Title: 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 Text Full Text PDF PubMed Scopus (140) Google Scholar). Activation of PLD occurs through interaction with the small G-proteins of the ADP-ribosylation factor (ARF) (5Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (820) Google Scholar, 6Hammond S.M. Altshuller Y.M. Sung T.-C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar) and Rac/Rho families (7Malcolm K.C. Ross A.H. Qui R.-G. Symons M. Exton J.H. J. Biol. Chem. 1994; 269: 25951-25954Abstract Full Text PDF PubMed Google Scholar) as well as with protein kinase C (PKC) (8Frohman M.A. Morris A.J. Curr. Biol. 1996; 6: 945-947Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 9Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (347) Google Scholar). The relative contribution of these factors to the activation of PLD is highly dependent on the cell type and signaling model examined. For example, stimulation of Rat-1 fibroblasts overexpressing the human insulin receptor (HIRcB cells) with insulin activates PLD exclusively through the ARF pathway (10Shome K. Vasudevan C. Romero G. Curr. Biol. 1997; 7: 387-396Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), whereas the activation of PLD by insulin in adipocytes appears to be primarily Rho-mediated (11Karnam P. Standaert M.L. Galloway L. Farese R.V. J. Biol. Chem. 1997; 272: 6136-6140Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Activation of PLD has been implicated in a wide variety of intracellular and extracellular processes, including actin polymerization, coatomer assembly, vesicle transport, neutrophil activation, and platelet aggregation (12Bi K. Roth M.G. Ktistakis N.T. Curr. Biol. 1997; 7: 301-307Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 13English D. Cell. Signalling. 1996; 8: 341-347Crossref PubMed Scopus (178) Google Scholar, 14Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (386) Google Scholar, 15Ha K.S. Exton J.H. J. Cell Biol. 1993; 123: 1789-1796Crossref PubMed Scopus (154) Google Scholar, 16Reinhold S.L. Prescott S.M. Zimmerman G.A. McIntyre T.M. FASEB J. 1990; 4: 208-214Crossref PubMed Scopus (170) Google Scholar).Activated PLD catalyzes the hydrolysis of phosphatidylcholine to generate PA. However, the downstream consequences of PA generation are not well understood. Although it is clear that the principal effects of PA in some systems may be mediated by its conversion to diacylglycerol (DAG) or lysophosphatidic acid (LPA), PA may also be a potent second messenger. Several laboratories have identified putative targets for PA in growth factor signal transduction, including a protein tyrosine phosphatase (17Zhao Z. Shen S.H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4251-4255Crossref PubMed Scopus (108) Google Scholar), phospholipase C-γ (18Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Abstract Full Text PDF PubMed Google Scholar), and Ras-GAP (19Tsai M.H. Yu C.L. Wei F.S. Stacy D.W. Science. 1989; 243: 522-526Crossref PubMed Scopus (232) Google Scholar). However, the physiological relevance of these interactions has not been established.Recently, Ghosh et al. (20Ghosh S. Strum J.C. Sciorra V.A. Daniel L. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar) reported that PA interacts directly with the serine-threonine kinase Raf-1, an important component of the MAPK signaling cascade. Here we report that the stimulation of the MAPK pathway by insulin is dependent on PLD activation, and this effect is mediated through the induction of Raf-1 translocation to the plasma membrane by PA. Furthermore, overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocks insulin-induced phosphorylation of MAPK. We also show that PA is required for complete activation of Raf-1 in response to insulin. However, PA alone cannot activate Raf kinase in vivo, does not have any effect on Raf kinase activity in vitro, and has no effects on the MAPK cascade. We also show that PA induces Raf-1 translocation to the plasma membrane and that the generation of PA is essential for the induction of Raf-1 translocation by insulin. PA also induced Raf-1 translocation to the plasma membrane in Ha-Ras(Q61L)-transformed Rat-1 fibroblasts, suggesting that PA may act concurrently with activated Ras in stimulating Raf-1 translocation. Raf-1 was found associated with intracellular vesicles containing the insulin receptor and clathrin after stimulation with insulin. Furthermore, Raf-1 association to endocytic vesicles was dependent on the generation of PA, suggesting a model in which Raf-1 migrates along with endocytic vesicles during receptor-mediated endocytosis via its interaction with PA.DISCUSSIONThe activation of Raf-1 kinase activity by growth factors requires its translocation from the cytoplasm to the plasma membrane where it is activated through a complex mechanism which includes the interaction with the GTP-bound form of Ras, and possibly phosphorylation by PKC, and tyrosine kinases (34Avruch J. Zhang X. