AHNAK, a Protein That Binds and Activates Phospholipase C-γ1 in the Presence of Arachidonic Acid

We have recently shown that phospholipase C-γ (PLC-γ) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-γ in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70–130 kDa. They were identified as fragments derived from an unusually large protein (∼700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathioneS-transferase fusion proteins. Both recombinant proteins activated PLC-γ1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-γ activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-γ1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent K m toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2can act as an additional trigger for PLC-γ activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation. We have recently shown that phospholipase C-γ (PLC-γ) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-γ in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70–130 kDa. They were identified as fragments derived from an unusually large protein (∼700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathioneS-transferase fusion proteins. Both recombinant proteins activated PLC-γ1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-γ activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-γ1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent K m toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2can act as an additional trigger for PLC-γ activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation. Activation of phosphoinositide-specific phospholipase C (PLC) 1The abbreviations used are: PLC, phosphoinositide-specific phospholipase C; cPLA2, cytosolic phospholipase A2; AA, arachidonic acid; GST, glutathioneS-transferase; BSA, bovine serum albumin; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PE, phosphatidylethanolamine; PS, phosphatidylserine; IP3, inositol 1,4,5-trisphosphate; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography is a key event in cellular signal transduction. PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), generating two second messengers, inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. To date, a total of 10 different isozymes of PLC have been identified in mammalian cells, which can be classified into three major subfamilies, β (β1 to β4), γ (γ1 and γ2), and δ (δ1 to δ4) isozymes, based on their primary structures (1Rhee S.G Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). Their structural differences correlate with varying mechanisms for their activation. Stimulation of β-isozymes by many agonists occurs through receptors coupled to heterotrimeric G-proteins and is mediated by the α-subunits of the Gq subfamily members and by βγ-subunits. In contrast, the γ-isozymes are activated when phosphorylated by various receptor-coupled protein-tyrosine kinases (1Rhee S.G Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). Several lines of evidence suggested alternative mechanisms for PLC-γ activation in the absence of tyrosine phosphorylation. Jones and Carpenter (2Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Abstract Full Text PDF PubMed Google Scholar) reported that phosphatidic acid could activate both tyrosine-phosphorylated and -unphosphorylated forms of PLC-γ to a similar extent. Since phosphatidic acid is the immediate product of phosphatidylcholine hydrolysis by phospholipase D, activation of phospholipase D in cells may lead to subsequent activation of PLC-γ. We (3Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 4Rameh L.E. Rhee S.G. Spokes K. Kazlauskas A. Cantley L.C. Cantley L.G. J. Biol. Chem. 1998; 273: 23750-23757Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) and others (5Falasca M. Logan S.K. Lehto V.P. Baccante G. Lemmon M.A. Schlessinger J. EMBO J. 1998; 17: 414-422Crossref PubMed Scopus (496) Google Scholar) have recently shown that the product of phosphatidylinositol 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP3), is an activator of PLC-γ. A considerable portion (30–50%) of IP3 generated in response to platelet-derived growth factor was not a consequence of tyrosine phosphorylation of PLC-γ but rather a secondary event following PIP3generation by platelet-derived growth factor-stimulated phosphatidylinositol 3-kinase (3Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 5Falasca M. Logan S.K. Lehto V.P. Baccante G. Lemmon M.A. Schlessinger J. EMBO J. 1998; 17: 414-422Crossref PubMed Scopus (496) Google Scholar). We have also shown that variously spliced forms of the microtubule-associated protein tau (6Hwang S.C. Jhon D.Y. Bae Y.S. Kim J.H. Rhee S.G. J. Biol. Chem. 1996; 271: 18342-18349Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) stimulate PLC-γ activity independently of tyrosine phosphorylation in the presence of unsaturated fatty acids, such as arachidonic acid (AA). Although the concentration of AA in resting cells is quite low, a large quantity of AA can be liberated from phosphatidylcholine by the action of cytosolic phospholipase A2 (cPLA2) upon cell activation (7Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar). Therefore, it is likely that certain stimuli that elicit cPLA2 activation may indirectly cause the activation of PLC-γ if the tau proteins are present. Tau proteins are exclusively expressed in neuronal cells (8Lee G. Cell Motil. Cytoskeleton. 1990; 15: 199-203Crossref PubMed Scopus (78) Google Scholar). Here, we report that non-neuronal cells also contain a protein that can activate PLC-γ in concert with AA, and we identify it as AHNAK. Our finding further bolsters the thesis that indirect activation of PLC-γ can occur in the absence of tyrosine phosphorylation. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) were purchased from Avanti Polar Lipids. AA and cholesterol were purchased from Calbiochem. PIP2 was obtained from Roche Molecular Biochemicals. [inositol-2-3H]PIP2and [2-3H]myo-inositol were purchased from NEN Life Science Products. PLC isozymes (PLC-β1, -γ1, -γ2, and -δ1) were purified from HeLa cells that had been transfected with recombinant vaccinia virus containing the entire coding sequence of the respective enzyme as described (9Park D. Jhon D.Y. Kriz R. Knopf J. Rhee S.G. J. Biol. Chem. 1992; 267: 16048-16055Abstract Full Text PDF PubMed Google Scholar). All manipulations were performed at 4 °C unless otherwise indicated. During purification, PLC-γ1-activating activity was measured at 30 °C for 10 min in 100 μl of a reaction mixture containing 36,000 cpm of [3H]PIP2, 30 μm PIP2, 120 μm PE, 30 μm PS, 30 μm cholesterol, 30 μm arachidonic acid, PLC-γ1 (10 ng), 3 mmCaCl2, 2 mm EGTA, 0.033% (w/v) sodium deoxycholate, 50 mm Hepes-NaOH (pH 7.0), and a source of activator. To maintain the stimulated activity in the linear range of the assay, we adjusted the amount of PLC to obtain an unstimulated, basal activity in the range of a 500–1200 cpm of [3H]IP3 generated. The purification procedure consisted of the following steps. Fresh bovine lungs (3.0 kg) were obtained from a local slaughterhouse and in 10 of a containing (pH mm EGTA, mm mm and with a The was at for 10 and the was further at for was at °C for 10 min and at for 30 The was through to The of was to a 30 that had been with mm Hepes-NaOH (pH mm and mm with the proteins were with 2 of a linear of m in the at a of 10 were and for PLC-γ1-activating activity were and The protein from the was to a that had been with (pH mm and were at a of with for min by a linear of in min were and for PLC-γ1-activating were and proteins (∼700 from the were by on gel The gel was with and protein were from the gel with a The proteins were with an and were with and proteins were by proteins (10 from three different in were with for and to on a with (w/v) acid. were at a of by a linear of in acid The were to amino acid The substrate was as of PS, cholesterol, and in a of as the with various of fatty acids were and were by in a of 50 (pH 7.0), 120 mm 10 mm and (w/v) sodium The was by 50 μl of the substrate with 50 μl of a containing PLC and the to be The mixture of a 30 each of PS, and 120 μm in 50 mm Hepes-NaOH (pH 120 mm 10 mm 0.033% 2 mm and μm unless otherwise for 10 min at 30 the were by the of μl of (w/v) acid and 100 μl of (w/v) bovine serum by The amount of in the to liberated was measured by a liquid In the of the the substrate was with in a containing 10 mm of sodium of were with AHNAK by with 100 μl of a 10 protein in 50 mm Hepes-NaOH (pH at 4 °C further with μl of for were with 50 mm (pH 100 mm with PLC-γ1 was in a total of 100 μl of a containing 50 mm Hepes-NaOH (pH 7.0), 120 mm 10 mm mm 2 mm EGTA, μm BSA, and 100 acid various of PLC-γ1 and proteins to be at for were with μl of the containing were by with 50 μl of for 30 to and to PLC-γ1 on the was by a Rhee S.