Phospholipase D and Its Product, Phosphatidic Acid, Mediate Agonist-dependent Raf-1 Translocation to the Plasma Membrane and the Activation of the Mitogen-activated Protein Kinase Pathway

Abstract

The primary known function of phospholipase D (PLD) is to generate phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine. However, the functional role of PA is not well understood. We report here evidence that links the activation of PLD by insulin and the subsequent generation of PA to the activation of the Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A (BFA), an inhibitor of the activation of ADP-ribosylation factor proteins, inhibited insulin-dependent production of PA and MAPK phosphorylation. The addition of PA reversed the inhibition of MAPK activation by BFA. Overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocked insulin-dependent activation of PLD and phosphorylation of MAPK. Real time imaging analysis showed that insulin induced Raf-1 translocation to cell membranes by a process that was inhibited by BFA. PA addition reversed the effects of BFA on Raf-1 translocation. However, PA did not activate Raf-1 in vitro or in vivo, suggesting that the primary function of PA is to enhance the recruitment of Raf-1 to the plasma membrane where other factors may activate it. Finally, we found that the recruitment of Raf-1 to the plasma membrane was transient, but Raf-1 remained bound to endocytic vesicles. The primary known function of phospholipase D (PLD) is to generate phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine. However, the functional role of PA is not well understood. We report here evidence that links the activation of PLD by insulin and the subsequent generation of PA to the activation of the Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A (BFA), an inhibitor of the activation of ADP-ribosylation factor proteins, inhibited insulin-dependent production of PA and MAPK phosphorylation. The addition of PA reversed the inhibition of MAPK activation by BFA. Overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocked insulin-dependent activation of PLD and phosphorylation of MAPK. Real time imaging analysis showed that insulin induced Raf-1 translocation to cell membranes by a process that was inhibited by BFA. PA addition reversed the effects of BFA on Raf-1 translocation. However, PA did not activate Raf-1 in vitro or in vivo, suggesting that the primary function of PA is to enhance the recruitment of Raf-1 to the plasma membrane where other factors may activate it. Finally, we found that the recruitment of Raf-1 to the plasma membrane was transient, but Raf-1 remained bound to endocytic vesicles. Growth factor-mediated activation of PLD 1The abbreviations used are: ARF, ADP-ribosylation factor; BFA, brefeldin A; CRD, cysteine-rich domain; DAG, diacylglycerol; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLD, phospholipase D; Raf-GFP, Raf-green fluorescent protein fusion protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-3-O-(thio)triphosphate. 1The abbreviations used are: ARF, ADP-ribosylation factor; BFA, brefeldin A; CRD, cysteine-rich domain; DAG, diacylglycerol; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLD, phospholipase D; Raf-GFP, Raf-green fluorescent protein fusion protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-3-O-(thio)triphosphate. has been well documented and occurs in response to a broad class of mitogens, including insulin, platelet-derived growth factor, epidermal growth factor, vasopressin, and phorbol esters (1Ben-Av P. Eli Y. Schmidt U.S. Tobias K.E. Liscovitch M. Eur. J. Biochem. 1993; 215: 455-463Crossref PubMed Scopus (39) Google Scholar, 2Donchenko V. Zannetti A. Baldini P.M. Biochim. Biophys. Acta. 1994; 1222: 492-500Crossref PubMed Scopus (44) Google Scholar, 3Price B.D. Morris J.D. Hall A. Biochem. J. 1989; 264: 509-515Crossref PubMed Scopus (39) Google Scholar, 4Yeo E.