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 35Daum G. Eisenmann-Tappe I. Fries H. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-479Abstract Full Text PDF PubMed Scopus (483) Google Scholar, 36Morrison D.K. Cutler Jr., R.E. Curr. Biol. 1997; 9: 174-179Crossref Scopus (534) Google Scholar). Whereas the precise nature of the events occurring at the plasma membrane remains unresolved, it is clear that the translocation of Raf-1 is crucial. Targeting Raf-1 to the plasma membrane by attaching a protein prenylation motif to the C terminus of Raf-1 is sufficient for activation of the kinase (37Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (877) Google Scholar), whereas trapping Raf-1 in the cytoplasm with cytosolic Ras prevents activation (22Lerner E.C. Qian Y. Blaskovich M.A. Fossum R.D. Vogt A. Sun J. Cox A.D. Der C.J. Hamilton A.D. Sebti S.M. J. Biol. Chem. 1995; 270: 26802-26806Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). However, translocation itself does not bring about the full activation of Raf-1. Mineo et al. (38Mineo C. Anderson R.G.W. White M.A. J. Biol. Chem. 1997; 272: 10345-10348Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) used a mutant Ras protein that is deficient in binding to wild-type Raf-1, but binds Raf-1(257L), to show that the interaction between Ras and Raf-1 stimulates Raf-1 kinase activity 3-fold better than targeting Raf-1 to the membrane alone. Furthermore, Roy et al. (39Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) showed that recruitment of Raf-1 to the plasma membrane by Ras was not sufficient for full activation of Raf-1 and that a second interaction between the cysteine-rich domain (CRD) on Raf-1 and the GTP-bound form of Ras was necessary for full activation. They also showed that deletion of the CRD from membrane-targeted Raf-1 abrogated Raf-1 kinase activity, suggesting that plasma membrane localization of Raf-1 by itself is insufficient for activation of Raf-1 but that a second regulatory event affecting the CRD must occur for Raf-1 activation. Thus, Raf-1 translocation and Raf-1 kinase activation are closely related but distinct phenomena.It has been assumed for some time that the interactions of Raf-1 with Ras are the primary mechanism driving the recruitment of Raf-1 to the cell membrane. Recently, other mechanisms that may play an important role in the recruitment of Raf-1 to the membrane have been investigated. For instance, Ghosh et al. (20Ghosh S. Strum J.C. Sciorra V.A. Daniel L. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar) have explored the interactions of Raf-1 with phosphatidylserine and PA in vitro. Phosphatidylserine appears to bind to the cysteine-rich domain (CRD) of Raf-1, which is analogous to the zinc finger on PKC. Luo et al. (40Luo Z. Diaz B. Marshall M.S. Avruch J. Mol. Cell. Biol. 1997; 17: 46-53Crossref PubMed Scopus (106) Google Scholar) replaced the Raf-1 CRD with the analogous zinc finger domain found on PKC and found that DAG activated this chimera independently of Ras activation, demonstrating that interaction of an effector with the CRD is critical in the activation of Raf-1. Other effectors, such as ceramide (41Huwiler A. Brunner J. Hummel R. Vervoordeldonk M. Stabel S. van der Bosch H. Pfeilschifter J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6959-6963Crossref PubMed Scopus (183) Google Scholar) and Rap1A (42Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), interact with Raf-1 at this site and consequently have effects on its activation. The PA-binding site proposed by Ghosh et al. (20Ghosh S. Strum J.C. Sciorra V.A. Daniel L. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar) does not lie in this crucial lipid binding regulatory domain on Raf-1 but on a second lipid-binding site near the catalytic domain of Raf-1. The influence of effector binding at this site on Raf-1 kinase activity, if any, has not been fully characterized at the present time. It has also been shown that inhibition of PLD-mediated generation of PA with ethanol inhibited phorbol ester-induced Raf-1 translocation to cell membranes (20Ghosh S. Strum J.C. Sciorra V.A. Daniel L. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). This suggests that the generation of PA by a receptor-sensitive PLD may play an important role in the recruitment and/or activation of Raf-1 kinase.The data reported here strongly support this view. By taking advantage of the fact that insulin-dependent PLD activation in HIRcB cells is mediated by ARF proteins in a BFA-sensitive manner (10Shome K. Vasudevan C. Romero G. Curr. Biol. 1997; 7: 387-396Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), we have shown that the blockade of PLD-dependent generation of PA disrupts the activation of Raf-1, the translocation of Raf-1 to membranes, and the phosphorylation of MAPK. We also demonstrated that overexpression of a catalytically inactive variant of PLD2 blocks insulin-induced activation of PLD and MAPK phosphorylation. In consistency with the hypothesis that the effects of BFA are a consequence of the blockade of the generation of PA by receptor-sensitive PLD, we have also shown that all the effects of BFA can be reversed by the addition of exogenous PA. However, our data show that PA alone cannot activate MAPK phosphorylation in live cells and that it cannot activate Raf-1 in vitro or in cultured cells. Taking all these data together, we conclude that the generation of PA is required but not sufficient for the activation of Raf-1 by insulin.On the other hand, we show here that PA is sufficient for induction of Raf-1 translocation and reverses the blockade of insulin-induced Raf-1 translocation by BFA to intracellular vesicles. These facts suggest a model in which PA directly facilitates Raf-1 translocation but does not activate the kinase and is insufficient to completely stimulate Raf-1 kinase activity in intact cells. Furthermore, we show that PA induces Raf-1 translocation in Ha-Ras(Q61L)-transformed cells, suggesting that PA and Ras may act concurrently and by parallel pathways in stimulating Raf-1 translocation to the plasma membrane. We therefore conclude that the main role of PA in the activation of the MAPK cascade is the induction of Raf-1 translocation to the cell membrane.Much of the attention on receptor-sensitive PLD has focused on PLD1, primarily because recombinant PLD1 may be activated by ARF, Rho, and PKCα in vitro (43Hammond S.M. Altshuller Y.M. Sung T.C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, 44Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 45Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. Cook S. Nozawa Y. Pystivich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). In vitro studies on recombinant PLD2, on the other hand, have demonstrated that purified PLD2 has a high basal activity that is largely insensitive to ARF and Rho (44Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 46Colley W. Sung T.C. Roll R. Hammond S.M. Altshuller Y.M. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar) and thus was thought to be an unlikely candidate for the receptor-sensitive PLD activity. However, our findings suggest that in HIRcB cells PLD2 is the main species involved in insulin-dependent PLD signaling. This conclusion is based on the fact that a catalytically inactive variant of PLD2 functions as a dominant negative and blocks insulin-induced phosphorylation of MAPK, whereas a catalytically inactive PLD1 does not. Interestingly, this further suggests that PLD2 is regulated by ARF in vivo, since the insulin-dependent PLD activity in HIRcB cells requires ARF activation (10Shome K. Vasudevan C. Romero G. Curr. Biol. 1997; 7: 387-396Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Recent evidence from the Sung et al. 3T. S. Sung, A. J. Morris, and M. A. Frohman, submitted for publication. agrees with this model. Although immunopurified PLD2 was found to be largely unresponsive to ARF, a preparation of crude membranes containing PLD2 overexpressed in COS-7 cells was activated by ARF preloaded with GTPγS, suggesting that PLD2 may be regulated by ARF in vivo. Furthermore, a PLD2 mutant lacking the N-terminal 308 amino acids displays both reduced in vitro and in vivobasal activity and is stimulated more than 10-fold by ARF. These results are consistent with a model for ARF-mediated PLD2 activation in response to insulin activation.Our data do not rule out the possibility that PA-stimulated Raf-1 translocation is mediated by metabolites of PA, specifically DAG or LPA. However, we do not believe this to be the case. DAG-mediated activation of PKC results in potent activation of Raf-1 (48van Dijk M.C.M. Muriana F.J.G. van der Hoeven P.C.J. de Widt J. Schaap D. Moolenaar W.H. van Blitterswijk W.J. Biochem. J. 1997; 323: 693-699Crossref PubMed Scopus (69) Google Scholar), and thus a significant conversion of PA to DAG would result in MAPK activation as well as potent activation of Raf-1. Since treatment of cells with PA alone failed to activate either Raf-1 or MAPK, we conclude that the generation of DAG does not play a significant role in the mechanism by which PA modulates the MAPK cascade. Likewise, a significant accumulation of LPA would also elicit a strong activation of the MAPK cascade (49van Dijk M.C.M. Postma F. Hilkmann H. Jalink K. van Blitterswijk W.J. Moolenaar W.H. Curr. Biol. 1998; 8: 386-392Abstract Full Text Full Text PDF PubMed Google Scholar). Therefore, conversion of PA to either LPA or DAG should have dramatic effects on the Raf-1-MAPK signaling cascade. Since these effects were not seen after the addition of PA in our experiments, it is likely that the effects of PA on Raf-1 translocation are due to a direct interaction between PA and Raf-1 and not a consequence of its conversion to other lipid second messengers.Previously, immunocytochemical characterization of Raf-1 translocation has been limited to a few studies in which Raf-1 was microinjected into Ras-transformed cells (37Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (877) Google Scholar, 50Traverse S. Cohen P. Paterson H. Marshall C. Rapp U. Grand R.J.A. Oncogene. 