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and We that the of cytosol of bovine HeLa cells to purified PLC-γ activity measured in the presence of We identified the bovine activator as tau (6Hwang S.C. Jhon D.Y. Bae Y.S. Kim J.H. Rhee S.G. J. Biol. Chem. 1996; 271: 18342-18349Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Since tau is exclusively expressed in neuronal cells, we that the activity in HeLa cell was to a protein with to tau and the of this non-neuronal PLC-γ activator. We various tissues and bovine lung cytosol for large purification of the PLC-γ activator. The in PLC-γ activity toward a substrate containing AA was the of bovine lung cytosol at °C for 10 min in a of PLC-γ activator not The cytosol was to on and to a activity that with a protein and of the protein with apparent molecular from to at purification gel on chromatography on to proteins. proteins were on a and four major protein between and were and the of each protein was for All of the proteins activated PLC-γ1 of the proteins in were with and on a The for and 3 were similar not suggesting that three proteins were two that were to three proteins were to and A of that the sequence of an unusually large protein named AHNAK in A. 1992; PubMed Scopus Google Scholar) coding for the of and were and respectively, in the different of AHNAK, the sequence of 2 was to AHNAK and and to AHNAK AHNAK is a protein of repeated motifs the purified activator proteins are fragments derived from AHNAK The of the purified AHNAK fragments to activate measured with a substrate containing PIP2 PE, PS, cholesterol, and sodium deoxycholate, was on unsaturated fatty shown in various unsaturated fatty acids were in PLC-γ in the presence of AHNAK when at a concentration of 30 had fatty acids were at concentrations to 100 AA was the of PLC-γ activity in the presence of the and was at μm 2 AA was when to as when into the substrate not The of the purified lung AHNAK and AA on PIP2 hydrolysis by -γ1, -γ2, and were in the presence of an amount of AHNAK Activation by AHNAK and AA were apparent with both PLC-γ1 and Stimulation of the activity of was and activity was not at AHNAK contains 30 of a of which are 128 amino acids in We if one several of this repeated are of We two fusion proteins 4 one containing one to AHNAK and the containing three and two to The two fusion proteins from could stimulate the activity of PLC-γ1 in a in the presence of AA, in concentrations to 100 had on the activity 4 was the but both stimulated apparent PLC-γ1 activity at concentrations as as 10 by the was at in the presence of 100 In the absence of AA, both forms were in PLC-γ1, with the of the AHNAK fragments purified from bovine We also the of -γ1, and isozymes the fusion protein Activation of PLC-γ1 was was also activated by the of AA and but to a with PLC-γ1, and the activity of was not at the with AHNAK fragments purified from bovine lung of PLC-γ1 by fusion proteins. of purified fusion proteins. the of by PLC-γ1 was measured in the presence of concentrations of 100 μm AA 100 μm AA with containing μm and mm The of PIP2 in the presence of 100 and 100 μm AA is by of various concentrations of recombinant AHNAK protein on the activity of PLC The of PLC-γ1 and were measured in the presence of 100 μm AA the concentrations of were as described in the to are expressed as activation obtained in the absence of In the of PLC-γ1 activity was on a of the was at an concentration of which is to and at concentrations The mechanism by which activity was not but the basal activity of PLC-γ1 in the absence of AHNAK and AA was not by the of Stimulation of PLC-γ1 activity by AA was also at different concentrations of of PLC isozymes are on S.G. PubMed Scopus Google and the activity of PLC-γ1 from of to a at of Jones G.A. Rhee S.G. Carpenter G. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). The PLC-γ1 activity stimulated by AA a similar AA had action at of the tau protein also PLC-γ1 and rather various PLC isozymes in the presence of AA (6Hwang S.C. Jhon D.Y. Bae Y.S. Kim J.H. Rhee S.G. J. Biol. Chem. 1996; 271: 18342-18349Abstract Full Text Full Text PDF PubMed Scopus (160) Google we tau and AHNAK a activation shown in AHNAK was at the PLC-γ1 activity in the presence of a concentration of that the tau interaction on PLC-γ with that of AHNAK. The of AHNAK and AA on PLC-γ activity at various concentrations of PIP2 was a substrate of PIP2 and In the presence of mm the activity of PLC-γ was with that measured with the substrate containing PIP2 PE, PS, cholesterol, and sodium not this substrate AHNAK and AA activated PLC-γ activity which with the activation in 4 with the substrate of PIP2, PE, PS, cholesterol, and sodium the concentrations of AHNAK and AA were The of PIP2 hydrolysis was measured with varying concentration of PIP2 in the presence and absence of and A of to the lines that the was not by the presence of concentrations of and AA, the apparent K m for PIP2 was from 4 μm to AHNAK could activate