ADP-ribosylation Factor and Rho Proteins Mediate fMLP-dependent Activation of Phospholipase D in Human Neutrophils

Abstract

Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the Gi family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required. Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the Gi family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required. Phospholipase D (PLD) 1The abbreviations used are: PLD, phospholipase D; PC, phosphatidylcholine; PA, phosphatidic acid; GTPγS, guanosine 5′-O-3-(thio)triphosphate; ARF, ADP-ribosylation factor; PI 3-kinase, phosphoinositide 3-kinase; fMLP, formylmethionylleucylphenylalanine; PI 3-kinase, phosphoinositide 3-kinase; PIPES, 1,4-piperazinediethanesulfonic acid; PEt, phosphatidylethanol; PAGE, polyacrylamide gel electrophoresis; PIP3, phosphoinositide trisphosphate; PMA, phorbol 12-myristate 13-acetate.is an important signal-transducing enzyme in a wide variety of cells and catalyzes the hydrolysis of phosphatidylcholine (PC) to produce the potential second messenger phosphatidic acid (PA) (1Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (350) Google Scholar, 2Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 3Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (388) Google Scholar, 4Cockcroft S. Prog. Lipid Res. 1997; 35: 345-370Crossref Scopus (56) Google Scholar). Studies in neutrophils and HL60 cells have identified a requirement for both cytosolic and membrane components for PLD activation when stimulated with the nonhydrolyzable analog of GTP, GTPγS. Through the use of reconstitution studies utilizing HL60 membranes (5Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (823) Google Scholar) or cytosol-depleted cells (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar), ARF1 and ARF3 have been identified as activators of GTPγS-stimulated PLD activity. The requirement for Rho in PLD activation was identified separately because RhoGDI, which extracts Rho from membranes, inhibited neutrophil PLD (7Bowman E.P. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1993; 268: 21509-21512Abstract Full Text PDF PubMed Google Scholar). Subsequent studies utilizing purified protein demonstrated that Rho proteins stimulated PLD directly (8Malcolm K.C. Ross A.H. Qiu R.-G. Symons M. Exton J.H. J. Biol. Chem. 1994; 269: 25951-25954Abstract Full Text PDF PubMed Google Scholar, 9Siddiqi A.R. Smith J.L. Ross A.H. Qiu R.-G. Symons M. Exton J.H. J. Biol. Chem. 1995; 270: 8466-8473Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 10Hammond 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). Although both ARF and Rho alone can stimulate PLD, other cytosolic factors have been shown to augment these responses in human neutrophils. A 50-kDa factor has been shown to act synergistically with both ARF (11Lambeth J.D. Kwak J.-Y. Bowman E.P. Perry D. Uhlinger D.J. Lopez I. J. Biol. Chem. 1995; 270: 2431-2434Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Bourgoin S. Harbour D. Desmarais Y. Takai Y. Beaulieu A. J. Biol. Chem. 1995; 270: 3172-3178Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and Rho in stimulating PLD (13Kwak J.-Y. Lopez I. Uhlinger D.J. Ryu S.H. Lambeth J.D. J. Biol. Chem. 1995; 270: 27093-27098Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Similarly, both G proteins also act synergistically with protein kinase Cα to activate PLD. Both synergistic (14Singer W.D. Brown H.A. Bokoch G.M. Sternweis P.C. J. Biol. Chem. 1995; 270: 14944-14950Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Kuribara H. Tago K. Yokozeki T. Sasaki T. Takai Y. Morii N. Narumiya S. Katada T. Kanaho Y. J. Biol. Chem. 1995; 270: 25667-25671Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and additive (16Martin A. Brown F.D. Hodgkin M.N. Bradwell A.J. Cook S.J. Hart M. Wakelam M.J.O. J. Biol. Chem. 1996; 271: 17397-17403Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Abousalham A. Liossis C. O'Brien L. Brindley D.N. J. Biol. Chem. 1997; 272: 1069-1075Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) responses have been reported for the simultaneous addition of ARF and Rho. A single PLD that is synergistically responsive to ARF/Rho/protein kinase Cα has been cloned and is now referred to as hPLD1 (10Hammond 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 roles of ARF and Rho are well documented with regard to PLD activation by GTPγS, but their roles in G protein-coupled receptors and receptor are not well and for In the of factor and PLD activity is reported to of phospholipase activation A. Exton J.H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Exton J.H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Ryu S.H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). in other including fMLP-stimulated neutrophils Y. A. Nozawa Y. J. Google Scholar), protein kinase a Rho proteins have been in the activation of PLD in human cells the receptor M. C. J. C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), and in stimulated with and K.C. Exton J.H. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). PLD activation is on ARF and Rho proteins, and an of PI 3-kinase has been K. C. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In we have the of ARF and Rho proteins to membranes by fMLP and the of these proteins to regulate PLD in human neutrophils. reported that activation was in HL60 cells of their cytosolic J. S. Biochem. J. 1993; PubMed Scopus Google Scholar). that both ARF and Rho proteins are by the fMLP receptor a pertussis heterotrimeric G protein. Although activation of PLD in intact neutrophils is inhibited by a of PI 3-kinase, wortmannin is not to the fMLP-stimulated of ARF and Rho to that the activation of PLD by the fMLP receptor is on ARF and Rho proteins in human neutrophils Gi proteins and is not on PI 3-kinase purified from from or from that from the ARF1 and myr-ARF1 proteins purified from as (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar). from The used have been J. Cunningham E. S. Biochem. J. 1996; PubMed Scopus Google Scholar). other as J. S. Biochem. J. 1993; PubMed Scopus Google Scholar). to S. PubMed Scopus (100) Google Scholar). of or a was with an of in to the was and of and for to neutrophils from other cells. by for of with streptolysin O. the and The proteins from the with and in and for ARF and proteins by in and and in The cells for with The cells by to and the cells with intact or PIPES, and for in and with for to in the in the or of fMLP a with of with of the was The was and in of The in and the with The to and and The with of and for the addition of are as the of Lipid and which was as B. S. Biochem. J. PubMed Scopus Google Scholar) was for of by of cells to the and reconstitution of PLD activity as (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar, B. Fensome A. S. J. Biochem. 1993; PubMed Scopus Google Scholar). neutrophils with streptolysin O for in the of in The cells and in and on The in the of and ARF proteins and fMLP as in the a the as neutrophils with pertussis toxin for as S. J. PubMed Scopus Google Scholar). as required. neutrophils with C3 toxin in the of for cells for in the of of intact cells was in the or of fMLP for or The cells with as the of the the cells in and with a for the cells in and a of was J. Cunningham E. S. Biochem. J. 1996; PubMed Scopus Google Scholar). the for to the membrane The membrane in and on with or was by translocation of ARF to membranes in of cells was with streptolysin O in the of and GTP, fMLP, or was as for the as for intact cells. To examine a requirement for ARF proteins in fMLP-stimulated PLD activity, human neutrophils with streptolysin O for to the cells of their cytosolic A that the of the ARF proteins from the and are in the loss is with the of fMLP to stimulate PLD activity in cytosol-depleted cells J. S. Biochem. J. 1993; PubMed Scopus Google and fMLP the to stimulate PLD activity that myr-ARF1 is also to the cells It was that myr-ARF1 alone the activity of PLD, and was on the of In the the of PLD activation by fMLP a and that reconstitution of PLD activity with myr-ARF1 and fMLP is the of the fMLP was used and myr-ARF1 was used ARF proteins are their and is to important for by the ARF factors M. M. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus (100) Google Scholar, S. B. S. S. M. 1996; PubMed Scopus Google Scholar). Both and ARF proteins for their to PLD activity in cytosol-depleted neutrophils. that is for the of PLD activity and also for the response with with (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar) and of (5Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (823) Google Scholar, D. J. M. J. M. S. A. 1994; PubMed Scopus Google Scholar), is not for of PLD a of is for with ARF1 for GTPγS-stimulated PLD activity (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar). of ARF proteins used to examine the requirement for for ARF, a of is used with for myr-ARF1 used is because of the of in E. by of myr-ARF1 and non-myr-ARF1 the of when also that the of PLD stimulated by these proteins is in when is the The fMLP receptor is to the pertussis heterotrimeric G proteins, and D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and are the of phospholipase and PI 3-kinase M. A. A. PubMed Scopus Google Scholar, B. S. T. I. M. D. S. B. B. S. Hsuan J.J. Science. 