Essential Ca2+-independent Role of the Group IVA Cytosolic Phospholipase A2 C2 Domain for Interfacial Activity

The cytosolic Group IVA phospholipase A2 (GIVAPLA2) translocates to intracellular membranes to catalyze the release of lysophospholipids and arachidonic acid. GIVAPLA2 translocation and subsequent activity is regulated by its Ca2+-dependent phospholipid binding C2 domain. Phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) also binds with high affinity and specificity to GIVAPLA2, facilitating membrane binding and activity. Herein, we demonstrate that GIVAPLA2 possessed full activity in the absence of Ca2+ when PI-4,5-P2 or phosphatidylinositol 3,4,5-trisphosphate were present. A point mutant, D43N, that is unable to bind Ca2+ also had full activity in the presence of PI-4,5-P2. However, when GIVAPLA2 was expressed without its Ca2+-binding C2 domain (ΔC2), there was no interfacial activity. GIVAPLA2 and ΔC2 both had activity on monomeric lysophospholipids. ΔC2, but not the C2 domain alone, binds to phosphoinositides (PIPns) in the same manner as the full-length GIVAPLA2, confirming the location of the PIPn binding site as the GIVAPLA2 catalytic domain. Moreover, proposed PIPn-binding residues in the catalytic domain (Lys488, Lys541, Lys543, and Lys544) were confirmed to be essential for PI-4,5-P2-dependent activity increases. Exploiting the effects of PI-4,5-P2, we have discovered that the C2 domain plays a critical role in the interfacial activity of GIVAPLA2 above and beyond its Ca2+-dependent phospholipid binding. The cytosolic Group IVA phospholipase A2 (GIVAPLA2) translocates to intracellular membranes to catalyze the release of lysophospholipids and arachidonic acid. GIVAPLA2 translocation and subsequent activity is regulated by its Ca2+-dependent phospholipid binding C2 domain. Phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) also binds with high affinity and specificity to GIVAPLA2, facilitating membrane binding and activity. Herein, we demonstrate that GIVAPLA2 possessed full activity in the absence of Ca2+ when PI-4,5-P2 or phosphatidylinositol 3,4,5-trisphosphate were present. A point mutant, D43N, that is unable to bind Ca2+ also had full activity in the presence of PI-4,5-P2. However, when GIVAPLA2 was expressed without its Ca2+-binding C2 domain (ΔC2), there was no interfacial activity. GIVAPLA2 and ΔC2 both had activity on monomeric lysophospholipids. ΔC2, but not the C2 domain alone, binds to phosphoinositides (PIPns) in the same manner as the full-length GIVAPLA2, confirming the location of the PIPn binding site as the GIVAPLA2 catalytic domain. Moreover, proposed PIPn-binding residues in the catalytic domain (Lys488, Lys541, Lys543, and Lys544) were confirmed to be essential for PI-4,5-P2-dependent activity increases. Exploiting the effects of PI-4,5-P2, we have discovered that the C2 domain plays a critical role in the interfacial activity of GIVAPLA2 above and beyond its Ca2+-dependent phospholipid binding. The Group IVA phospholipase A2 (GIVAPLA2) 1The abbreviations used are: GIVAPLA2, the wild type (749-amino acid) Group IVA phospholipase A2 (cytosolic phospholipase A2 α); PLA2, phospholipase A2; BSA, fatty acid-free bovine serum albumin; ΔC2, α/β hydrolase domain of GIVAPLA2 (amino acids 134–756, including C-terminal His tag); His GIVAPLA2, GIVAPLA2 with a C-terminal extension of one Tyr and six His residues; HRP, horseradish peroxidase; Lyso-PC, 1-palmitoyl-l-lyso-3-phosphatidylcholine; Lyso-PLA, lysophospholipase; MAFP, methyl arachidonyl fluorophosphonate; PAPC, 1-palmitoyl-2-arachidonoyl-phosphatidylcholine; PI, phosphatidylinositol; PIP, PI monophosphate; PIP2, PI bisphosphate; PIP3, PI 3,4,5-trisphosphate; PI-4,5-P2, PI 4,5-bisphosphate; PI-4-P, PI 