Association of N- and C-terminal Domains of Phospholipase D Is Required for Catalytic Activity

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)4D, denoted HKD. RPLD1 contains two HKD domains, located in the N- and C-terminal regions. Deletion mutants of rPLD1 that contained only an N- or C-terminal HKD domain exhibited no catalytic activity when expressed in COS 7 cells. However, when N-terminal fragments containing one of the HKD domains were cotransfected with a C-terminal fragment containing the other HKD domain, PLD activity was restored. Furthermore, immunoprecipitation assays showed that the N- and C-terminal halves of rPLD1 were physically associated when expressed in COS 7 cells. In addition, deletion of 168 amino acids from the N terminus of rPLD1 significantly enhanced basal PLD activity while inhibiting the response to phorbol ester. Likewise, the coexpression of this truncated N-terminal half with the C-terminal half resulted in increased PLD activity. In summary, this study provides direct evidence that the enzymatic activity of rPLD1 requires the presence of the HKD domains in both the N- and C-terminal regions of the molecule. More importantly, the two halves of rPLD1 can associate, and this may be essential to bring the two HKD domains together to form an active catalytic center. These findings provide new insights into the catalytic mechanism of enzymes of the PLD superfamily. Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)4D, denoted HKD. RPLD1 contains two HKD domains, located in the N- and C-terminal regions. Deletion mutants of rPLD1 that contained only an N- or C-terminal HKD domain exhibited no catalytic activity when expressed in COS 7 cells. However, when N-terminal fragments containing one of the HKD domains were cotransfected with a C-terminal fragment containing the other HKD domain, PLD activity was restored. Furthermore, immunoprecipitation assays showed that the N- and C-terminal halves of rPLD1 were physically associated when expressed in COS 7 cells. In addition, deletion of 168 amino acids from the N terminus of rPLD1 significantly enhanced basal PLD activity while inhibiting the response to phorbol ester. Likewise, the coexpression of this truncated N-terminal half with the C-terminal half resulted in increased PLD activity. In summary, this study provides direct evidence that the enzymatic activity of rPLD1 requires the presence of the HKD domains in both the N- and C-terminal regions of the molecule. More importantly, the two halves of rPLD1 can associate, and this may be essential to bring the two HKD domains together to form an active catalytic center. These findings provide new insights into the catalytic mechanism of enzymes of the PLD superfamily. Phospholipase D (PLD) 1The abbreviations used are: PLD, phospholipase D; PKC, protein kinase C; PMA, 4β-phorbol 12-myristate 13-acetate. 1The abbreviations used are: PLD, phospholipase D; PKC, protein kinase C; PMA, 4β-phorbol 12-myristate 13-acetate.catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline (1Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (914) Google Scholar). It also carrys out a phosphatidyl transfer reaction which is used as a specific measure of PLD activity (2Kanfer J.N. Can. J. Biochem. 1980; 58: 1370-1380Crossref PubMed Scopus (170) Google Scholar). PLD activity has been detected in almost all organisms and is involved in a variety of signal transduction cascades (3Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (385) Google Scholar, 4Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). PLD activity has been shown to be regulated by small G proteins, PKC, protein-tyrosine kinases, and intracellular Ca2+. It was first cloned from plant (5Wang X. Xu L. Zheng L. J. Biol. Chem. 1994; 269: 20312-20317Abstract Full Text PDF PubMed Google Scholar) followed by yeast (6Rose K. Rudge S.A. Frohman M.A. Morris A.J. Engebrecht J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12151-12155Crossref PubMed Scopus (196) Google Scholar). To date, two types of mammalian PLD genes, termed PLD1 and PLD2, have been cloned (7Hammond S.M. Altshuller Y.M. Sung T.-C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Crossref PubMed Scopus (595) Google Scholar, 8Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu G. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 9Park S.-K. Provost J.P. Bae C.D. Ho W.