correlation of palmitoylation and phosphorylation has been documented in the studies of the rat bradykinin B2 receptor in Chinese hamster ovary cells (37), although palmitoylation and phosphorylation of the receptor were found to be mutually exclusive. In the case with rPLD1, we found that palmitoylation of rPLD1 is, on the contrary, required for an extensive Ser/Thr phosphorylation of the enzyme (Fig. 4). N-terminally truncated rPLD1 or full-length rPLD1(C240A,C241A) that lacked palmitoylation also lacked extensive Ser/Thr phosphorylation. However, analysis of wild type and various other rPLD1 constructs showed that extensive Ser/Thr phosphorylation of the enzyme is not necessary for its palmitoylation (Fig. 4). These results suggest that palmitoylation and phosphorylation of rPLD1 are carried out sequentially. Palmitoylation of rPLD1 may help to specify its location in a membrane fraction, where the kinase is available for phosphorylation of the enzyme. Our previous finding that the Ser/Thr-phosphorylated rPLD1 is detected exclusively in the membrane fraction (29) is consistent with our current findings and presumptions.

It was surprising to find that rPLD1 that lacked the N-terminal 168 amino acids was not palmitoylated in COS7 cells, since both Cys240 and Cys241 are present in the molecule. rPLD1 that lacks the first 50 amino acids exhibits wild type enzyme properties (Refs. 10 and 11 and this study). Therefore, it is likely that amino acids 50-168 are important for the lipid modification. These amino acids have also been proposed to play important roles in regulating the properties of rPLD1 (Refs. 10 and 11 and Fig. 2 of this study), which include inhibition of the basal activity, mediation of the activation by PKC, and Ser/Thr phosphorylation of the enzyme. The N-terminal 168 amino acids may be involved in maintaining a structural feature that is important for the regulation; thus, truncation of these amino acids could cause a conformational change that makes the phosphorylation or palmitoylation sites inaccessible to the kinase or palmitoyltransferase. It is also possible that these amino acids may be directly involved in recruiting the enzymes that modify rPLD1, since a potential PX domain has been defined for human PLD1 within these amino acids, which is usually involved in protein-protein interactions (12). The possibility also exists that the N-terminal Xpress epitope tag is close to the palmitoylation sites and thus suppresses the lipid modification.

Characterization of both the phosphorylation and palmitoylation suggests that both of the posttranslational modifications are not required for the catalytic activity of the enzyme. However, these modifications may play roles in membrane association of the enzyme. Our previous studies showed that Ser/Thr-phosphorylated rPLD1 locates exclusively in the membrane fraction (29). In this study, we found that although the palmitoylation-deficient rPLD1(C240A,C241A) can associate with the membrane, the association is weakened compared with wild type enzyme. The palmitate group by itself has been shown to promote more stable membrane association of a peptide (38). It is possible that palmitoylation of rPLD1 plays a role in tethering the enzyme to membranes by increasing the hydrophobicity of the enzyme. The fact that palmitoylation-deficient rPLD1(C240A,C241A) is partially released from the membrane fraction by 1 M NaCl treatment suggests that in addition to hydrophobic interactions, ionic interactions may contribute to the association of rPLD1 with membranes. We also found that rPLD1(C240A,C241A) was very sensitive to treatment with 0.1 M Na2CO3 (pH 11), and less than 30% of this double cysteine mutant remained membrane-associated after this treatment (Fig. 6B). Na2CO3 (pH 11) at a concentration of 0.1 M primarily acts to denature protein structure without disrupting the organization of the bilayer and, thus, often dissociates peripheral membrane proteins from membranes (36). Since wild type rPLD1 remains membrane-associated when treated with 0.1 M Na2CO3 (pH 11) (Fig. 6B), it seems that palmitoylation may play a role in membrane association of the enzyme. This function of palmitoylation has been documented for PSD-95 (postsynaptic density-95), a protein enriched in brain that mediates synaptic plasticity. Wild type PSD-95 has been shown to partition as an integral membrane protein, and this membrane association of PSD-95 requires palmitoylation at N-terminal cysteines (39).

In addition to its role in membrane association, palmitoylation helps specify the membrane location of many proteins (35). PLD activity has been detected in many subcellular membranes, including the nuclear envelope, endoplasmic reticulum, Golgi apparatus, secretory vesicles, plasma membrane, and a specific subdomain on plasma membranes, the caveolae (40). Studies of palmitoylation-deficient human PLD1 showed that the double Cys mutant was located at the plasma membrane, while the wild type PLD1 was located on intracellular membranes (32). It is possible that, although palmitoylation and Ser/Thr phosphorylation may not be essential for the catalytic activity of rPLD1, they may help to specify its location within the cell. Interestingly, like N-terminally truncated rPLD1, N-terminally truncated SPO14, a yeast PLD isoform, also lacks a Ser/Thr phosphorylation modification, while the wild type SPO14 is Ser/Thr-phosphorylated (41). This N-terminally deleted SPO14 retains catalytic activity; however, the activity is mislocalized, and the yeast strain carrying this SPO14 mutant cannot sporulate (21).

In summary, we have found that rPLD1 is palmitoylated at Cys240 and Cys241 and that this lipid acylation requires the presence of the N-terminal 168 amino acids and the interdomain association of the N- and C-terminal halves of rPLD1. Although palmitoylation of rPLD1 is not essential for its catalytic activity or its response to PKC, it is required for an extensive Ser/Thr phosphorylation modification of the enzyme and its stable association with membranes. In addition, we found that the cysteine residues, Cys310 and Cys612, play important roles in regulating the enzymatic activity of rPLD1. However, the biochemical mechanisms of these effects and the physiological functions of palmitoylation and Ser/Thr phosphorylation of rPLD1 require further investigation.

    ACKNOWLEDGEMENT

We thank Judy Nixon for typing the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Investigator of the Howard Hughes Medical Institute. To whom all correspondence should be addressed. Tel.: 615-322-6494; Fax: 615-322-4381; E-mail: john.exton@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009425200

2 Since palmitic acid can be converted to other fatty acids and metabolites in cells, the identity of the acyl group(s) incorporated into rPLD1 cannot be stated with certainty in the absence of further studies requiring a large amount of enzyme protein. The involvement of cysteine residues and the susceptibility to hydroxylamine indicate a thioester linkage, and the rapid labeling of human PLD1 in COS cells incubated with [3H]palmitic acid (33) suggests that the fatty acid may be incorporated intact.

    ABBREVIATIONS

The abbreviations used are: PLD, phospholipase D; rPLD, rat PLD; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PMA, phorbol 12-myristate 13-acetate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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