4-phosphate; PI-3-P, PI 3-phosphate; PI-3,4-P2, PI 3,4-bisphosphate; PIPn, phosphoinositide; TBS-T/BSA, Tris-buffered saline-Tween 20/BSA solution; WT, wild type. plays a central role in intracellular phospholipid hydrolysis. Although it is only one of many different mammalian phospholipase A2s (1Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Google Scholar), it is the rate-limiting provider of lysophospholipid and the free polyunsaturated fatty acids such as arachidonic acid that go on to form platelet-activating factor and eicosanoids, respectively (2Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Google Scholar, 3Bonventre J.V. Huang Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Google Scholar, 4Fujishima H. Sanchez Mejia R.O. Bingham III, C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4807Google Scholar, 5Gijon M.A. Spencer D.M. Siddiqi A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Google Scholar). These various downstream products are central to many physiological processes as well as many pathological conditions (6Sapirstein A. Bonventre J.V. Biochim. Biophys. Acta. 2000; 1488: 139-148Google Scholar, 7Funk C.D. Science. 2001; 294: 1871-1875Google Scholar). The activity of GIVAPLA2 in mammalian cells is regulated by at least two major mechanisms that can act separately or in conjunction with each other. The is by intracellular Ca2+ to the translocation of GIVAPLA2 the to its in the and membranes S. T. E. Leslie C.C. J. Biol. Chem. Scholar, A.R. J. Biol. Chem. Scholar, Spencer D.M. A. Leslie C.C. J. Biol. Chem. 2001; Scholar). Ca2+-dependent is by the C2 domain of GIVAPLA2, binds two Ca2+ with a affinity E.A. 1997; Scholar, S. J. Biol. Chem. Scholar). The that to translocation of the C2 domain to membranes also to membrane of E.A. D.M. J. Biol. Chem. Scholar, E.A. Scholar, Scholar, S. J. Biol. Chem. 1999; Scholar), the catalytic α/β hydrolase domain to with its A. J. H. J. 1999; Scholar). The major for GIVAPLA2 is at one or A. Scholar, Y. S. F. III, A. T. J. Biol. Chem. 2000; 275: Scholar, Y. S. J. H. J. Biol. Chem. 2001; Scholar). that to of GIVAPLA2 by the activity of the A. Scholar, Y. S. F. III, A. T. J. Biol. Chem. 2000; 275: Scholar, Y. S. J. H. J. Biol. Chem. 2001; Scholar). Ca2+ and such as phosphoinositides have in the of GIVAPLA2 activity. by and K. H. S. H. K. Biochim. Biophys. Acta. and Leslie and C.C. Biochim. Biophys. Acta. on and GIVAPLA2, that the activity of with that GIVAPLA2 activity is by but and by with phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) D.A. Dennis E.A. J. Biol. Chem. Scholar). that GIVAPLA2 binds in a to PI-4,5-P2 with high affinity and specificity D.A. Dennis E.A. J. Biol. Chem. Scholar). the presence of PI-4,5-P2 and the absence of GIVAPLA2 both to and was in D.A. Dennis E.A. J. Biol. Chem. Scholar). to we that of both phosphatidylinositol and PI-4,5-P2 with and were for release by cells J. M.A. Li Dennis E.A. J. 2000; Scholar, M.A. J. Dennis E.A. Biophys. 2000; Scholar). no in intracellular was the of the PIPn J. M.A. Li Dennis E.A. J. 2000; Scholar). have of GIVAPLA2 activity in without in the of intracellular Ca2+ M.A. Spencer D.M. Siddiqi A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Google Scholar, J. M.A. Li Dennis E.A. J. 2000; Scholar, M.A. J. Dennis E.A. Biophys. 