Molecular Analysis of Mammalian Phospholipase D2

Abstract

The mammalian phosphatidylcholine-specific phospholipase D (PLD) enzymes PLD1 and PLD2 have been proposed to play roles in signal transduction and membrane vesicular trafficking in distinct subcellular compartments. PLD1 is activated in a synergistic manner in vitro by protein kinase C-α, ADP-ribosylation factor 1 (ARF1), and Rho family members. In contrast, PLD2 is constitutively active in vitro. We describe here molecular analysis of PLD2. We show that the NH2-terminal 308 amino acids are required for PLD2's characteristic high basal activity. Unexpectedly, PLD2 lacking this region becomes highly responsive to ARF proteins and displays a modest preference for activation by ARF5. Chimeric analysis of PLD1 and PLD2 suggests that the ARF-responsive region is in the PLD carboxyl terminus. We also inserted into PLD2 a region of sequence unique to PLD1 known as the "loop" region, which had been proposed initially to mediate effector stimulation but that subsequently was shown instead to be required in part for the very low basal activity characteristic of PLD1. The insertion decreased PLD2 activity, consistent with the latter finding. Finally, we show that the critical role undertaken by the conserved carboxyl terminus is unlikely to involve promoting PLD association with membrane surfaces. The mammalian phosphatidylcholine-specific phospholipase D (PLD) enzymes PLD1 and PLD2 have been proposed to play roles in signal transduction and membrane vesicular trafficking in distinct subcellular compartments. PLD1 is activated in a synergistic manner in vitro by protein kinase C-α, ADP-ribosylation factor 1 (ARF1), and Rho family members. In contrast, PLD2 is constitutively active in vitro. We describe here molecular analysis of PLD2. We show that the NH2-terminal 308 amino acids are required for PLD2's characteristic high basal activity. Unexpectedly, PLD2 lacking this region becomes highly responsive to ARF proteins and displays a modest preference for activation by ARF5. Chimeric analysis of PLD1 and PLD2 suggests that the ARF-responsive region is in the PLD carboxyl terminus. We also inserted into PLD2 a region of sequence unique to PLD1 known as the "loop" region, which had been proposed initially to mediate effector stimulation but that subsequently was shown instead to be required in part for the very low basal activity characteristic of PLD1. The insertion decreased PLD2 activity, consistent with the latter finding. Finally, we show that the critical role undertaken by the conserved carboxyl terminus is unlikely to involve promoting PLD association with membrane surfaces. phospholipase D ADP-ribosylation factor mammalian ARF yeast ARF hemagglutinin protein kinase C-α guanosine 5′-3-O-(thio)triphosphate. Phosphatidylcholine-specific phospholipase D (PLD)1 cDNAs have been cloned from a wide variety of species ranging from bacteria to humans (reviewed in Refs. 1Morris A.J. Engebrecht J. Frohman M.A. Trends Pharmacol. Sci. 1996; 17: 182-185Abstract Full Text PDF PubMed Scopus (176) Google Scholar and 2Frohman M.A. Sung T.-C. Morris A.J. Daniels L. Phospholipase D. Landes, Washington, D. C.1999Google Scholar). Isolation of the first animal PLD cDNA sequence (human PLD1) and subsequent studies revealed that an evolutionarily related PLD superfamily was widespread and encoded several regions of conserved sequence (1Morris A.J. Engebrecht J. Frohman M.A. Trends Pharmacol. Sci. 1996; 17: 182-185Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 3Hammond 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: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 5Ponting C.P. Kerr I.D. Protein Sci. 1996; 5: 914-922Crossref PubMed Scopus (283) Google Scholar, 6Koonin E.V. Trends Biochem. Sci. 1996; 21: 242-243Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 7Sung T.C. Roper R. 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 (308) Google Scholar). Two distinct mammalian PLD genes approximately 50% identical have been isolated from humans, rats, and mice (3Hammond 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: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 8Colley W.C. Altshuller Y.M. Sue-Ling C.K. Copeland N.G. Gilbert D.J. Jenkins N.A. Branch K.D. Bollag R.J. Bollag W.B. Frohman M.A. Biochem. J. 1997; 326: 745-753Crossref PubMed Scopus (115) Google Scholar, 9Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 10Katayama K. Kodaki T. Nagamachi Y. Yamashita S. Biochem. J. 1998; 329: 647-652Crossref PubMed Scopus (60) Google Scholar, 11Steed P.M. Clark