-T. Exton J.H. J. Biol. Chem. 1997; 272: 29268-29271Google Scholar, 10Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 11Colley W.C. Sung T.-C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 12Colley W.C. Altshuller Y.M. Sue-Ling C.K. Copeland N.G. Gilbert D.J. Jenkins N.A. Branch K.D. Tsirka S.E. Bollag R.J. Bollag W.B. Frohman M.A. Biochem. J. 1997; 326: 745-753Crossref PubMed Scopus (115) Google Scholar, 13Lopez I. Arnold R.S. Lambeth J.D. J. Biol. Chem. 1998; 273: 12846-12852Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 14Min D.S. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 7044-7051Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 15Park S.-K. Min D.S. Exton J.H. Biochem. Biophys. Res. Comun. 1998; 244: 364-367Crossref PubMed Scopus (54) Google Scholar, 16Plonk S.G. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 4823-4826Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). PLD1 has a low basal activity and responds to PKC and small G protein of the ADP-ribosylation factor and Rho families. On the other hand, PLD2 is constitutively active and shows little response to stimuli. Data base searches using PLD1 and other sequences reveal that PLD belongs to a superfamily (17Morris A.J. Engebrecht J. Frohman M.A. Trends Pharmacol. Sci. 1996; 17: 182-185Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 18Ponting C.P. Kerr I.D. Protein Sci. 1996; 5: 914-922Crossref PubMed Scopus (280) Google Scholar, 19Koonin E.G. Trends Biochem. Sci. 1996; 21: 242-243Abstract Full Text PDF PubMed Scopus (139) Google Scholar) with four highly conserved regions (17Morris A.J. Engebrecht J. Frohman M.A. Trends Pharmacol. Sci. 1996; 17: 182-185Abstract Full Text PDF PubMed Scopus (174) Google Scholar). The most prominent conserved sequences reside in regions I and IV and contain the invariant motif, HXK(X)4D, denoted HKD. The HKD motif is found in other enzymes (17Morris A.J. Engebrecht J. Frohman M.A. Trends Pharmacol. Sci. 1996; 17: 182-185Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 18Ponting C.P. Kerr I.D. Protein Sci. 1996; 5: 914-922Crossref PubMed Scopus (280) Google Scholar, 19Koonin E.G. Trends Biochem. Sci. 1996; 21: 242-243Abstract Full Text PDF PubMed Scopus (139) Google Scholar), including phosphatidyltransferases, poxvirus envelope proteins, a Yersinia murine toxin, and several endonucleases, including Nuc (20Zhao Y. Stuckey J.A. Lohse D.L. Dixion J.E. Protein Sci. 1997; 6: 2655-2658Crossref PubMed Scopus (40) Google Scholar). Mutation of the HKD motifs residing in region I or IV renders human PLD1 and mouse PLD2 inactive (21Sung T.-C. Roper R.L. Zhang Y. Rudge S.A. Temel R. Hammond S.M. Morris A.J. Moss B. Engebrecht J. Frohman M.A. EMBO J. 1997; 16: 4519-4530Crossref PubMed Scopus (301) Google Scholar). Studies of Nuc also suggest that the histidine in the HKD domain is directly involved in the catalytic reaction by forming a phosphoenzyme intermediate (22Gottlin E.B. Rudolph A.E. Zhao Y. Matthews H.R. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9202-9207Crossref PubMed Scopus (128) Google Scholar). However, the mechanism(s) by which the two HKD domains are organized spatially to form an active catalytic center is not clear. Vaccinia virus protein VP37 is also a member of the PLD superfamily. Mutagenesis studies reveal that the partially conserved HKD motif is essential for its function (21Sung T.-C. Roper R.L. Zhang Y. Rudge S.A. Temel R. Hammond S.M. Morris A.J. Moss B. Engebrecht J. Frohman M.A. EMBO J. 1997; 16: 4519-4530Crossref PubMed Scopus (301) Google Scholar). Interestingly, although VP37 protein contains only one HKD domain, it can be immunoprecipitated as a dimer (23Schmutz C. Rindisbacher L. Galmiche M.C. Wittek R. Virology. 1995; 213: 19-27Crossref PubMed Scopus (29) Google Scholar). This observation raises the intriguing possibility that the two HKD motifs in PLD can be brought together to form a single active site by self-association. In this study, we generated a series of truncated rPLD1 mutants that contained either the HKD motif in the N-terminal half (region I) or in the C-terminal half (region IV). We investigated the PLD activity of the truncated fragments alone or in combination and explored the possibility of association of these fragments in COS 7 cells. Plasmid Construction—The full-length rPLD1 and its series of truncation mutants with coding regions corresponding to amino acids 169–1036, 320–1036, 585–1036, 1–584, 169–584, and 320–584 were created