2000; Scholar, M.A. Spencer D.M. Leslie C.C. J. Biol. Chem. Scholar, Sapirstein A. N. Bonventre J.V. J. Biol. Chem. 2001; Scholar). we the of GIVAPLA2 be regulated by Ca2+ and that GIVAPLA2 activity in the presence of many have that the that bind Ca2+ also full activity in the presence of PI-4,5-P2. binding to Ca2+ is not for GIVAPLA2 we that the presence of the C2 domain is for interfacial activity. for the a role of the C2 in that it is to GIVAPLA2 in or at a membrane we have confirmed that the site domain alone, and not the C2 a PIPn binding site residues at and PI-4,5-P2 and were Science. GIVAPLA2 S. J. Scholar), to GIVAPLA2 J.V. S. J. Biol. Chem. Scholar), IVA J. J. Biol. Chem. Scholar), and a with the of Group IVA J. Biol. Chem. 1999; were and GIVAPLA2 wild type and the to at residues and A. were of GIVAPLA2 wild type and the two and S. J. Biol. Chem. were of of and were by PI-3-P, PI-3,4-P2, and phosphatidylinositol 3,4,5-trisphosphate were a were were was was were was and were was was the and and affinity were was was and were horseradish was was and were membrane was Group IVA and the of GIVAPLA2 with a C-terminal His extension as His GIVAPLA2 to it the acid was a in the by the with and the GIVAPLA2 and it the GIVAPLA2 expressed in in S. J. Scholar, E. A.R. Scholar, Leslie C.C. Biophys. Scholar, E. F. III, Leslie C.C. J. Biol. Chem. Scholar). was used for was in the to The Ca2+-binding was the with for and A PIPn-binding site proposed on and to D.A. Dennis E.A. J. Biol. Chem. was with with the same as The were and The and conditions for are in the were confirmed by to the C2 two different were used to the C2 domain a and and to it to be a His The C2 domain acids of GIVAPLA2 with and The and conditions for the C2 domain are also in the to the hydrolase domain without the C2 domain (ΔC2), the was with as in E.A. D.M. J. Biol. Chem. to a its the at the ΔC2 of residues and the same C-terminal as the full-length Group IVA and for GIVAPLA2 in cells were cells with were and with and His GIVAPLA2, D43N, ΔC2, the C2 or the point to be to the were to and one that was confirmed to the and The was as but the were with as in a a was and were at a of of was the cells that were in cells that had with were and with on for and for at at to cells and of His GIVAPLA2 such that the was and on confirmed the high of was the His affinity The a was used to for The were to the with but not by The and were to by was also to a and was with not as a for of that The and that the were and at a high by The in had full activity on such that were used the or at in the with the activity of GIVAPLA2 D.A. Dennis E.A. J. Biol. Chem. Scholar), were in of at fatty acid free bovine serum and The were of and in a of The was as D.A. Dennis E.A. J. Biol. Chem. Scholar, S. D.A. C.C. Dennis E.A. J. Chem. Scholar). The were in and to form a of The not Ca2+ to the of the was of in a of the as the form at a and that in the The were to form to that the were the and the the and the each were to of the Ca2+ or was in the The had with to the and the The in the were in a for The was by the of GIVAPLA2 of that Ca2+ and by the to and the as a the was and the fatty acids were a Dennis E.A. as D.A. Dennis E.A. J. Biol. Chem. Scholar, S. D.A. C.C. Dennis E.A. J. Chem. Scholar). The were in the of activity only of of were no was were and were the with was to the activity with the that the PI-4,5-P2 PI-4,5-P2, and D.A. Dennis E.A. J. Biol. Chem. Scholar). PI-4,5-P2 the activity of GIVAPLA2 by to the of used was to and the was to The were in manner to that The of and of were in to hydrolysis. of that the was not by the The in the with different of the GIVAPLA2 or or the of the activity with PI-4,5-P2 to without PI-4,5-P2 is at a wild type is with each to for the of the and the and E.A. D.M. J. Biol. Chem. Scholar, C.C. J. Biol. Chem. Scholar), were BSA, and in a of The Lyso-PC, is the critical of Dennis E.A. Scholar). The was to the above that the were in without Ca2+ or The Ca2+ or was to the the was The was only confirming the in the was in to and there were no of The was used to the fatty acids as that acid was to the to the of the of acid the form of the was to and the was to no acid was in the are as the a