K.L. Boyar W.C. Lasala D.J. FASEB J. 1998; 12: 1309-1317Crossref PubMed Scopus (100) Google Scholar, 12Lopez I. Arnold R.S. Lambeth J.D. J. Biol. Chem. 1998; 273: 12846-12852Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). PLD proteins examined thus far from nonmammalian species exhibit constitutive activity when assayed in vitro, and their regulation in vivo is effected through mechanisms such as phosphorylation and translocation (13Rudge S.A. Morris A.J. Engebrecht J. J. Cell Biol. 1998; 140: 81-90Crossref PubMed Scopus (120) Google Scholar, 14Wang X.M. Trends Plant Sci. 1997; 2: 261-266Abstract Full Text PDF Scopus (30) Google Scholar). Mammalian PLD2 is similarly constitutively active in vitro (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 9Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 11Steed P.M. Clark K.L. Boyar W.C. Lasala D.J. FASEB J. 1998; 12: 1309-1317Crossref PubMed Scopus (100) Google Scholar, 15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar) and is regulated in vivo through unknown mechanisms (15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar, 16Slaaby R. Jensen T. Hansen H. Frohman M.A. Seedorf K. J. Biol. Chem. 1998; (in press): 273Google Scholar). In contrast, PLD1 exhibits a low basal activity when expressed in tissue culture cell lines or as a recombinant, purified protein in vitro and is directly stimulated by the presence of recombinant, purified protein kinase C-α (PKC-α) or ARF or Rho small GTP-binding protein family members (3Hammond 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: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 9Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 17Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. 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 (496) Google Scholar). Each class of effectors can act alone to stimulate PLD1 and in combination to elicit a synergistic activation, suggesting that for each there is a separate site of interaction on PLD1 (10Katayama K. Kodaki T. Nagamachi Y. Yamashita S. Biochem. J. 1998; 329: 647-652Crossref PubMed Scopus (60) Google Scholar, 17Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. 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 (496) Google Scholar). In our initial studies on this topic, we undertook site-directed mutagenesis of regions held in common among PLDs from different species (conserved regions II, III, and IV; Ref. 7Sung T.C. Roper R. 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 (308) Google Scholar). We found that these regions were critical for catalysis in vitro and for PLD function in vivo and developed a hypothetical model for the catalytic cycle involving a covalent phosphatidyl-enzyme intermediate (7Sung T.C. Roper R. 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 (308) Google Scholar). However, although many of the mutants displayed diminished or no enzymatic activity, none of them exhibited selective responsiveness to ARF, Rho, or PKC-α (7Sung T.C. Roper R. 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 (308) Google Scholar). We subsequently undertook molecular analysis of PLD1 and found that the amino terminus conferred PKC-α responsiveness. 2Sung, T.-C., Zhang, Y., Morris, A. J., and Frohman, M. A. (1999) J. Biol. Chem. 274, in press. In addition, we showed that both the amino terminus and the central loop region mediated a negative regulatory effect that maintained PLD1's low basal activity and finally that a conserved carboxyl-terminal region is critical for PKC-α-mediated catalysis.2 In this report, we targeted for analysis the NH2-terminal region of PLD2 and unexpectedly generated a strongly ARF-responsive isoform, which we characterize and discuss in the context of PLD2 regulation in vivo. All phospholipids were purchased from Avanti Polar Lipids. Phosphatidylinositol 4,5-bisphosphate was isolated as described (3Hammond 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: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar).l-α-[choline-methyl-3H]dipalmitoyl phosphatidylcholine was obtained from American Radiolabeled Chemicals, and [32P]phosphoric acid was from ICN pharmaceuticals. All other reagents were obtained from previously noted standard sources and were of analytical grade unless otherwise specified (3Hammond 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: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar). Site-directed mutagenesis of expression plasmids was carried out using the Quik-Change kit (Stratagene). Plasmids were sequenced to confirm the intended mutation and the integrity