-J. Exton J.H. J. Biol. Chem. 1995; 270: 3980-3988Abstract Full Full PubMed Scopus Google of PLD occurs the of the ADP-ribosylation factor 1993; Full PubMed Scopus Google Scholar, J. Morris J. Biol. Chem. 1995; 270: Full Full PubMed Scopus Google and M. Exton J.H. J. Biol. Chem. 1994; Full PubMed Google well protein kinase Morris Biol. Full Full PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google The of factors to the activation of PLD is on the cell and of the insulin insulin PLD the Biol. Full Full PubMed Scopus Google the activation of PLD by insulin in to P. J. Biol. Chem. Full Full PubMed Scopus Google of PLD has been in a of and including and Biol. Full Full PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J.H. PubMed Scopus Google Scholar, Exton J.H. J. Biol. 1993; PubMed Scopus Google Scholar, J. PubMed Scopus Google PLD the hydrolysis of to generate However, the of PA generation not well understood. is that the effects of PA in may by to or lysophosphatidic acid PA may a PA in growth factor including a protein A. 1993; PubMed Scopus Google phospholipase J. Biol. Chem. 1993; Full PubMed Google and 1989; PubMed Scopus Google However, the of has not been J. Biol. Chem. Full Full PubMed Scopus Google that PA the kinase an of the MAPK cascade. we report that the of the MAPK by insulin is on PLD and is the of Raf-1 translocation to the plasma membrane by of a catalytically inactive variant of PLD2, but not PLD1, phosphorylation of MAPK. We that PA is activation of Raf-1 in response to However, PA activate kinase in vivo, not on kinase in and has effects on the MAPK cascade. We that PA Raf-1 translocation to the plasma membrane and that the generation of PA is the of Raf-1 translocation by PA induced Raf-1 translocation to the plasma membrane in suggesting that PA may in Raf-1 translocation. Raf-1 was found the insulin and Raf-1 to endocytic was on the generation of PA, suggesting a in Raf-1 endocytic via activation of Raf-1 kinase by growth factors translocation the to the plasma membrane where is a the the of and phosphorylation by PKC, and J. Biochem. 1994; Full PubMed Scopus Google Scholar, J. Biochem. 1994; Full PubMed Scopus Google Scholar, Biol. Scopus Google the of the the plasma membrane is that the translocation of Raf-1 is Raf-1 to the plasma membrane by a protein to the of Raf-1 is activation of the kinase 1994; PubMed Scopus Google Raf-1 in the activation Y. A. J. J. Biol. Chem. 1995; 270: Full Full PubMed Scopus Google However, translocation not the activation of J. Biol. Chem. Full Full PubMed Scopus Google used a protein that is in to but to that the and Raf-1 Raf-1 kinase Raf-1 to the membrane A. J. J. Biol. Chem. Full Full PubMed Scopus Google showed that recruitment of Raf-1 to the plasma membrane by was not activation of Raf-1 and that a the cysteine-rich on Raf-1 and the of was showed that of the Raf-1 Raf-1 kinase suggesting that plasma membrane of Raf-1 by is activation of Raf-1 but that a the Raf-1 Raf-1 translocation and Raf-1 kinase activation but has been time that the of Raf-1 the primary the recruitment of Raf-1 to the cell other that may an role in the recruitment of Raf-1 to the membrane been J. Biol. Chem. Full Full PubMed Scopus Google the of Raf-1 and PA in to to the cysteine-rich of is to the on J. Biol. PubMed Scopus Google the Raf-1 the found on and found that of that of an the is in the activation of A. J. M. J. A. PubMed Scopus Google and M. J. Biol. Chem. Full Full PubMed Scopus Google Raf-1 and effects on The by J. Biol. Chem. Full Full PubMed Scopus Google not in on Raf-1 but on a the of The of on Raf-1 kinase has not been the has been that inhibition of generation of PA inhibited phorbol Raf-1 translocation to cell membranes J. Biol. Chem. Full Full PubMed Scopus Google that the generation of PA by a PLD may an role in the recruitment activation of Raf-1 here of the that insulin-dependent PLD activation in is by in a Biol. Full Full PubMed Scopus Google we that the of generation of PA the activation of the translocation of Raf-1 to and the phosphorylation