with each in or binding was and was to the A for each and a at were used for each of the The were for in of and The was with a in of The was with the with of for The was for in of with of by or the His the the was for with of the in of the the was a of as by the the was the the was but not and with for to the The was two to and to the had of the absence of GIVAPLA2 is to bind to and be on lysophospholipid Dennis E.A. Biochim. Biophys. Acta. Scholar), membrane PI-4,5-P2 D.A. Dennis E.A. J. Biol. Chem. Scholar), and membranes of the A. Scholar). have that in the presence of and of GIVAPLA2 activity in D.A. Dennis E.A. J. Biol. Chem. Scholar). we have the activity of on GIVAPLA2 in the absence of the presence of and phosphatidylinositol in not activity for GIVAPLA2 in the absence of Ca2+ as in However, in the presence of PI-3-P, PI-4-P, PI-3,4-P2, PI-4,5-P2, or PIP3, there was GIVAPLA2 activity in the absence of Ca2+ Moreover, the with PI-4,5-P2 and were to one with and without Ca2+ and were both the that but and also activity in the absence of to These GIVAPLA2 without Ca2+ were only and of the with but are above the activity of GIVAPLA2 on without Ca2+ or with and of without Ca2+ have the free to we Ca2+ of GIVAPLA2 to that the can the domain A of various GIVAPLA2 is in the D43N, ΔC2, and His GIVAPLA2, we activity in the PI-4,5-P2 activity with and without The in demonstrate that in the presence of PI-4,5-P2, the same activity as His its to bind the activity the same with or without The ΔC2 not have activity with or without in to the activity. with and that the activity of is with His GIVAPLA2 in the presence of PI-4,5-P2, it was critical to without PI-4,5-P2. in D43N, ΔC2, and a of ΔC2 and the C2 domain have no activity above on in the presence of and ΔC2 were not to have activity in Ca2+-dependent conditions on E.A. D.M. J. Biol. Chem. Scholar, J. Biol. Chem. 1999; Scholar). activity was conditions for His GIVAPLA2 in the absence of both PI-4,5-P2 and Ca2+ not and of D43N, ΔC2, and we Dennis E.A. Biochim. Biophys. Acta. and C.C. J. Biol. Chem. had that GIVAPLA2 activity on of the activity of and ΔC2 on in the absence of PI-4,5-P2. in His GIVAPLA2 and both have high activity on to ΔC2 and at and His GIVAPLA2 and D43N, there was no in activity in the presence or absence of Ca2+ not The activity of ΔC2 on was His GIVAPLA2 and D43N, that the C2 domain is for activity on as well as on The of interfacial or activity for ΔC2 have that it was of catalytic activity. had that ΔC2 the to bind to and membranes with or without Ca2+ in E.A. D.M. J. Biol. Chem. Scholar, S. J. Biol. Chem. 1999; Scholar). was also that both full-length GIVAPLA2 and the ΔC2 possessed activity on monomeric E.A. D.M. J. Biol. Chem. Scholar), the catalytic residues are on domain to that ΔC2 as a was with in His GIVAPLA2, D43N, and ΔC2 have activity on monomeric to and the of are with activity the of His GIVAPLA2 and on were at and interfacial activity. However, ΔC2 a in activity to the critical at there was no to A and and not activity for of the activity for ΔC2 in The activity of ΔC2 His GIVAPLA2 and at is with by in the of that be by the GIVAPLA2 and D43N, but not the activity of ΔC2 is the two full-length the was well above the at conditions of have the monomeric activity of GIVAPLA2 is to the same site as the activity. we the site mutant, The had no monomeric activity. the conditions activity as as have for The of of activity by that GIVAPLA2 catalytic on J.V. S. J. Biol. Chem. Scholar). of to of GIVAPLA2 binds to was on a The have of various and to of The have used to the PIPn binding of various PIPn and PIPn S. J. 2000; Scholar, S. J. Science. 2001; Scholar, Huang Science. 