by polymerase chain reaction amplification. The forward primers to amplify the rPLD1 with coding region starting at amino acid 1, 169, 320, or 585 were 5′-GGGGTACCTATGTCACTAAGAAGTGAGGC, 5′-GGGGTACCTGAAAATGCAATCCAGGAGG, 5′-GGGGTACCTGGAGGCGCCATCGAGGAG, and 5′-GGGGTACCTAACACCGGCTCCATCCGA, respectively. The reverse primers that were used to amplify the rPLD1 constructs that end either at amino acid 584 or at 1036 were 5′-GGCTCTAGATTAGGAGGCGCTGTCGACGCT, and 5′-GGCTCTAGATTAAGTCCAAACCTCCATGGG, respectively. The polymerase chain reaction fragments were then subcloned in frame with the N-terminal express-tag at KpnI/XbaI sites in the polylinker region of the PcDNA 3.1 vector (Invitrogen). All the constructs were sequenced to verify the coding regions of rPLD1. COS 7 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum in humidified 10% CO2. The cells were transfected with FuGENE 6 (Boehringer Mannheim) according to manufacturer's instruction. After overnight serum starvation (0.5% fetal bovine serum in Dulbecco's modified Eagle's medium), PLD assays were carried out (24Malcolm K.C. Elliott C.M. Exton J.H. J. Biol. Chem. 1996; 271: 13135-13139Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). COS 7 cells were harvested after transfection and overnight starvation as described above. The cells were washed twice with ice-cold lysis buffer A (25 mmHepes, 10% glycerol, 1 mm of EDTA, 1 mm of EGTA, 1 mm dithiothreitol, and 1 mmphenylmethylsulfonyl fluoride and protease inhibitor mixture tablets (Boehringer Mannheim) (1 tablet/50 ml of lysis buffer). The cells were then resuspended in lysis buffer A and passed through 27-gauge needles five times. The cell lysate was then centrifuged at 120,000 ×g for 45 min at 4 °C to separate the cytosol and the crude membrane fraction. The membrane fraction was washed four times with the lysis buffer A and then passed through a 27-gauge needle until the pellet was resuspended. The Bradford method was used for the quantitation of protein concentration of the cytosol and the membrane fractions. Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to membranes (Immobilon-P, Millipore). The blots were blocked with 5% non-fat milk and incubated with the appropriate primary antibodies followed by horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were detected using enhanced chemiluminescence. COS 7 cells cultured on a 100-mm dish were transfected and starved as described above. The cells were washed twice with ice-cold phosphate-buffered saline and then resuspended in the lysis buffer B (25 mm Hepes, 10% glycerol, 50 mm KCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1% SDS, 1% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride and the protease inhibitor mixtures described above). The cell suspension was then passed through a 27-gauge needle five times, and the resulting cell lysate 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 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 spun, and the resulting supernatant incubated with 3 μl of mouse monoclonal anti-express antibodies (Invitrogen) and 20 μl of protein A beads overnight. The immunoprecipitates were washed four times with the lysis buffer B, and the resulting products resuspended in SDS sample buffer and analyzed by Western blotting with monoclonal anti-V5 antibodies (Invitrogen). Rat PLD1 (rPLD1) contains four conserved regions with one HKD domain in region I and the other in region IV (Fig. 1). Mutagenesis of either HKD domain inactivates the enzyme (21Sung T.-C. Roper R.L. Zhang Y. Rudge S.A. Temel R. Hammond S.M. Morris A.J. Moss B. Engebrecht J. Frohman M.A. EMBO J. 1997; 16: 4519-4530Crossref PubMed Scopus (301) Google Scholar), suggesting the integrity of the HKD domains is essential. In order to directly address this and the possibility that self-association of rPLD is involved in its catalytic mechanism, a series of deletion mutants of rPLD1 containing only one of the HKD motifs were generated and tagged at the N terminus with Express-epitope. As demonstrated in Fig. 1, the rPLD1 molecule was N-terminally truncated and/or split at amino acid 584 to generate N- or C-terminal fragments. This position was selected because the surrounding amino acids are not conserved or involved in alternative splicing (8Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu G. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 11Colley W.C. Sung T.