PLC-γ1 at concentrations as as 4 we that AHNAK a with To was the of and PLC-γ1 was in the containing PLC-γ1 was from the with a and by of PLC-γ1 to AHNAK was was with a that had been with The to was by the of but not by the of GST, to the containing PLC-γ we that the was on is shown in in the presence of AA the was at PLC-γ1 concentrations as as and in a PLC-γ1 in the absence of AA, the was at 30 results that PLC-γ1 with AHNAK, and the role of AA is to the We could also the of and PLC-γ1 in the presence of AA not this from high to of PLC-γ1 to the of cytosolic of HeLa cells, which are in AHNAK and PLC-γ1, were with a substrate containing and of was stimulated by the of AA, but a fatty acid was The of recombinant AHNAK to the reaction mixture not further the of it that cells a amount of activator for PLC and also that in the of HeLa cells this activation activity in the absence of AA was to PLC-γ1 as as PLC isozymes and that are to in HeLa cells cells not contain and G. Since AHNAK and AA stimulated to a but 3 and we the of PLC-γ1 to the activity by derived from the PLC-γ1 D. R. M.A. Carpenter G. A. 1997; PubMed Scopus Google Scholar). The cells expressed amount of PLC-γ1 and of D. R. M.A. Carpenter G. A. 1997; PubMed Scopus Google Scholar, J. Carpenter G. Biol. 1998; PubMed Scopus Google Scholar). cells were obtained by cells with vaccinia virus the PLC-γ1 of PLC-γ1 by the was by were obtained from cells with virus the cells, and the activity of the cytosolic was measured in the presence and absence of AA The PLC activity in the cells was and not by the of In contrast, the PLC activity in the cytosol from cells was that from cells and by the of results that the is the of AA were from HeLa cells that had been with for and used as substrate for AA the of inositol by PLC-γ1, but the of recombinant AHNAK not cause further In a the were with to proteins and used as the AA not cause of PLC-γ the of AHNAK AA of PLC-γ results suggest that the AHNAK to activate PLC-γ1 in the presence of AA and that AHNAK and AA were of the activity of PLC-γ1 toward cell In the we tau, AHNAK PLC-γ in the presence of AHNAK was as a protein to the of bovine cells and J. Cell Biol. PubMed Scopus Google Scholar). The protein was also independently identified by and as the product of a is in and several of cell lines and named AHNAK to A. 1992; PubMed Scopus Google Scholar). that AHNAK was from a and two of and that the amino acids and the amino acids, the amino acid sequence derived from the two which about of the entire AHNAK coding it was suggested that the AHNAK protein can be into three structural the amino acids, a large of about amino acids with multiple repeated motifs, and the amino acids the AHNAK was to J. M. PubMed Scopus Google Scholar). The that the of and recently been The sequence a of amino acids for AHNAK. suggested the of AHNAK is of 30 repeated The repeated is 128 amino acids in but of the are 128 The motifs are on to each with to amino acid of the amino acid sequence the repeated a of of a a and to of amino acid The AHNAK suggesting that the activator proteins purified from bovine lung are fragments of AHNAK rather To this of HeLa cell were serum the purified activator A major protein with an of was with several of when were in the presence of a mixture of when the were in the absence of the were with but for several to the was not and the of molecular was that AHNAK is to AHNAK to be an and protein in various cellular AHNAK was not in the of HeLa cells and phosphorylated on and J. Cell Biol. 1993; PubMed Scopus Google Scholar). In contrast, was mainly in the and to a in the in cell and it in M. K. K. J. J. Cell 1993; Google Scholar). was also that different could different in a In AHNAK was mainly in the when cells were in but to the with a in the of phosphorylation upon an in concentrations with Cell PubMed Scopus Google Scholar). The of as as the of phosphorylation of the protein in several cells was growth and J. Cell Biol. 1993; PubMed Scopus Google Scholar). on and cellular of AHNAK, been that at the cellular of AHNAK. Our report is the such Activation of PLC-γ isozymes by AHNAK was in many from activation by tau (6Hwang S.C. Jhon D.Y. Bae Y.S. Kim J.H. Rhee S.G. J. Biol. Chem. 1996; 271: 18342-18349Abstract Full Text Full Text PDF PubMed Scopus (160) Google is in their primary structures that both proteins are with two each in that may a mechanism of activation. have that tau can with three of the activation reaction PLC-γ 1998; PubMed Scopus Google PIP2 C.C. Chen J. K.S. J. 1997; Full Text PDF PubMed Scopus Google and AA J. 1997; Google Scholar). Tau