2001; 294: Scholar), including phospholipase T. A. T. J. Biol. Chem. Scholar). The binding for a with of was no binding was for GIVAPLA2 GIVAPLA2 is to the fatty of a by the fatty of the lysophospholipid C.C. J. Biol. Chem. Scholar, K. Dennis E.A. J. Biol. Chem. 1997; Scholar). on the the have the with the to the of the the we two GIVAPLA2 was with of of GIVAPLA2 with the and with there was at PIPn as in to effects of the MAFP, was The for not with GIVAPLA2 not The location and of each on the is in a in The of the that conditions GIVAPLA2 PI-3-P, PI-4-P, and phosphatidylinositol to a in was also with PI-4,5-P2, PI-3,4-P2, phosphatidylinositol and with PIP3, PI, and was for or The same were for His GIVAPLA2 with or the not domain was to bind to ΔC2 and the C2 domain were with MAFP, with and with the The that ΔC2, but not the C2 the same as the GIVAPLA2 was in the same manner and to the for the GIVAPLA2 not Although it was for and His GIVAPLA2, was not and not with the for ΔC2 and the C2 domain. of Ca2+ was for The was used to the of different GIVAPLA2 to bind to the various was and However, it be used as a of of binding or specificity of binding for are for including the of the various in the and the for each that the on the were not as a on the factor to is the of the of each PIPn-binding such as and have phospholipid binding in various S. S. S. Scholar, J. 1999; Scholar). of also bind to the such as M.A. J. Proc. Natl. Acad. Sci. U. S. A. Scholar). GIVAPLA2 is it not bind but binds to only in D.A. Dennis E.A. J. Biol. Chem. Scholar). Moreover, GIVAPLA2 at least membrane binding the the C2 and the PIPn binding GIVAPLA2 binds to the it is that one or both of the binding are in to the the for or These be on the by the of of the and be with of the for the of and S. J. Biol. Chem. that residues were in the binding and activity PI-4,5-P2. The two and were on phospholipid conditions GIVAPLA2 activity with PI-4,5-P2 was to without S. J. Biol. Chem. Scholar). and had activity and S. J. Biol. Chem. Scholar). both of had no in activity with to PI-4,5-P2 but each had a S. J. Biol. Chem. Scholar). of the PI-4,5-P2 in by the we to the in conditions that the of PI-4,5-P2 or D.A. Dennis E.A. J. Biol. Chem. Scholar). the and activity of and in the and PI-4,5-P2 that the activity for both with the S. J. Biol. Chem. Scholar). The in the presence of only PI-4,5-P2 that and only with for the These S. J. Biol. Chem. Scholar). we have confirmed that residues to be for the PIPn-binding a D.A. Dennis E.A. J. Biol. Chem. Scholar), we that the PIPn-binding site of GIVAPLA2 be at a of residues acids and have of to the or The and The had no in or activity not and the same with PI-4,5-P2 as for GIVAPLA2 in the D.A. Dennis E.A. J. Biol. Chem. Scholar). The are with for a that was to have no effects on the or activity S. J. Biol. Chem. Scholar). of on PIPn effects and GIVAPLA2 we a mutant, that was not a His GIVAPLA2 that was by the Y. S. F. III, A. T. J. Biol. Chem. 2000; 275: Scholar). that in the the had of the activity of the His GIVAPLA2 activity of the His the is with the activity GIVAPLA2 A. Scholar, Y. S. F. III, A. T. J. Biol. Chem. 2000; 275: Scholar, J.V. J. Biol. Chem. Scholar). also that in the PI-4,5-P2 the had of the activity of the His GIVAPLA2 the His GIVAPLA2 and the were both by in the presence of PI-4,5-P2. no or and GIVAPLA2 at are E. F. III, Leslie C.C. J. Biol. Chem. Scholar). site was Y. S. J. H. J. Biol. Chem. 2001; Scholar). was not in the His GIVAPLA2 used in on of the same E. F. III, Leslie C.C. J. Biol. Chem. and is also in the effects of PI-4,5-P2. that and or of can the activity of GIVAPLA2 activity without GIVAPLA2 activity with and on the of the high activity of GIVAPLA2 is a of GIVAPLA2 that we have the GIVAPLA2 activity PI-4,5-P2 were when the was or Lyso-PC, as in