-C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Full-length or deletion mutants of rPLD1 were expressed in COS 7 cells, and the PLD activity was measured. Although Express-tagged full-length rPLD1 showed activity comparable with that of rPLD1 with no tag, all the deletion mutants that contained only one HKD domain showed no catalytic activity compared with vector (Fig.2 A). The lack of PLD activity of the deletion mutants was also observed when cells were incubated with 100 nm PMA, which stimulates PLD through the activation of PKC (1Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (914) Google Scholar) (Fig. 2 B, cf. Fig.2 A). To explore the possibility that the deletion mutants had no PLD activity because they could not target to the membrane, COS 7 cells expressing wild type or truncated forms of rPLD1 were fractionated into cytosol and crude membranes as described under “Experimental Procedures.” The fractions were analyzed by Western blotting with monoclonal anti-Express antibody. Except for a trace amount of the C-terminal fragment rPLD1-(585–1036) present in the cytosol (data not shown), all the deletion mutants of rPLD1 were detected exclusively in the membrane fraction, as was the case for full-length rPLD1 (Fig. 2 C). Thus, rPLD1 deletion mutants that contained only one of the HKD domains were competent to target to membranes. Although deletion mutants containing only one HKD motif exhibited no PLD activity, it seemed possible that an active catalytic center could be restored when a mutant containing the N-terminal HKD motif was cotransfected with a mutant containing the other HKD domain. To examine this possibility, rPLD1-(1–584), rPLD1-(169–584), or rPLD1-(320–584) was cotransfected with rPLD1-(585–1036) in COS 7 cells and the PLD activity measured. Full-length rPLD1 and rPLD1 carrying a 168- or a 319-amino acid deletion at the N terminus were also transfected separately as positive controls. When rPLD1-(1–584) was cotransfected with rPLD1-(585–1036). PLD activity comparable with that of the full-length enzyme was detected (Fig. 3 A). When these cells were treated with PMA, the PLD activity was also stimulated, as seen for the wild type PLD (Fig. 3 B). Restoration of PLD activity was also observed when the deletion mutants rPLD1-(169–584) and rPLD1-(320–585) were cotransfected with rPLD1-(585–1036). These combinations exhibited higher basal activity than seen with wild type rPLD1 (Fig. 3 A), but showed no response to PMA (Fig.3 B). These findings were consistent with earlier observations (15Park S.-K. Min D.S. Exton J.H. Biochem. Biophys. Res. Comun. 1998; 244: 364-367Crossref PubMed Scopus (54) Google Scholar) that these N-terminal deletions increase the basal catalytic activity of the enzyme, but abolish its response to phorbol ester. The foregoing results indicated that PLD activity required the presence of both HKD motifs and that the N- and C-terminal halves of rPLD1 might interact to bring the two HKD motifs together to form an active catalytic site. In order to investigate the association between the N-terminal and C-terminal halves of rPLD1, immunoprecipitation was performed. For the of immunoprecipitation and several of the mutants described in Fig. 1 were generated as C-terminal These and We first these deletion mutants could also the PLD activity when cotransfected with the Express-tagged in COS 7 cells. In PLD activity comparable with that of the full-length enzyme was (data not shown), suggesting that the N-terminal fragments were to form a with the Express-tagged C-terminal half of the The immunoprecipitation were to examine the N-terminal fragments of rPLD1 tagged with could be immunoprecipitated by anti-Express which the C-terminal of rPLD1 tagged with The COS 7 cells were and the cell were immunoprecipitated with anti-Express antibodies and analyzed by Western blotting with When were carried out in which was cotransfected with either the vector or a expressing a no protein was detected by However, when was cotransfected with or with corresponding to the of and were detected by respectively. of and was not the of is to that of IgG and can be by SDS-polyacrylamide gel the N-terminal of rPLD can with its C-terminal All PLD to contain two of the HKD motif, with one in the N-terminal half of the enzyme and the other in the C-terminal half (17Morris A.J. Engebrecht J. Frohman M.A. Trends Pharmacol. Sci. 1996; 17: 182-185Abstract Full Text PDF PubMed Scopus (174) Google Scholar). In