of MAPK. We that of a catalytically inactive variant of activation of PLD and MAPK phosphorylation. the that the effects of BFA a of the of the generation of PA by PLD, we that the effects of BFA reversed by the addition of However, that PA activate MAPK phosphorylation in and that activate Raf-1 in vitro or in we that the generation of PA is but not the activation of Raf-1 by the other we here that PA is of Raf-1 translocation and the of Raf-1 translocation by BFA to vesicles. a in PA Raf-1 translocation but not activate the kinase and is to Raf-1 kinase in we that PA Raf-1 translocation in suggesting that PA and may and by in Raf-1 translocation to the plasma We that the role of PA in the activation of the MAPK is the of Raf-1 translocation to the cell of the on PLD has on PLD1, may by ARF, and in vitro J. Morris J. Biol. Chem. 1995; 270: Full Full PubMed Scopus Google Scholar, J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Y. Morris J. Biol. Chem. Full Full PubMed Scopus Google vitro on PLD2, on the other that has a that is to and J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Morris Biol. Full Full PubMed Scopus Google and was to an the PLD However, that in is the in insulin-dependent PLD is on the that a catalytically inactive variant of a and phosphorylation of MAPK, a catalytically inactive that is by in vivo, the insulin-dependent PLD in activation Biol. Full Full PubMed Scopus Google evidence the A. J. and M. A. was found to to ARF, a of membranes in was by GTPγS, suggesting that may by in a the in vitro and in and is by a activation in response to insulin not the that Raf-1 translocation is by of PA, or However, we not to the activation of in activation of Raf-1 J. Biochem. J. PubMed Scopus Google and a of PA to in MAPK activation well activation of of PA to activate Raf-1 or MAPK, we that the generation of not a role in the by PA the MAPK cascade. a of a activation of the MAPK Biol. Full Full PubMed Google of PA to or effects on the cascade. effects not the addition of PA in is that the effects of PA on Raf-1 translocation to a PA and Raf-1 and not a of to other of Raf-1 translocation has been to a in Raf-1 was 1994; PubMed Scopus Google Scholar, P. 1993; Google Scholar, Y. J. 1995; PubMed Scopus Google However, has been to growth Raf-1 translocation. we the of Raf-1 translocation to the plasma membrane in that growth kinase activation and that is an growth Raf-1 to translocation of to the plasma membrane in we a of the plasma membrane to the the of the cell Raf-1 not the the plasma membrane to the by a the in to the of translocation in response to insulin or PA we here that the of Raf-1 to the plasma membrane is However, Raf-1 not the plasma the insulin and Raf-1 by that Raf-1 the insulin in in response to insulin and The of that that Raf-1 endocytic insulin, and the insulin a Raf-1 and the of in the Raf-1 and the to on the of is on the that BFA blocked the of the insulin and the of Raf-1 in the endocytic vesicles. we that PA is of the insulin and that the Raf-1 and is by a PA and is of by J. Biol. PubMed Scopus Google Scholar, Morris J. Biol. Chem. Full Full PubMed Scopus Google Scholar, M. 1995; Full PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar, V. V. M. P. Full Full PubMed Scopus Google Scholar, M. Full Full PubMed Scopus Google that PA may an of vesicles. that generation of PA, in a the generation of PIP2, may membrane and the of a a the membranes of the PA and may a a of by Y. J. Biol. Chem. Full Full PubMed Scopus Google of or showed that inhibition of blocked MAPK phosphorylation. that suggesting that Raf-1 vesicles. a activation of MAPK. We that the activation of the MAPK by insulin of the insulin Raf-1 is endocytic in and PLD activation to and Raf-1 translocation to the role of generation of to PA has the the recruitment of Raf-1 to the plasma membrane where is by factors on the plasma and PKC, and of endocytic in may in the recruitment of of to the plasma membrane and to the membranes of endocytic vesicles. Raf-1 PA, and that and that of Y. J. Biol. Chem. Full Full PubMed Scopus Google that the of is activation of the MAPK by Raf-1 in we that is by phosphatidic Growth factor-mediated activation of PLD 1The abbreviations used are: ARF, ADP-ribosylation factor; BFA, brefeldin A; CRD, cysteine-rich domain; DAG, diacylglycerol; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLD, phospholipase D; Raf-GFP, Raf-green fluorescent protein fusion protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-3-O-(thio)triphosphate. 