1995; 269: PubMed Scopus Google Scholar). To the of activation of the PLD activity by the fMLP receptor in the also a activation of Gi proteins, we the of pertussis toxin we as reported J. Biol. Chem. 263: Full Text PDF PubMed Google Scholar, M. J. PubMed Scopus Google Scholar), pertussis toxin to of the fMLP-stimulated PLD activity in intact cells toxin also fMLP-stimulated PLD activity when the and the streptolysin O are in which the cytosolic proteins are To a requirement for Gi proteins in the of PLD activity by we the effect of pertussis toxin on the PLD activity in cytosol-depleted neutrophils Although the of PLD activity in the of and myr-ARF1 was not inhibited by pertussis toxin the fMLP-stimulated activity was inhibited that of myr-ARF1 by the fMLP receptor is and that in the pathway to myr-ARF1 activation has to the heterotrimeric Gi studies in a variety of cells have that not only ARF but also Rho can activate PLD activity (1Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (350) Google Scholar, 2Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 3Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (388) Google Scholar, 4Cockcroft S. Prog. Lipid Res. 1997; 35: 345-370Crossref Scopus (56) Google Scholar). It has been reported that in HL60 Rho is to a in PLD activation (16Martin A. Brown F.D. Hodgkin M.N. Bradwell A.J. Cook S.J. Hart M. Wakelam M.J.O. J. Biol. Chem. 1996; 271: 17397-17403Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). these as an To examine the of Rho proteins in the we Rho proteins of cells. with streptolysin O for of and the and the for Rho The of in the cells The of Rho in the was with that in the cells these the of the ARF proteins was to of the cells Rho proteins are to cytosolic when cells are by J. Cunningham E. S. Biochem. J. 1996; PubMed Scopus Google Scholar), that proteins not as proteins in which the is as is the in cells. In addition to proteins also in the cells not the of Rho proteins, the of PLD activity by fMLP is in the cytosol-depleted cells which that Rho proteins not a in fMLP-stimulated PLD has been shown to a of PLD activity by but when it is with ARF, a synergistic activation is (10Hammond 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). it was that a factor in the myr-ARF1-reconstituted response stimulated by fMLP in the cells. C3 Rho proteins, To a for Rho proteins, cells with C3 The PLD activity was when Rho proteins the Rho to the PLD response in the of ARF and proteins act synergistically to regulate fMLP-stimulated PLD activity. ARF can stimulate PLD activity in the of Rho Rho the of ARF. ARF and are in the are in a and activation in the of ARF and Rho with membranes S. Biochem. J. PubMed Scopus Google Scholar, S. J. M. J. 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To for we wortmannin the translocation of ARF and Rho proteins in fMLP-stimulated intact cells. to wortmannin not the translocation of ARF proteins to membranes and inhibited translocation that wortmannin alone the of Rho proteins in the membranes To that wortmannin was in the of PLD activity in intact cells in we that wortmannin inhibited the fMLP-stimulated PLD activation in intact cells as reported A. Biochem. J. 1993; PubMed Scopus Google Scholar). only a by wortmannin was in cells in which cytosolic proteins are the of not the reconstitution of PLD activity by myr-ARF1 in the cytosol-depleted neutrophils, and was not inhibited by wortmannin not The of of PI 3-kinase in the activation of fMLP-stimulated PLD in the cytosol-depleted cells is also in with M. J. A. S. Biochem. J. 1997; PubMed Scopus Google Scholar). have shown that fMLP is to stimulate PI 3-kinase activation in cytosol-depleted cells because of the loss of PI 3-kinase and from the cells. these the that is an requirement in the of ARF and Rho proteins in human neutrophils. it is that in intact cells PI 3-kinase has a because wortmannin is an in intact cells. In intact cells the activation of PLD activity is and the activity in cytosol-depleted cells is and have a on the of To the of of PI 3-kinase in the of ARF and to membranes, we of the that of PLD activity (1Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (350) Google Scholar, 2Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 3Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (388) Google Scholar, 4Cockcroft S. Prog. Lipid Res. 1997; 35: 345-370Crossref Scopus (56) Google Scholar) is not inhibited by wortmannin M. Nakashima S. Y. Nozawa Y. Biochem. 1997; PubMed Scopus Google Scholar). The of to stimulate PLD activity by the activation of protein kinase translocation of ARF proteins to membranes as reported for cells M.A. A. 1993; PubMed Scopus Google Scholar). In human neutrophils, we that both ARF and Rho to membranes in which no PI 3-kinase activation the of of ARF and to cells with to ARF and Rho to The cells with streptolysin O for and The cytosol-depleted cells with or GTPγS. In the stimulated a small of PLD activity. In the the activity was with which the of protein kinase to the The addition of to these cells an response with cells Activation of G protein-coupled receptors and receptor in intact cells leads to of PLD activity, but the components that to PLD activation to PLD has and and by a acid and both are by ARF and Rho is In HL60 a that is to neutrophils, the that is is hPLD1 D.J. Frohman M.A. Biochem. J. 1997; PubMed Scopus Google Scholar). In we that ARF and Rho proteins are in PLD in fMLP-stimulated human neutrophils. permeabilization the cells of ARF proteins but not and the of fMLP to stimulate PLD activity not restores PLD activity in these cytosol-depleted cells was because we shown that ARF proteins PLD when was used as a of ARF are to a response to that of protein in PLD activation (5Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (823) Google Scholar, S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar). is for of when the is used M. M. