Evidence for and Characterization of Ca2+ Binding to the Catalytic Region of Arabidopsis thaliana Phospholipase Dβ

Most types of plant phospholipase D (PLD) require Ca2+ for activity, but how Ca2+ affects PLD activity is not well understood. We reported previously that Ca2+ binds to the regulatory C2 domain that occurs in the N terminus of the Ca2+-requiring PLDs. Using Arabidopsis thaliana PLDβ and C2-deleted PLDβ (PLDβcat), we now show that Ca2+ also interacts with the catalytic regions of PLD. PLDβcat exhibited Ca2+-dependent activity, was much less active, and required a higher level of Ca2+ than the full-length PLDβ. Ca2+ binding of the proteins was stimulated by phospholipids; phosphatidylserine was the most effective among those tested. Scatchard plot analysis of Ca2+ binding data yielded an estimate of 3.6 high affinity (Kd = 29 μm) binding sites on PLDβ. The Ca2+-PLDβcat interaction increased the affinity of the protein for the activator, phosphatidylinositol 4,5-bisphosphate, but not for the substrate, phosphatidylcholine. This is in contrast to the effect of Ca2+ binding to the C2 domain, which stimulates phosphatidylcholine binding but inhibits phosphatidylinositol 4,5-bisphosphate binding of the domain. These results demonstrate the contrasting and complementary effects of the Ca2+- and lipid-binding properties of the C2 and catalytic domains of plant PLD and provide insight into the mechanism by which Ca2+ regulates PLD activity. Most types of plant phospholipase D (PLD) require Ca2+ for activity, but how Ca2+ affects PLD activity is not well understood. We reported previously that Ca2+ binds to the regulatory C2 domain that occurs in the N terminus of the Ca2+-requiring PLDs. Using Arabidopsis thaliana PLDβ and C2-deleted PLDβ (PLDβcat), we now show that Ca2+ also interacts with the catalytic regions of PLD. PLDβcat exhibited Ca2+-dependent activity, was much less active, and required a higher level of Ca2+ than the full-length PLDβ. Ca2+ binding of the proteins was stimulated by phospholipids; phosphatidylserine was the most effective among those tested. Scatchard plot analysis of Ca2+ binding data yielded an estimate of 3.6 high affinity (Kd = 29 μm) binding sites on PLDβ. The Ca2+-PLDβcat interaction increased the affinity of the protein for the activator, phosphatidylinositol 4,5-bisphosphate, but not for the substrate, phosphatidylcholine. This is in contrast to the effect of Ca2+ binding to the C2 domain, which stimulates phosphatidylcholine binding but inhibits phosphatidylinositol 4,5-bisphosphate binding of the domain. These results demonstrate the contrasting and complementary effects of the Ca2+- and lipid-binding properties of the C2 and catalytic domains of plant PLD and provide insight into the mechanism by which Ca2+ regulates PLD activity. It has been long recognized that Ca2+ is a stimulator of plant PLD 1The abbreviations used are: PLD, phospholipase D; PH, pleckstrin homology; PX, phox-homolgy; PIP2, phosphatidyl 4,5-bisphosphate; GST, glutathione-S-transferase; STE, sodium chloride/Tris/EDTA; MES, 2-(N-morpholino)ethanesulfonic acid; PG, phosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.1The abbreviations used are: PLD, phospholipase D; PH, pleckstrin homology; PX, phox-homolgy; PIP2, phosphatidyl 4,5-bisphosphate; GST, glutathione-S-transferase; STE, sodium chloride/Tris/EDTA; MES, 2-(N-morpholino)ethanesulfonic acid; PG, phosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. activity. However, regulation of plant PLD by Ca2+ has been a source of debate because the common plant PLD requires millimolar amounts of Ca2+ for activity in vitro (1Heller M. Adv. Lipid Res. 1978; 16: 267-326Crossref PubMed Google Scholar, 2Wang X. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 211-231Crossref PubMed Scopus (222) Google Scholar). Recent characterization of multiple plant PLDs has made it clear that most plant PLDs are capable of significant enzymatic activity at micromolar levels of Ca2+ near those encountered in the cell (3Pappan K. Qin W. Dyer J.H. Zheng L. Wang X. J. Biol. Chem. 1997; 272: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 4Pappan K. Zheng S. Wang X. J. Biol. Chem. 1997; 272: 7048-7054Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 5Pappan K. Wang X. Arch. Biochem. Biophys. 