1The abbreviations used are: ARF, ADP-ribosylation factor; BFA, brefeldin A; CRD, cysteine-rich domain; DAG, diacylglycerol; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLD, phospholipase D; Raf-GFP, Raf-green fluorescent protein fusion protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-3-O-(thio)triphosphate. has been well documented and occurs in response to a broad class of mitogens, including insulin, platelet-derived growth factor, epidermal growth factor, vasopressin, and phorbol esters (1Ben-Av P. Eli Y. Schmidt U.S. Tobias K.E. Liscovitch M. Eur. J. Biochem. 1993; 215: 455-463Crossref PubMed Scopus (39) Google Scholar, 2Donchenko V. Zannetti A. Baldini P.M. Biochim. Biophys. Acta. 1994; 1222: 492-500Crossref PubMed Scopus (44) Google Scholar, 3Price B.D. Morris J.D. Hall A. Biochem. J. 1989; 264: 509-515Crossref PubMed Scopus (39) Google Scholar, 4Yeo E.-J. Exton J.H. J. Biol. Chem. 1995; 270: 3980-3988Abstract Full Full PubMed Scopus Google of PLD occurs the of the ADP-ribosylation factor 1993; Full PubMed Scopus Google Scholar, J. Morris J. Biol. Chem. 1995; 270: Full Full PubMed Scopus Google and M. Exton J.H. J. Biol. Chem. 1994; Full PubMed Google well protein kinase Morris Biol. Full Full PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google The of factors to the activation of PLD is on the cell and of the insulin insulin PLD the Biol. Full Full PubMed Scopus Google the activation of PLD by insulin in to P. J. Biol. Chem. Full Full PubMed Scopus Google of PLD has been in a of and including and Biol. Full Full PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J.H. PubMed Scopus Google Scholar, Exton J.H. J. Biol. 1993; PubMed Scopus Google Scholar, J. PubMed Scopus Google PLD the hydrolysis of to generate However, the of PA generation not well understood. is that the effects of PA in may by to or lysophosphatidic acid PA may a PA in growth factor including a protein A. 1993; PubMed Scopus Google phospholipase J. Biol. Chem. 1993; Full PubMed Google and 1989; PubMed Scopus Google However, the of has not been J. Biol. Chem. Full Full PubMed Scopus Google that PA the kinase an of the MAPK cascade. we report that the of the MAPK by insulin is on PLD and is the of Raf-1 translocation to the plasma membrane by of a catalytically inactive variant of PLD2, but not PLD1, phosphorylation of MAPK. We that PA is activation of Raf-1 in response to However, PA activate kinase in vivo, not on kinase in and has effects on the MAPK cascade. We that PA Raf-1 translocation to the plasma membrane and that the generation of PA is the of Raf-1 translocation by PA induced Raf-1 translocation to the plasma membrane in suggesting that PA may in Raf-1 translocation. Raf-1 was found the insulin and Raf-1 to endocytic was on the generation of PA, suggesting a in Raf-1 endocytic via activation of Raf-1 kinase by growth factors translocation the to the plasma membrane where is a the the of and phosphorylation by PKC, and J. Biochem. 1994; Full PubMed Scopus Google Scholar, J. Biochem. 