of the surrounding sequences for at least 100 base pairs using Sequenase (U.S. Biochemical Corp.). Deletion mutants and chimeric PLD1/PLD2 cDNAs were constructed using convenient restriction sites or polymerase chain reaction-based strategies and were sequenced at all junctions to ensure that the reading frame was maintained. For two constructs, a Ras membrane localization sequence was appended to the 3′-end of the open reading frame using a linker encoding the sequence PGCMSCKCVLS. COS-7 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. For transfections, the cells were grown in 35-mm dishes and then switched into Opti-MEM I (Life Technologies, Inc.). For in vivo assays, the cells were washed into serum and phosphoric acid-free Dulbecco's modified Eagle's medium after transfection and labeled with 5 μCi of [32P]phosphoric acid (Pi) per dish for 18 h (19van Blitterswijk W.J. Hilkmann H. EMBO J. 1993; 12: 2655-2662Crossref PubMed Scopus (61) Google Scholar). Recombinant mammalian ARF, RhoA, Rac-1, and PKC-α were purified and activated using GTPγS or phorbol 12-myristate 13-acetate as described previously (17Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. 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 (496) Google Scholar). Yeast ARF, mutant mammalian ARFs, and chimeric mammalian/yeast ARFs were generated, prepared, and activated as described previously (20Liang J.O. Sung T.-C. Morris A.J. Frohman M.A. Kornfeld S. J. Biol. Chem. 1997; 272: 33001-33008Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Mammalian PLD activity was measured using the in vitro head group release assay and the in vivo transphosphatidylation assay as described previously (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 17Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. 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 (496) Google Scholar, 21Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 22Wakelam M.J. Cooke S.J. Currie S. Palmer S. Plevin R. Biochem. Soc. Trans. 1991; 19: 321-324Crossref PubMed Scopus (14) Google Scholar). PLD cDNAs were transiently overexpressed in COS-7 cells as described previously using the mammalian expression vector, pCGN (3Hammond 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: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar), or immunoaffinity-purified from baculovirus-infected Sf9 cells as described previously (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar,15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar, 17Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. 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 (496) Google Scholar). The transfection efficiency was observed to be approximately 5–20%. Western blots were performed as described previously, using affinity-purified rabbit anti-PLD peptide antisera to detect PLD1 and PLD2 (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar, 17Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. 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 (496) Google Scholar) or the monoclonal antibody 12CA5 to detect HA-tagged PLD1 and PLD2 (7Sung T.C. Roper R. 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 (308) Google Scholar). Conservation of protein sequence between yeast PLD and mammalian PLD1 begins at amino acid 325 of PLD1 (amino acid 308 of PLD2). We previously found that the nonconserved region (denoted the amino terminus) of PLD1 mediates responsiveness to PKC but is not required for PLD1's intrinsic enzymatic activity or for small GTP-binding protein stimulation of PLD1 (Fig.1).2 We also observed that deletion of either the amino terminus or the unique "loop" sequence from PLD1 increased its basal activity, suggesting that these regions in combination were responsible for at least part of the difference in the PLD1 and PLD2 states of activation in vitro.2 These findings prompted us to investigate the function of the PLD2 NH2 terminus, since PLD2 exhibits high basal activity and is not PKC-responsive. The amino termini are well conserved between the different mammalian PLD2 proteins but exhibit little similarity to the PLD1 amino termini, particularly over the first 80 amino acids. Weak homology is observed from amino acid 83 to 196, including a phox domain (Fig. 1, Ref. 2Frohman M.A. Sung T.