1999; 368: 347-353Crossref PubMed Scopus (49) Google Scholar, 6Qin W. Pappan K. Wang X. J. Biol. Chem. 1997; 272: 28267-28273Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 7Wang C. Wang X. Plant Physiol. 2001; 127: 1102-1112Crossref PubMed Scopus (130) Google Scholar). Arabidopsis thaliana has 12 PLDs that are grouped into PLDα, -β, -γ, -δ, and -ζ according to the sequence similarities, gene architectures, and domain structures (8Qin C. Wang X. Plant Physiol. 2002; 128: 1057-1068Crossref PubMed Scopus (226) Google Scholar). Except for PLDζ, all other PLDs characterized to date in A. thaliana require Ca2+ for activity. PLDβ, -γ, and -δ are active in micromolar ranges of Ca2+ (6Qin W. Pappan K. Wang X. J. Biol. Chem. 1997; 272: 28267-28273Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 7Wang C. Wang X. Plant Physiol. 2001; 127: 1102-1112Crossref PubMed Scopus (130) Google Scholar), and PLDα, which gives rise to the common plant PLD activity, is active at micromolar levels of Ca2+ under acidic conditions with mixed lipid vesicles (5Pappan K. Wang X. Arch. Biochem. Biophys. 1999; 368: 347-353Crossref PubMed Scopus (49) Google Scholar). Ca2+ increases the membrane association of PLD, which has been suggested as a mechanism for rapid activation of PLD in plant wound response (9Ryu S.B. Wang X. Biochim. Biophys. Acta. 1996; 1303: 243-250Crossref PubMed Scopus (122) Google Scholar). A positive correlation between increased cytoplasmic Ca2+ levels and increased PLD activity was indicated when the Ca2+ levels of carnation petals were perturbed using various Ca2+-ATPase inhibitors and calmodulin antagonists (10de Vrije T. Munnik T. J. Exp. Bot. 1997; 48: 1631-1637Crossref Scopus (40) Google Scholar). Recent studies have provided more insight into the mechanism of Ca2+ regulation of PLD activity. Amino acid sequence analysis indicates that most PLDs contain a C2 domain in their N-terminal regulatory regions, except for PLDζ, which has the pleckstrin homology (PH) and phox homology (PX) domains (8Qin C. Wang X. Plant Physiol. 2002; 128: 1057-1068Crossref PubMed Scopus (226) Google Scholar). C2 domains are Ca2+/phospholipid binding folds that consist of ∼130 amino acid residues (11Ponting C.P. Parker P.J. Protein Sci. 1996; 5: 162-166Crossref PubMed Scopus (154) Google Scholar, 12Shao X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (292) Google Scholar). C2 domains have been identified in a number of proteins involved in signal transduction or membrane trafficking, and these domains often mediate a Ca2+-dependent binding of proteins to phospholipids (13Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 14Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The binding of Ca2+ to plant PLD C2 domains has been demonstrated with isolated C2 domains from A. thaliana PLDβ and PLDα (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). That study also showed that the Ca2+ binding induced conformational changes of the C2 domain and promoted the binding of the C2 domain to PC. Thus, Ca2+-binding of the C2 domain underlies, at least in part, the biochemical basis of Ca2+-dependent PLD activity. PLDβ requires both Ca2+ and phosphatidylinositol 4,5-bisphosphate (PIP2) for activity (3Pappan K. Qin W. Dyer J.H. Zheng L. Wang X. J. Biol. Chem. 1997; 272: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 6Qin W. Pappan K. Wang X. J. Biol. Chem. 1997; 272: 28267-28273Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). PLD C2 domains also bind PIP2; however, Ca2+ weakens the PIP2-C2 interaction (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Thus, the inverse relationship between Ca2+- and PIP2-binding of the C2 domain suggests a complex, multi-step process of PLD activation. Further work has identified another PIP2-binding region in the PLD catalytic fold (16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google Scholar), which consists of two duplicated HxKxxxxD motifs and which lies in the C-terminal two-thirds of the protein (6Qin W. Pappan K. Wang X. J. Biol. Chem. 1997; 272: 28267-28273Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 17Pointing C.P. Kerr I.D. Protein Sci. 1996; 5: 914-922Crossref PubMed Scopus (280) Google Scholar, 18Gottlin E.B. Rudolph A.Z. Zhao Y. Mattews H.R. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9202-9207Crossref PubMed Scopus (128) Google Scholar). The PIP2-bound catalytic domain increases the enzyme's affinity for its substrate PC, and Ca2+ stimulates the PLDβ-PIP2 interaction (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google Scholar). These observations suggest that Ca2+ probably interacts with the catalytic region. The present study explores the potential interaction of Ca2+ with the catalytic regions. We have determined that the C2-deleted PLDβ (PLDβcat) binds Ca2+ but is less activated than the full-length enzyme. Furthermore, Ca2+ stimulates PIP2- but not PC-binding of PLDβcat, properties that both complement and contrast with the lipid binding properties of the C2 domain. Construction of GST-PLDβ and GST-PLDβcat and GST-βC2 Proteins—Three GST fusion proteins GST-PLDβ, GST-PLDβcat, and GST-PLDβC2 (Fig. 1) were used in this study. To construct the C2-deleted PLDβcat, a DNA fragment encompassing the catalytic domain of PLDβ (amino acid residues 158–829) was generated by PCR using the PLDβ cDNA in pBluescript SK as a DNA template, T7 primer as 3′ primer, and a synthetic olignucleotide as 5′ primer, which included an EcoRI restriction site at its 5′ end. The PCR-amplified DNA fragment was digested with EcoRI restriction enzyme and ligated directly into the pGEX-2T vector (Pharmacia). The creation of the GST-PLDβC2 construct has been described previously (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). All of the constructs were transformed into Escherichia coli BL21 for protein expression. and of in for of the constructs was induced by to = and at activity were by at for and with A and The was in A that and and was on for was to and the were were by at for The was used or at The of GST fusion proteins was using a of a previously reported Biochem. PubMed Scopus Google Scholar). The was in and The was on for and were to a of and The were on for and at for The was to a and was to a of The GST fusion proteins was mixed with at for The GST fusion proteins to were with of GST activity was and was as The of GST fusion protein to the was by the GST fusion protein with in by the protein of the with a protein GST-PLDβ and GST-PLDβcat were by to a and to at in and as described previously K. T. S. J. Biochem. 95: PubMed Scopus Google Scholar). The were with of and in the or of that was from a that was The membrane was at for with which it was with and The was to binding of the GST-PLDβ proteins and GST was using a described with J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.H. S. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of GST-PLDβ, GST-PLDβcat, or GST to was with and Ca2+ and of activity, as in a of To the effect of and on Ca2+ various phospholipids were and to the as in the To Ca2+ binding GST-PLDβ was in the of PS, of and amounts of and binding was binding was by the of by the of binding it was that of was to all GST-PLDβ Ca2+ sites not of GST fusion at for with to fusion proteins was with the affinity by at for The was with of a All were with to the fusion proteins was by and the were to the of GST fusion proteins used on the basis of the and of binding data were to two binding J. Biochem. PubMed Scopus Google a of sites or two of according to the for a of = or for two of = these is the of Ca2+ to of fusion to the number of binding is the association and is the Ca2+ The data were by both using a least in which yielded for and K. The was as and for and were reported as the of the PLD activity was determined as described previously X. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 211-231Crossref PubMed Scopus (222) Google Scholar). The included MES, lipid and of protein in a of The lipid vesicles were made of in a of The of was using of into the was by previously for binding to PLD (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google was used to binding to PLDβ and its in the binding of of and of were mixed with GST fusion proteins to in a binding and of Ca2+ in a of The Ca2+ were made by of the Ca2+ with The was at for with The were with of the binding the of to the was by GST to was used to All were at least activity was as of To binding as a of Ca2+ a was using lipid made of of mixed with of using in a of C2-deleted PLDβ and of A. thaliana full-length PLDβ and the C2-deleted PLDβcat were as GST fusion proteins (Fig. The GST-PLDβ and GST-PLDβcat of and (Fig. The PLDβ activity was stimulated by Ca2+ and a at (Fig. a Ca2+ level to that required for PLDβ in and for PLDβ the GST fusion K. Zheng S. Wang X. J. Biol. Chem. 1997; 272: 7048-7054Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Qin W. Pappan K. Wang X. J. Biol. Chem. 1997; 272: 28267-28273Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The PLDβcat also PC, but at a The activation of PLDβcat of the activity of PLDβ. Furthermore, activity of PLDβcat at millimolar levels of Ca2+ (Fig. the of to under conditions indicates that the level of activity with PLDβcat is PLD activity. These results that PLDβcat, which the C2 domain, and amino acid residues to but requires much higher levels of Ca2+ for activity. to C2-deleted of PLDβ and PLDβcat were determined by a PLDβ and PLDβcat demonstrated a to bind Ca2+ in the of phosphatidylserine (Fig. but not in its not To the Ca2+ binding exhibited by PLDβ, affinity binding were in the of various of Ca2+ and PLDβ fusion protein to Ca2+ binding by PLDβ was (Fig. and Scatchard analysis (Fig. an that was of two of and binding sites J. Biochem. PubMed Scopus Google Scholar). the two of binding sites or the PLDβ Ca2+ binding data in the two with 3.6 high affinity (Kd = 29 μm) and affinity (Kd = binding by binding by as protein is stimulated by acidic phospholipids J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.H. S. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biochemistry. PubMed Scopus Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), and demonstrated a for Ca2+ To the effect of on Ca2+ GST-PLDβ fusion proteins were with Ca2+ and various of (Fig. the of PS, PLDβcat The of increased the affinity of PLDβcat for Ca2+ in a Ca2+ to PLDβ, PLDβcat, and in a and at for of these fusion proteins (Fig. The Ca2+-binding process to most for in the of PS, PLDβcat more than as much Ca2+ as PLDβ was to have the Ca2+-binding in the of The fusion proteins were with phospholipids to or not other phospholipids Ca2+ binding (Fig. The GST-PLDβ fusion proteins exhibited less Ca2+ binding when or were for PS, at a in the binding stimulated PLDβcat binding to Ca2+ to of the level using PS, stimulated much less Ca2+ were also used to the effect of on Ca2+ binding by the GST-PLDβ fusion the effect on Ca2+ binding to effects on Ca2+ binding by PLDβ and PLDβcat, this of the Ca2+ binding level using The Ca2+ binding to PLDβ promoted by these phospholipids is of because the lipid required for PLDβ activity is a substrate of this enzyme K. S. Wang X. Arch. Biochem. Biophys. 