1994; Full PubMed Scopus Google Scholar, Biol. Scopus Google the of the the plasma membrane is that the translocation of Raf-1 is Raf-1 to the plasma membrane by a protein to the of Raf-1 is activation of the kinase 1994; PubMed Scopus Google Raf-1 in the activation Y. A. J. J. Biol. Chem. 1995; 270: Full Full PubMed Scopus Google However, translocation not the activation of J. Biol. Chem. Full Full PubMed Scopus Google used a protein that is in to but to that the and Raf-1 Raf-1 kinase Raf-1 to the membrane A. J. J. Biol. Chem. Full Full PubMed Scopus Google showed that recruitment of Raf-1 to the plasma membrane by was not activation of Raf-1 and that a the cysteine-rich on Raf-1 and the of was showed that of the Raf-1 Raf-1 kinase suggesting that plasma membrane of Raf-1 by is activation of Raf-1 but that a the Raf-1 Raf-1 translocation and Raf-1 kinase activation but has been time that the of Raf-1 the primary the recruitment of Raf-1 to the cell other that may an role in the recruitment of Raf-1 to the membrane been J. Biol. Chem. Full Full PubMed Scopus Google the of Raf-1 and PA in to to the cysteine-rich of is to the on J. Biol. PubMed Scopus Google the Raf-1 the found on and found that of that of an the is in the activation of A. J. M. J. A. PubMed Scopus Google and M. J. Biol. Chem. Full Full PubMed Scopus Google Raf-1 and effects on The by J. Biol. Chem. Full Full PubMed Scopus Google not in on Raf-1 but on a the of The of on Raf-1 kinase has not been the has been that inhibition of generation of PA inhibited phorbol Raf-1 translocation to cell membranes J. Biol. Chem. Full Full PubMed Scopus Google that the generation of PA by a PLD may an role in the recruitment activation of Raf-1 here of the that insulin-dependent PLD activation in is by in a Biol. Full Full PubMed Scopus Google we that the of generation of PA the activation of the translocation of Raf-1 to and the phosphorylation of MAPK. We that of a catalytically inactive variant of activation of PLD and MAPK phosphorylation. the that the effects of BFA a of the of the generation of PA by PLD, we that the effects of BFA reversed by the addition of However, that PA activate MAPK phosphorylation in and that activate Raf-1 in vitro or in we that the generation of PA is but not the activation of Raf-1 by the other we here that PA is of Raf-1 translocation and the of Raf-1 translocation by BFA to vesicles. a in PA Raf-1 translocation but not activate the kinase and is to Raf-1 kinase in we that PA Raf-1 translocation in suggesting that PA and may and by in Raf-1 translocation to the plasma We that the role of PA in the activation of the MAPK is the of Raf-1 translocation to the cell of the on PLD has on PLD1, may by ARF, and in vitro J. Morris J. Biol. Chem. 1995; 270: Full Full PubMed Scopus Google Scholar, J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Y. Morris J. Biol. Chem. Full Full PubMed Scopus Google vitro on PLD2, on the other that has a that is to and J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Morris Biol. Full Full PubMed Scopus Google and was to an the PLD However, that in is the in insulin-dependent PLD is on the that a catalytically inactive variant of a and phosphorylation of MAPK, a catalytically inactive that is by in vivo, the insulin-dependent PLD in activation Biol. Full Full PubMed Scopus Google evidence the A. J. and M. A. was found to to ARF, a of membranes in was by GTPγS, suggesting that may by in a the in vitro and in and is by a activation in response to insulin not the that Raf-1 translocation is by of PA, or However, we not to the activation of in activation of Raf-1 J. Biochem. J. PubMed Scopus Google and a of PA to in MAPK activation well activation of of PA to activate Raf-1 or MAPK, we that the generation of not a role in the by PA the MAPK cascade. a of a activation of the MAPK Biol. Full Full PubMed Google of PA to or effects on the cascade. effects not the addition of PA in is that the effects of PA on Raf-1 translocation to a PA and Raf-1 and not a of to other of Raf-1 translocation has been to a in Raf-1 was 1994; PubMed Scopus Google Scholar, P. 1993; Google Scholar, Y. J. 1995; PubMed Scopus Google However, has been to growth Raf-1 translocation. we the of Raf-1 translocation to the plasma membrane in that growth kinase activation and that is an growth Raf-1 to translocation of to the plasma membrane in we a of the plasma membrane to