GIVAPLA2, the activity on was that for activity and as for the the activity without PI-4,5-P2 was there was activity and also Dennis E.A. Biochim. Biophys. Acta. Scholar). The and of the or that the activity of Scholar, Dennis E.A. 1999; Scholar, Dennis E.A. Biochim. Biophys. Scholar). have at of by the of the same in two different the GIVAPLA2 activity on and the were without the The of GIVAPLA2 to of lysophospholipids is to be and also C.C. J. Biol. Chem. and Dennis E.A. Biochim. Biophys. Acta. Scholar). were with PI-4,5-P2 there was a in GIVAPLA2 activity in the absence or presence of as in These were in the for PI-4,5-P2 in the S. J. Biol. Chem. Scholar, A. A. J. J. 2001; Scholar). is in to the that a in activity in the presence of PI-4,5-P2 The in the absence of PI-4,5-P2 was Ca2+-dependent and and also Dennis E.A. Biochim. Biophys. Acta. Scholar), in to the that no Ca2+ The of to have had a on the activity of GIVAPLA2, the activity Dennis E.A. J. Biol. Chem. Scholar, 2000; Scholar). in the presence of Ca2+ and absence of PI-4,5-P2, the activity by with the of However, when PI-4,5-P2 was the the was a that in the presence of the PI-4,5-P2 effects on GIVAPLA2 activity on the of the The same effects on activity be in the absence of Ca2+ A and However, without PI-4,5-P2, the with a of activity. that in the absence of activity also on the of the in the presence of PI-4,5-P2 with no the by a in to the These that the of the is also for PI-4,5-P2-dependent activity. that the or of the be it lysophospholipid or can the activity of GIVAPLA2 as well as the effects of Ca2+ and PI-4,5-P2 on its activity. of the effects of PI-4,5-P2, and the of a for GIVAPLA2 was Y. Dennis E.A. 1999; that can mammalian The is to GIVAPLA2 activity in of or and Y. Dennis E.A. 1999; Scholar). GIVAPLA2 is the its are in the intracellular membranes such as and Spencer D.M. A. Leslie C.C. J. Biol. Chem. 2001; Scholar). is well that the translocation of GIVAPLA2 to its membranes can be regulated by intracellular Spencer D.M. A. Leslie C.C. J. Biol. Chem. 2001; and the effects of Ca2+ are the C2 we have that the C2 domain of GIVAPLA2 is for interfacial activity. a role for the C2 domain in the of the catalytic domain. role also be for Ca2+-dependent interfacial activity but be by the Ca2+-dependent membrane binding role of the C2 domain. for the translocation and of GIVAPLA2 without a in intracellular M.A. Spencer D.M. Siddiqi A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Google Scholar, J. M.A. Li Dennis E.A. J. 2000; Scholar, M.A. J. Dennis E.A. Biophys. 2000; Scholar, M.A. Spencer D.M. Leslie C.C. J. Biol. Chem. Scholar, Sapirstein A. N. Bonventre J.V. J. Biol. Chem. 2001; Scholar). with D.A. Dennis E.A. J. Biol. Chem. Scholar), the the that PI-4,5-P2, and have role to Ca2+ in the membrane affinity of membrane affinity activity by and membrane are in and and have in a of the membrane N. Biochim. Biophys. Acta. Scholar, S. Biol. Scholar, J. Scholar, A. A. J. Biol. Chem. Scholar). The activity for GIVAPLA2 in at PI-4,5-P2 is a for many membranes S. J. S. J.P. Scholar). of PI-4,5-P2 have in various intracellular membranes such as and cytosolic in and cells J. and at the cytosolic membranes of cells S. J. Biol. Chem. Scholar). the the PI-4,5-P2 the separately for GIVAPLA2 in the same cells S. J. Biol. Chem. Scholar). of a and GIVAPLA2 the arachidonic acid release S. J. Biol. Chem. Scholar), that there be a physiological GIVAPLA2 and PI-4,5-P2. we that GIVAPLA2 can have its activity on or with or without with PI-4,5-P2 D.A. Dennis E.A. J. Biol. Chem. and C.C. J. Biol. Chem. Scholar, Dennis E.A. Biochim. Biophys. Acta. Scholar), is to be a that can to full GIVAPLA2 activity in However, PI-4,5-P2, as the of S. J. S. J.P. Scholar, K. S. S. 