1998; PubMed Scopus (128) Google Scholar). The that Ca2+ binding by PLDβcat suggests that Ca2+ interaction with PLDβ. the of PLDβcat and with for Ca2+ binding to membrane proteins is to the interaction of proteins and studies have that association of with Ca2+ binding of the C2 domain but inhibits binding of PIP2, a of PLDβ (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). this the effect of Ca2+ on the interaction of and with PLDβ, PLDβcat, and were directly (Fig. the of PLDβ, PLDβcat, and at a the Ca2+ the of to PLDβ and increased but was for at millimolar levels Ca2+ PLDβcat binding to (Fig. The binding for the much catalytic activity and the increased Ca2+ of PLDβ, PLDβcat, and all at levels in the of Ca2+ (Fig. in Ca2+ binding of binding by PLDβ increased with Ca2+ levels to of the (Fig. Further increases in Ca2+ the of to PLDβ. PLDβcat a but with a (Fig. These results that the catalytic region is for the binding of by PLD, and Ca2+ regulates this binding in a PLD in and regulation X. Plant Biol. 2002; 5: PubMed Scopus Google Scholar, Y. Biochim. Biophys. Acta. 2002; PubMed Scopus Google Scholar, J.H. Rev. Physiol. Biochem. 2002; PubMed Google Scholar). The PLD in is much more than that in other The A. thaliana PLD has 12 PLD (8Qin C. Wang X. Plant Physiol. 2002; 128: 1057-1068Crossref PubMed Scopus (226) Google Scholar, X. Plant Biol. 2002; 5: PubMed Scopus Google Scholar), two PLD are in and in J.H. Rev. Physiol. Biochem. 2002; PubMed Google Scholar, J. J. 1999; PubMed Scopus Google Scholar). Furthermore, of the 12 A. thaliana PLDs contain the C2 domain, which are to plant PLDs. The two A. thaliana PLDs have domains that are also in PLDs. All of the plant PLDs required Ca2+ for activity, the activity of A. thaliana is of The present study that A. thaliana PLDβ has multiple Ca2+ binding regions, the C2 domain and the C-terminal catalytic region. Ca2+ binding to the catalytic region interaction of PLD with the activator, PIP2, which is required for PLDβ activity (3Pappan K. Qin W. Dyer J.H. Zheng L. Wang X. J. Biol. Chem. 1997; 272: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). work has that binding to the catalytic region the enzyme's affinity for the substrate, (16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google Scholar, C. Wang C. Wang X. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, Ca2+ interaction with the catalytic region is to with binding and lipid The that Ca2+ is to binding at least in part, Ca2+ is required for the activity of the PLDs but not the PLDs. stimulates the activity of all other A. thaliana PLDs (6Qin W. Pappan K. Wang X. J. Biol. Chem. 1997; 272: 28267-28273Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 7Wang C. Wang X. Plant Physiol. 2001; 127: 1102-1112Crossref PubMed Scopus (130) Google Scholar, C. Wang X. Plant Physiol. 2002; 128: 1057-1068Crossref PubMed Scopus (226) Google and also and PLDs J.H. Rev. Physiol. Biochem. 2002; PubMed Google Scholar, J. J. 1999; PubMed Scopus Google Scholar). and domains are to with a binding has been identified in the catalytic region of the and PLDs J. J. 1999; PubMed Scopus Google Scholar). This is required for the PLD activity J. J. 1999; PubMed Scopus Google and is in the A. thaliana (8Qin C. Wang X. Plant Physiol. 2002; 128: 1057-1068Crossref PubMed Scopus (226) Google Scholar). However, PLDβ and other Ca2+-dependent PLDs two of the amino acid residues involved in binding (16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google Scholar). these PLDs Ca2+ to the binding of to the PLD catalytic as in (16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google and present The PLDβcat exhibited a much catalytic activity and a much higher of Ca2+ to activation with the enzyme. binding by PLDβcat was Thus, the catalytic activity of PLDβcat probably results from an binding of substrate vesicles by the of the C2 domain. The present results also suggest that Ca2+ binding by the C2 domain the binding of PLDβ to its substrate in as for phospholipase J. Biol. Chem. Full Text PDF PubMed Google Scholar). A phospholipase that the C2 domain to with and activity in however, it as as the full-length phospholipase J. Biol. Chem. Full Text PDF PubMed Google Scholar). Ca2+ binds to PLDβ in a in the of Ca2+ binding is much phospholipids Ca2+ binding to to most but and also to Ca2+ This by the of these acidic their at to Ca2+ Ca2+ binding has been reported for other proteins Biochemistry. PubMed Scopus Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar, T.C. Biochemistry. PubMed