-C. Morris A.J. Daniels L. Phospholipase D. Landes, Washington, D. C.1999Google Scholar) that has been proposed to mediate a wide variety of protein-protein interactions (23Ponting C.P. Protein Sci. 1996; 5: 2353-2357Crossref PubMed Scopus (268) Google Scholar). Many membrane-associated proteins require free amino termini to successfully interact with membrane surfaces, particularly if they encode prenylation sequences. PLD2 does not encode such sequences, and it was known from earlier studies that a free amino terminus is not required, since the protein appears to behave normally when fused to an NH2-terminal HA-epitope peptide (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 7Sung T.C. Roper R. 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 (308) Google Scholar, 15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar). To assess the potential role of the PLD2 amino terminus, a 308-amino acid NH2-terminal deletion analogous to the 325-amino acid NH2-terminal deletion previously described for PLD1 was constructed, transfected into COS-7 cells, and assayed for basal and stimulated PLD activities as described under "Experimental Procedures" (Fig. 2). As shown in Fig.2 B, the PLD activities in lysates from COS-7 cells transfected by a control plasmid (Gbx-2, a homeobox protein) and stimulated by a variety of effectors, or transfected with PLD1 but not stimulated by exogenous effectors, are relatively low (see also Fig.2 D, where it is shown that anti-PLD antisera can detect the overexpressed but not the endogenous proteins). In contrast, lysates from PLD1-transfected cells exhibit a dramatic increase in activity when stimulated by mammalian ARF1, Rho family members, or PKC-α. Lysates from PLD2-transfected cells exhibit a high level of basal activity, which increases slightly (less than 2-fold) in the presence of the exogenous effectors, in particular ARF1. A portion of this increase can be accounted for by stimulation of endogenous PLD1 by the effectors. The remaining very modest ARF-dependent increase is variably observed both for PLD2 expressed in COS-7 cell lysates and for immunopurified native and baculovirus-generated PLD2 (see TableI and Refs. 4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 12Lopez I. Arnold R.S. Lambeth J.D. J. Biol. Chem. 1998; 273: 12846-12852Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, and 15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar). The finding that purified PLD2 is largely constitutively active in vitrohas led to the proposal that PLD2 is regulated in vivothrough negative regulatory mechanisms that are released upon agonist stimulation (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar, 16Slaaby R. Jensen T. Hansen H. Frohman M.A. Seedorf K. J. Biol. Chem. 1998; (in press): 273Google Scholar). The modest and variable response of PLD2in vitro to ARF suggested that ARF stimulation of PLD2in vivo might play a more significant role in some unknown context.Table IStimulation of purified recombinant full-length and NH2-terminally truncated PLD2 by ARF1AdditionsActivityIncreasecpm−foldPLD2Δ(1–308)11941.0 ± 0.7+0.1 μm ARF1, GTPγS47514.0 ± 0.9+0.5 μm ARF1, GTPγS16,04713.4 ± 3.1+0.5 μm ARF113671.1 ± 0.2+1 μg of bovine serum albumin12501.0 ± 0.1PLD212,1301.0 ± 0.2+0.1 μm ARF1, GTPγS17,9751.5 ± 0.1+0.5 μm ARF1, GTPγS22,7801.9 ± 0.1+0.5 μmARF112,4291.0 ± 0.2+1 μg of bovine serum albumin93140.8 ± 0.3PLD2 and PLD2Δ(1–308) were expressed and purified from baculovirus-infected Sf9 cells as previously described (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar). Hydrolysis of 3H-labeled phosphatidylcholine was assayedin vitro in the presence of the PLD proteins alone or in combination with activators or controls as depicted above. The cpm shown above represent an averaging of four experiments and were adjusted by subtracting the counts observed in the assay blank (∼1030 cpm). Each experiment was performed in duplicate, and the average duplicate variability was 2%. Open table in a new tab PLD2 and PLD2Δ(1–308) were expressed and purified from baculovirus-infected Sf9 cells as previously described (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 15Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (387) Google Scholar). Hydrolysis of 3H-labeled phosphatidylcholine was assayedin vitro in the presence of the PLD proteins alone or in combination with activators or controls as depicted above. The cpm shown above represent an averaging of four experiments and were adjusted by subtracting the counts observed in the assay blank (∼1030 cpm). Each experiment was performed in duplicate, and the average duplicate variability was 2%. Unexpectedly, deletion of the amino terminus of PLD2 (PLD2Δ(1–308)) eliminates 85% of the high basal activity in COS-7 cell lysates and renders the resulting truncated protein almost as ARF1-responsive as PLD1 (Fig. 2 B). No response to PKC-α was detected, consistent with the prior observation that the PKC-α-responsive region in PLD1 is in its NH2 terminus. Less anticipated was the observation that no response to Rho family members was observed (Fig. 2 B), although the site of interaction of PLD1 with Rho family members is in the carboxyl terminus (7Sung T.C. Roper R. 