this study, we deletion to generate a series of rPLD1 mutants to investigate the of the PLD transfection of COS 7 cells, we found truncated mutants of rPLD that contained only one HKD domain exhibited no activity, although all the mutants the membrane fraction. However, PLD activity was restored when the N- and C-terminal halves of rPLD1 that contained the HKD domains were Thus, the N- and C-terminal halves of rPLD1 were to form a that exhibited catalytic activity. immunoprecipitation we found that the N- and C-terminal of rPLD1 physically associated in These results that PLD activity requires the presence and association of the HKD domains from both the N- and C-terminal halves of the PLD is a but the domain involved in the membrane has not been In this study, we found that deletion mutants of rPLD1 that contain only the N- or C-terminal of rPLD1 molecule were to target to the membrane fraction. These results suggest that the of PLD with membrane may be by two or It is to that the is an essential for PLD S.-K. Provost J.P. Bae C.D. Ho W.-T. Exton J.H. J. Biol. Chem. 1997; 272: 29268-29271Google Scholar, 14Min D.S. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 7044-7051Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, S. J. Biol. Chem. 1995; 270: PubMed Scopus Google Scholar), and the between this and amino acids has been to be involved in membrane and protein L. Biol. 1998; PubMed Scopus Google Scholar, S.G. Biol. 1994; PubMed Scopus Google Scholar). this mechanism may be involved in the association of PLD with membranes. showed that the N- and C-terminal halves of rPLD1 in but the of the and the domains involved are not clear. The association between the two halves not other protein rPLD1 is active to phosphatidylcholine when appropriate are (8Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu G. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 14Min D.S. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 7044-7051Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Thus, the N-terminal of rPLD1 most directly with its C-terminal rPLD1 This of the possibility that the association could be regulated by in coexpression of rPLD1-(169–584) or rPLD1-(320–584) with could the PLD activity and showed that can interact with rPLD1-(585–1036) (Fig. the domain in the N-terminal that is essential for the with the C-terminal be located in the first amino but is required to its and that of the site in the C-terminal studies of rPLD1 showed that deletion of the first 168 or amino acids of rPLD1 not but enhanced the basal activity of the enzyme (15Park S.-K. Min D.S. Exton J.H. Biochem. Biophys. Res. Comun. 1998; 244: 364-367Crossref PubMed Scopus (54) Google Scholar). However, no response to PMA or was observed (15Park S.-K. Min D.S. Exton J.H. Biochem. Biophys. Res. Comun. 1998; 244: 364-367Crossref PubMed Scopus (54) Google Scholar). it was that amino acids at the N-terminal end of rPLD1 were involved in by PKC (15Park S.-K. Min D.S. Exton J.H. Biochem. Biophys. Res. Comun. 1998; 244: 364-367Crossref PubMed Scopus (54) Google Scholar). This was in the present study using a When rPLD1-(169–584), or was with rPLD1-(585–1036) in COS 7 cells, the basal PLD activity was (Fig. 3 A). However, no of the PLD activity was observed when PMA was (Fig. 3 B). a in the N-terminal end of rPLD1 to contain an for PLD activity that can be by direct with or by The self-association of rPLD1 found in this study may be a mechanism by of the superfamily to an active catalytic center. of the enzymes in the PLD superfamily contain two HKD motifs (17Morris A.J. Engebrecht J. Frohman M.A. Trends Pharmacol. Sci. 1996; 17: 182-185Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 18Ponting C.P. Kerr I.D. Protein Sci. 1996; 5: 914-922Crossref PubMed Scopus (280) Google Scholar, 19Koonin E.G. Trends Biochem. Sci. 1996; 21: 242-243Abstract Full Text PDF PubMed Scopus (139) Google Scholar), and the motifs are essential for catalytic Thus, it is that the two of HKD domains are brought together to form an active catalytic site through the self-association. the N-terminal half and C-terminal half of rPLD1 can it is possible that rPLD1 can through a to Thus, the two HKD motifs could be brought together by or of rPLD1. These two possible of rPLD1 could be regulated and PLD could function as a or dimer in and is to the domains of PLD that in membrane catalytic activity, and with We for in the of this