Scopus Google Scholar). protein and bind Ca2+ in the of acidic phospholipids but in their Biochemistry. PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar). of these proteins with on membrane which has an effect on the of Ca2+ required for interaction Biochemistry. PubMed Scopus Google Scholar). A binding has been to the Ca2+-dependent binding of protein to and its Ca2+-dependent activation J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). to this levels of Ca2+ a interaction between protein and higher Ca2+ the enzyme. high and affinity Ca2+ conformational changes in protein reported in this and other studies (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google suggest a and enzyme activation for PLDβ. The present study that Ca2+ has effects on binding to the C2 domain and the catalytic region. Ca2+ inhibits binding to near Ca2+ stimulates PLDβcat binding to as it millimolar Ca2+ binding (Fig. Ca2+ has a but effect on binding to the enzyme (Fig. with the at a the of PLDβ activity (Fig. level Ca2+ binding by PLDβ but a PLDβ activity. the other millimolar Ca2+ both the activity and binding of Ca2+ binding by the of PLDβ a conformational (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google that to the of amino acid residues of the binding region the active site (16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google Scholar). However, the significant of binding by Ca2+ suggests that this directly the catalytic domain to of this interaction by high of Ca2+ probably between the Ca2+ and the acidic residues of the binding region for binding to PLDβ, but not PLDβcat, bind its its activity mixed vesicles is less at millimolar Ca2+ are clear to the data from these in vitro studies to the binding and regulation of PLDβ in is a to that these studies are to the activation and regulation of plant PLDβ in it is that of Ca2+ to an as or the of the plant to Ca2+ is from to that the of Ca2+ to Ca2+ in the which increases in binding and PLDβ activity are These increases of Ca2+ are to with Ca2+ or those on the membrane P.J. Bot. PubMed Scopus Google and this is in with of PLDβ activity (Fig. this (Fig. the C2 domain binds the membrane in a with and and this is in with that Ca2+ the of the C2 domain (15Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). it binds the membrane with its residues with PIP2, with it binds the membrane with its binding the membrane (Fig. The binding and of membrane phospholipids by the C2 and catalytic regions to enzyme the membrane PLDβ interacts with the C2 domain the binding region to to the membrane in an increased Ca2+ binding affinity of the C2 domain that of the catalytic region suggests that PLDβ is active at Ca2+ this is to the in levels are than those present in the substrate vesicles used for in vitro activity Plant Physiol. PubMed Scopus Google Scholar). studies demonstrate the activation of PLDβ by or (3Pappan K. Qin W. Dyer J.H. Zheng L. Wang X. J. Biol. Chem. 1997; 272: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 4Pappan K. Zheng S. Wang X. J. Biol. Chem. 1997; 272: 7048-7054Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google levels of these in response to and Plant Physiol. 2001; PubMed Scopus Google Scholar). increased levels the binding of PLDβcat to PC, domain binding to is not stimulated by at (16Zheng L. Shan J. Krishnamoorthi R. Wang X. Biochemistry. 2002; 41: 4546-4553Crossref PubMed Scopus (45) Google Scholar). Thus, changes in the of Ca2+ and in response to and the binding of both and substrate to the catalytic domain of PLDβ and its activity (Fig. This mechanism a basis for regulation of PLD activity, which is suggested to under as W. Wang C. Qin C. T. Wang X. Plant PubMed Scopus Google Scholar), W. M. W. R. Wang X. PubMed Scopus Google Scholar), and in response to a plant W. Qin C. Zhao J. Wang X. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Ca2+ and as in various and characterization of their interaction with PLD into the in activation and of Ca2+-dependent PLDs.