was shown to with but not with of PLC, from of cells 1998; PubMed Scopus Google Scholar). Tau was also shown to with PIP2, of PIP2 was by tau in a similar to that by the protein, C.C. Chen J. K.S. J. 1997; Full Text PDF PubMed Scopus Google Scholar). of by fatty acids was also from the that by tau was stimulated by fatty acids, with AA as the and fatty acids J. 1997; Google Scholar). We have shown that AHNAK can with PLC-γ1 in the presence of that AHNAK and tau as the receptors for unsaturated fatty acids, PLC-γ1 is not by fatty acids, the activity of the enzyme was by fatty acids in the absence of the activator proteins. a consequence of interaction with the AHNAK, the apparent K m for PIP2 be that that on the not but rather J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is also the for PLC-γ1 Jones G.A. Rhee S.G. Carpenter G. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). The K we obtained contain two the for and the for PIP2 molecule are to the apparent K into two is apparent that the of AHNAK AA results in of the which can be by in the for for PIP2, for In this it is to that the activation of PLC-γ tyrosine phosphorylation is also attributable to an in the for substrate but not to an in the Jones G.A. Rhee S.G. Carpenter G. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). is also to that the of activation the and activation was with as as with in the presence of deoxycholate, that both tau and AHNAK are to be phosphorylation may a role in of their as PLC-γ Tau is a substrate of various protein protein protein and protein in J. 1997; PubMed Scopus Google and can be AHNAK is also shown to be J. Cell Biol. 1993; PubMed Scopus Google Scholar, Cell PubMed Scopus Google Scholar). In a of a of agonists to their receptors the activation of PLC-γ through tyrosine phosphorylation. evidence that the activation mechanism for PLC-γ is not as as Activation of phospholipase D may also lead to activation of PLC-γ1 through of phosphatidic acid (2Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Abstract Full Text PDF PubMed Google Scholar). We and others have recently shown that purified PLC-γ and that receptors coupled to phosphatidylinositol 3-kinase are of of PLC-γ indirectly in cells through the of (3Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 4Rameh L.E. Rhee S.G. Spokes K. Kazlauskas A. Cantley L.C. Cantley L.G. J. Biol. Chem. 1998; 273: 23750-23757Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 5Falasca M. Logan S.K. Lehto V.P. Baccante G. Lemmon M.A. Schlessinger J. EMBO J. 1998; 17: 414-422Crossref PubMed Scopus (496) Google Scholar, G. M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Our (6Hwang S.C. Jhon D.Y. Bae Y.S. Kim J.H. Rhee S.G. J. Biol. Chem. 1996; 271: 18342-18349Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) and suggest that of AA can be trigger for PLC-γ activation in the presence of tau AHNAK proteins. AA is mainly from phosphatidylcholine by the action of cPLA2 (7Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar) and as the of various The in activate cells by AA is also to several to can activate protein C 1992; PubMed Scopus Google R. PubMed Scopus Google and K. M. M. A. 1998; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google it protein D. A. PubMed Scopus Google Scholar) and to J. G. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). PLC-γ is to the of AA Activation of cPLA2 (7Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (747) Google and it can be a secondary event following PLC activation, activation by receptors PLC-γ activation by growth factor in may activate PLC-γ if AHNAK tau are constituting a in the hydrolysis of the PIP2 is a activator of cPLA2 M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, hydrolysis of PIP2 by PLC the activity of cPLA2, constituting a in of AA In results suggest that of AA can PLC-γ activation, and we have also shown that HeLa cells the for activation. Several in have suggested that AA PLC activity independently of to of with AA PLC and this response Google Scholar). They also that activation of was in the of and in cells Life PubMed Scopus Google Scholar). AA stimulated in and from bovine cells, and were M. 1990; PubMed Scopus Google Scholar). The fatty acid was also shown to and in M.A. J. 1990; PubMed Scopus Google Scholar) and to by an in J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and in a cell S.C. M. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). The was shown to be independent of J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We that if not of now can be by the of AHNAK and