2001; Scholar), is to PI-4,5-P2 and PIP3, and also but not activity. is the of the PIPn J. M.A. Li Dennis E.A. J. 2000; Scholar, S. J. S. J.P. Scholar), it is that it to the activity of The of and PI-4,5-P2 was in cells by and by J. M.A. Li Dennis E.A. J. 2000; Scholar). GIVAPLA2 to release arachidonic acid without in intracellular J. M.A. Li Dennis E.A. J. 2000; Scholar). The of PIPn in the cells have at the intracellular membranes to GIVAPLA2 Spencer D.M. A. Leslie C.C. J. Biol. Chem. 2001; Scholar), as one or PI have to to membranes S. Biol. Scholar, J. 2000; Scholar). The full activity of in PI-4,5-P2 and high activity confirmed that and His GIVAPLA2 can have full activity in the absence of Ca2+ be for GIVAPLA2 the C2 domain is The ΔC2 not have or activity on interfacial with or without PI-4,5-P2, or The activity of ΔC2 that the catalytic site was but activity on to the in ΔC2, free C2 domain was a the C2 domain and not have activity or without that the two not with each to form These for the that C2 domain is for GIVAPLA2 interfacial activity of its to bind binding were to the C2 domain or the ΔC2 the PIPn binding The PIPn-binding in the for the GIVAPLA2, His GIVAPLA2, and ΔC2, the C2 domain no PIPn binding. that the site but not the C2 binds These the that a GIVAPLA2 PIPn binding site is on the site domain S. J. Biol. Chem. Scholar). have confirmed with and that the PI-4,5-P2 binding site residues Lys541, Lys543, and are in the ΔC2 S. J. Biol. Chem. Scholar). that the catalytic domain also bind to in a Ca2+-dependent manner A. Scholar), the PIPn binding residues are in GIVAPLA2 but not in its or have that two are not by PI-4,5-P2, in to GIVAPLA2 K. D.A. Dennis E.A. J. 2000; Scholar). The location of the PIPn binding site on the GIVAPLA2 catalytic domain to the PI-4,5-P2 binding site in the C2 domain of S. J. J. Biol. Chem. Scholar). Although the C2 domain not be for PIPn it to be for a interfacial by facilitating or site is that various of C2 domain residues are separately in PIPn and the Ca2+-dependent membrane binding. the presence of the PIPn-binding residues on the catalytic domain and the of the catalytic domain to bind to on The C2 domain to membrane affinity A. Scholar, E.A. T. J. J. Biol. Chem. Scholar), the critical role of the C2 domain in interfacial activity. have that including PI-4,5-P2, the activity of GIVAPLA2 by K. H. S. H. K. Biochim. Biophys. Acta. Scholar, C.C. Biochim. Biophys. Acta. Scholar, S. J. Biol. Chem. Scholar, A. A. J. J. 2001; Scholar). to that PI-4,5-P2 the activity of GIVAPLA2 in of by or and in by to D.A. Dennis E.A. J. Biol. Chem. Scholar). A is that when PI-4,5-P2 is to that have the effects are as in D.A. Dennis E.A. J. Biol. Chem. Scholar). PI-4,5-P2 is to that have high the effects of PI-4,5-P2 as in and K. H. S. H. K. Biochim. Biophys. Acta. Scholar, C.C. Biochim. Biophys. Acta. Scholar, S. J. Biol. Chem. Scholar, A. A. J. J. 2001; Scholar). the high of activity in the of of PI-4,5-P2 to activity as in and C.C. Biochim. Biophys. Acta. Scholar, S. J. Biol. Chem. Scholar, and A. A. J. J. 2001; the effects of PI-4,5-P2 are not by the D.A. Dennis E.A. J. Biol. Chem. Scholar), a binding The PIPn-binding also to by a or be in to the membrane affinity of GIVAPLA2 in the presence of PI-4,5-P2 D.A. Dennis E.A. J. Biol. Chem. Scholar). that in to the role of to Ca2+ there is a role of to or a a is to the discovered of the C2 domain for interfacial activity is The of the C2 domain and of GIVAPLA2 by PI-4,5-P2 in and A. J. H. J. 1999; Scholar, S. J. Biol. Chem. and to the of GIVAPLA2 activity and are to and for the GIVAPLA2 and are to for of GIVAPLA2 WT, and are to for a of GIVAPLA2 and are to for the PI-3-P, PI-3,4-P2, and used in and for many and and for the of the with