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 (308) Google Scholar), 3T.-C. Sung, Y. M. Altshuller, A. J. Morris, and M. A. Frohman, unpublished results. and this region is well conserved between PLD1 and PLD2. Nonetheless, since it is extraordinarily unlikely that PLD2 would encode a cryptic capacity for response to ARF in vitro unless it was employed in some context in vivo, the finding suggests three potential hypotheses. First, ARF might stimulate PLD2Δ(1–308) indirectly by acting on some other factor present in the COS-7 cell lysates. Second, ARF might derepress full-length PLD2 inhibited via some unknown mechanism in vivo. Third, PLD2 might undergo truncationin vivo in some setting, generating an ARF-responsive isoform. We next examined PLD2Δ(1–308) activity in vivo (Fig.2 C). PLD2Δ(1–308) exhibited moderate activity in vivo in unstimulated cells, although less so than wild-type PLD2 (see also Fig. 6 A). Nonetheless, the relatively robust activity observed suggests that the truncated protein is subcellularly localized to sites containing activated ARF (in contrast to PLD1) or that negative regulatory regions active only in vivo are present in the NH2 terminus. Since PLD1 and PLD2 subcellularly localize to different regions (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar) and since PLD1 encodes an in vivo specific negative regulatory region in its amino terminus,2 both hypotheses are tenable. Finally, we examined PLD2Δ(1–308) protein expression (Fig.2 D). Immunoreactive protein is observed near the predicted size of 66 kDa, in addition to proteolyzed fragments, which are also observed for wild-type PLD2, but not PLD1 or PLD1Δ(1–325). The proteolyzed fragments observed for PLD2Δ(1–308) are unlikely to be catalytically active, since both larger NH2-terminal deletions or deletion of sequence from the terminus protein not In the full-length PLD2 COS-7 cell catalytically active NH2-terminally truncated proteins be present that to the ARF responsiveness To out of ARF1, we expressed PLD2Δ(1–308) in and immunoaffinity-purified it using an (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). full-length PLD2 and PLD2Δ(1–308) in this manner are largely not shown and Ref. 4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). As shown in full-length PLD2 is stimulated by (less than PLD2Δ(1–308) exhibits a The stimulation is observed at relatively low of is and is not by protein serum Since both the and PLD proteins were expressed and were these that ARF directly with PLD1 and PLD2 localize to different subcellular regions (4Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). least mammalian ARFs and these also localize to different subcellular regions (reviewed in Refs. J. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar and K. Zhang C. I. R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). are three of the ARFs, by ARF1, and is the and to the and to and to the membrane under some to the membrane and for the other ARFs are not well and the for ARF1, and The mechanisms of of the ARFs also and are and are largely agonist stimulation and subsequent In contrast, is and the of it is in the of We previously that PLD1 is activated by mammalian more than by yeast Ref. J.O. Sung T.-C. Morris A.J. Frohman M.A. Kornfeld S. J. Biol. Chem. 1997; 272: 33001-33008Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We examined this response for PLD2Δ(1–308) and unexpectedly found that PLD2Δ(1–308) is more stimulated by than by (Fig. A). The preference is not to the deletion of the amino terminus, since similarly to wild-type it to finding prompted the of the of the mammalian to To we examined activation by and and found that was a relatively of both PLDs but that stimulated PLD2Δ(1–308) more than (Fig. B). finding was examined in more by a (Fig. C). We that although and stimulated both PLDs activated PLD2Δ(1–308) at a than and with as In contrast, and stimulation of PLD1 were not stimulated slightly more than in three separate but the of the difference was variable and than was observed for PLD2Δ(1–308) (Fig. and not The finding that and stimulated PLD1 was previously to a central portion of as the region responsible for stimulation of PLD1 (20Liang J.O. Sung