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. S. S. M. 1996; PubMed Scopus Google Scholar, S. S. S. J. B. M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). is the factors and the that on ARF the of The factor for ARF has been shown to on proteins but not ARF S. B. S. S. M. 1996; PubMed Scopus Google Scholar, S. S. S. J. B. M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). ARF does not from the cells permeabilization Although the of was to a response to fMLP in in the of ARF a for identified of cells with C3 inhibited the reconstitution of PLD activity, the Rho of fMLP-stimulated PLD activation by ARF and Rho has been for the and the the that are in the activation of PLD in intact cells with a in ARF is in and the in that although ARF can activate PLD activity in the of Rho activation only in the of ARF. that fMLP receptors can activate ARF and Rho was from the that fMLP stimulates their translocation to the membrane The from studies are with of Wakelam and (16Martin A. Brown F.D. Hodgkin M.N. Bradwell A.J. Cook S.J. Hart M. Wakelam M.J.O. J. Biol. Chem. 1996; 271: 17397-17403Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), reported that Rho was not a of PLD in HL60 cells (16Martin A. Brown F.D. Hodgkin M.N. Bradwell A.J. Cook S.J. Hart M. Wakelam M.J.O. J. Biol. Chem. 1996; 271: 17397-17403Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). their studies not an with fMLP as a but used GTPγS, and for the To the pathway to ARF and activation by the fMLP we have the that to ARF and activation It is that the fMLP receptor is to the pertussis G proteins and and that the of intact cells with pertussis toxin in the of fMLP-stimulated PLD activity J. Biol. Chem. 263: Full Text PDF PubMed Google Scholar, M. J. PubMed Scopus Google Scholar). that the activity was also to pertussis toxin the that the heterotrimeric G proteins of the Gi family can to ARF and or activate ARF and the that PI 3-kinase activation important in because it is that an of PI 3-kinase, can PLD activity in intact cells. regulate the PI 3-kinase directly in these cells. is because factors for ARF proteins have been identified with as factors for in and have been cloned S. B. S. S. M. 1996; PubMed Scopus Google Scholar, A. J.J. A. Science. 1997; PubMed Scopus Google Scholar, B. L. S. H. B. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. S. J. M. S. A. 1997; PubMed Scopus Google Scholar). and have and have been shown to Rho factors are proteins with and with a that as the the that wortmannin activation of PLD activity in intact human neutrophils, the activation of the PI 3-kinase pathway by protein the to ARF an is that these are for the because proteins with protein K. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). the requirement of PI 3-kinase as an to ARF and activation in In the we that wortmannin no effect on the PLD activity stimulated by of is in with in which we that in the cytosol-depleted fMLP of is because of of the PI 3-kinase M. J. A. S. Biochem. J. 1997; PubMed Scopus Google Scholar). The second was to examine the translocation of ARF and to membranes was inhibited by wortmannin in intact cells. ARF translocation was and that of Rho was inhibited that PI 3-kinase is not in the activation of ARF and and the is that protein directly with the of the are the of the factors that are in neutrophils. A does not fMLP-stimulated PLD indicating that the factor is not of neutrophil is the to the cells the potential to activate of their as and the PI 3-kinase and phospholipase activation by fMLP is not on PLD and phospholipase have that of ARF to the membranes is on and that of Rho In human neutrophils, we have shown that the PLD is the and that these with the membrane the PLD in the H. J. N. S. Biochem. J. 1997; PubMed Scopus Google Scholar). The requirement for neutrophils to that a PLD, has to the membrane for ARF to The illustrating the components that in the activation of PLD activity is in fMLP Gi proteins, and the ARF and Rho factors directly for of ARF and Rho to the membrane the PLD is with activation of PLD activity. also the translocation of ARF and Rho proteins, but in PI 3-kinase does to the for the activation of the these factors to a family of proteins, it is that cells a of and components required. which a receptor PI 3-kinase to activate ARF and Rho and fMLP, which G protein-coupled to the The the translocation of ARF and Rho is not for the of C3