the the of the cell Raf-1 not the the plasma membrane to the by a the in to the of translocation in response to insulin or PA we here that the of Raf-1 to the plasma membrane is However, Raf-1 not the plasma the insulin and Raf-1 by that Raf-1 the insulin in in response to insulin and The of that that Raf-1 endocytic insulin, and the insulin a Raf-1 and the of in the Raf-1 and the to on the of is on the that BFA blocked the of the insulin and the of Raf-1 in the endocytic vesicles. we that PA is of the insulin and that the Raf-1 and is by a PA and is of by J. Biol. PubMed Scopus Google Scholar, Morris J. Biol. Chem. Full Full PubMed Scopus Google Scholar, M. 1995; Full PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar, V. V. M. P. Full Full PubMed Scopus Google Scholar, M. Full Full PubMed Scopus Google that PA may an of vesicles. that generation of PA, in a the generation of PIP2, may membrane and the of a a the membranes of the PA and may a a of by Y. J. Biol. Chem. Full Full PubMed Scopus Google of or showed that inhibition of blocked MAPK phosphorylation. that suggesting that Raf-1 vesicles. a activation of MAPK. We that the activation of the MAPK by insulin of the insulin Raf-1 is endocytic in and PLD activation to and Raf-1 translocation to the role of generation of to PA has the the recruitment of Raf-1 to the plasma membrane where is by factors on the plasma and PKC, and of endocytic in may in the recruitment of of to the plasma membrane and to the membranes of endocytic vesicles. Raf-1 PA, and that and that of Y. J. Biol. Chem. Full Full PubMed Scopus Google that the of is activation of the MAPK by Raf-1 in we that is by phosphatidic The activation of Raf-1 kinase by growth factors translocation the to the plasma membrane where is a the the of and phosphorylation by PKC, and J. Biochem. 1994; Full PubMed Scopus Google Scholar, J. Biochem. 1994; Full PubMed Scopus Google Scholar, Biol. Scopus Google the of the the plasma membrane is that the translocation of Raf-1 is Raf-1 to the plasma membrane by a protein to the of Raf-1 is activation of the kinase 1994; PubMed Scopus Google Raf-1 in the activation Y. A. J. J. Biol. Chem. 1995; 270: Full Full PubMed Scopus Google However, translocation not the activation of J. Biol. Chem. Full Full PubMed Scopus Google used a protein that is in to but to that the and Raf-1 Raf-1 kinase Raf-1 to the membrane A. J. J. Biol. Chem. Full Full PubMed Scopus Google showed that recruitment of Raf-1 to the plasma membrane by was not activation of Raf-1 and that a the cysteine-rich on Raf-1 and the of was showed that of the Raf-1 Raf-1 kinase suggesting that plasma membrane of Raf-1 by is activation of Raf-1 but that a the Raf-1 Raf-1 translocation and Raf-1 kinase activation but has been time that the of Raf-1 the primary the recruitment of Raf-1 to the cell other that may an role in the recruitment of Raf-1 to the membrane been J. Biol. Chem. Full Full PubMed Scopus Google the of Raf-1 and PA in to to the cysteine-rich of is to the on J. Biol. PubMed Scopus Google the Raf-1 the found on and found that of that of an the is in the activation of A. J. M. J. A. PubMed Scopus Google and M. J. Biol. Chem. Full Full PubMed Scopus Google Raf-1 and effects on The by J. Biol. Chem. Full Full PubMed Scopus Google not in on Raf-1 but on a the of The of on Raf-1 kinase has not been the has been that inhibition of generation of PA inhibited phorbol Raf-1 translocation to cell membranes J. Biol. Chem. Full Full PubMed Scopus Google that the generation of PA by a PLD may an role in the recruitment activation of Raf-1 The here of the that insulin-dependent PLD activation in is by in a Biol. Full Full PubMed Scopus Google we that the of generation of PA the activation of the translocation of Raf-1 to and the phosphorylation of MAPK. We that of a catalytically inactive variant of activation of PLD and MAPK phosphorylation. the that the effects of BFA a of the of the generation of PA by PLD, we that the effects of BFA reversed by the addition of However, that PA activate MAPK phosphorylation in and that activate Raf-1 in vitro or in we that the generation of PA is but not the activation of Raf-1 by the other we here that PA is of Raf-1 translocation and the of Raf-1 translocation by BFA to vesicles. a in PA Raf-1 translocation but not activate the kinase and is to Raf-1 kinase in we that PA Raf-1 translocation in suggesting that PA and may and by in Raf-1 translocation to the plasma We that the role of PA in the activation of the MAPK is the of Raf-1 translocation to the cell of the on PLD has on PLD1, may by ARF, and in vitro J. Morris J. Biol. Chem. 1995; 270: Full Full PubMed Scopus Google Scholar, J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Y. Morris J. Biol. Chem. Full Full PubMed Scopus Google vitro on PLD2, on the other that has a that is to and J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Morris Biol. Full Full PubMed Scopus Google and was to an the PLD However, that in is the in insulin-dependent PLD is on the that a catalytically inactive variant of a and phosphorylation of MAPK, a catalytically inactive that is by in vivo, the insulin-dependent PLD in activation Biol. Full Full PubMed Scopus Google evidence the A. J. and M. A. was found to to ARF, a of membranes in was by GTPγS, suggesting that may by in a the in vitro and in and is by a activation in response to insulin not the that Raf-1 translocation is by of PA, or However, we not to the activation of in activation of Raf-1 J. Biochem. J. PubMed Scopus Google and a of PA to in MAPK activation well activation of of PA to activate Raf-1 or MAPK, we that the generation of not a role in the by PA the MAPK cascade. a of a activation of the MAPK Biol. Full Full PubMed Google of PA to or effects on the cascade. effects not the addition of PA in is that the effects of PA on Raf-1 translocation to a PA and Raf-1 and not a of to other of Raf-1 translocation has been to a in Raf-1 was 1994; PubMed Scopus Google Scholar, P. 1993; Google Scholar, Y. J. 1995; PubMed Scopus Google However, has been to growth Raf-1 translocation. we the of Raf-1 translocation to the plasma membrane in that growth kinase activation and that is an growth Raf-1 to translocation of to the plasma membrane in we a of the plasma membrane to the the of the cell Raf-1 not the the plasma membrane to the by a the in to the of translocation in response to insulin or PA we here that the of Raf-1 to the plasma membrane is However, Raf-1 not the plasma the insulin and Raf-1 by that Raf-1 the insulin in in response to insulin and The of that that Raf-1 endocytic insulin, and the insulin a Raf-1 and the of in the Raf-1 and the to on the of is on the that BFA blocked the of the insulin and the of Raf-1 in the endocytic vesicles. we that PA is of the insulin and that the Raf-1 and is by a PA and is of by J. Biol. PubMed Scopus Google Scholar, Morris J. Biol. Chem. Full Full PubMed Scopus Google Scholar, M. 1995; Full PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar, V. V. M. P. Full Full PubMed Scopus Google Scholar, M. Full Full PubMed Scopus Google that PA may an of vesicles. that generation of PA, in a the generation of PIP2, may membrane and the of a a the membranes of the PA and may a a of by Y. J. Biol. Chem. Full Full PubMed Scopus Google of or showed that inhibition of blocked MAPK phosphorylation. that suggesting that Raf-1 vesicles. a activation of MAPK. We that the activation of the MAPK by insulin of the insulin Raf-1 is endocytic in and PLD activation to and Raf-1 translocation to the role of generation of to PA has the the recruitment of Raf-1 to the plasma membrane where is by factors on the plasma and PKC, and of endocytic in may in the recruitment of of to the plasma membrane and to the membranes of endocytic vesicles. Raf-1 PA, and that and that of Y. J. Biol. Chem. Full Full PubMed Scopus Google that the of is activation of the MAPK by Raf-1 in we that is by phosphatidic We the and the We a of