1993; 8: 3175-3181PubMed Google Scholar, 51Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (520) Google Scholar). However, little work has been done to characterize growth factor-induced Raf-1 translocation. Here, we have studied the dynamics of insulin-induced Raf-1 translocation to the plasma membrane in live cells. Our data demonstrate that Raf-GFP undergoes growth factor-induced kinase activation and translocation, indicating that Raf-GFP is an appropriate model for growth factor-induced Raf-1 dynamics. In order to assess translocation of Raf-GFP to the plasma membrane in live cells, we imaged a confocal section of the plasma membrane adjacent to the cover glass. To select this section, several images along the z axis of the cell were collected. Because Raf-1 does not enter the nucleus, the plasma membrane sections adjacent to the coverslip were identified by choosing a confocal section below the nucleus. These sections were imaged at 37 °C in order to examine the kinetics of Raf translocation in response to insulin or PA stimulation.By using this experimental approach, we show here that the association of Raf-1 to the plasma membrane is transient. However, Raf-1 does not simply dissociate from the plasma membrane. Dual staining immunogold labeling for the insulin receptor and Raf-1 resolved by electron microscopy shows that Raf-1 co-localized with the insulin receptor in intracellular vesicular structures in response to stimulation with both insulin and PA. The structure of these vesicles suggests that they are endosomes. To confirm that Raf-1 binds endocytic vesicles, cells were treated with insulin, and vesicles containing the insulin receptor were isolated using a specific anti-insulin receptor antibody. Both Raf-1 and the heavy chain of clathrin were present in these preparations. Consistent with our model, the association between Raf-1 and the isolated vesicles also appeared to be dependent on the presence of PA. This conclusion is based on the observation that BFA blocked the internalization of the insulin receptor and abolished the localization of Raf-1 in the immunoisolated endocytic vesicles. Therefore, we propose that PA is required for endocytosis of the insulin receptor and that the association between Raf-1 and endosomes is mediated by a direct interaction between PA and Raf-1. This is consistent with current models of vesicle formation mediated by acidic phospholipids (32Ktistakis N.T. Brown H.A. Waters M.G. Sternweis P.C. Roth M.G. J. Cell Biol. 1996; 134: 295-306Crossref PubMed Scopus (328) Google Scholar, 33Chung J.-K. Sekiya F. Kang H.-S. Lee C. Han J.-S. Kim S.R. Bae Y.S. Morris A.J. Rhee S.G. J. Biol. Chem. 1997; 272: 15980-15985Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 52Liscovitch M. Cantley L.C. Cell. 1995; 81: 659-662Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 53Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar, 54Takei K. Haucke V. Slepnev V. Farsad K. Salazar M. Chen H. De Camilli P. Cell. 1998; 94: 131-141Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 55Matsuoka K. Orci L. Amherdt M. Bednarek S. Hamamoto S. Schekman R. Yeung T. Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) which suggest that PA may form an integral part of newly formed vesicles. These models suggest that PLD-mediated generation of PA, through participation in a positive feedback loop concurrently with the generation of PIP2, may sufficiently perturb membrane structure and facilitate the formation of a vesicle from a planar membrane. Consequently, the membranes of newly formed vesicles are enriched with the acidic phospholipids PA and PIP2. This acidic surface may serve as a binding matrix for a number of signaling molecules such as Raf-1.Recent work by Daaka et al. (47Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar) also supports this model. By using dominant suppressor mutants of β-arrestin or dynamin, they showed that inhibition of G-protein-coupled receptor endocytosis blocked MAPK phosphorylation. Furthermore, they isolated Raf-1-containing vesicles that co-isolated with clathrin-coated vesicles, suggesting that Raf-1 associates with clathrin-coated vesicles. Our data suggest a very similar model for insulin-mediated activation of MAPK. We suggest that the activation of the MAPK cascade by insulin also requires endocytosis of the insulin receptor. Raf-1 is associated with endocytic vesicles in insulin-treated cells, and PLD activation appears to be necessary for receptor-mediated endocytosis and Raf-1 translocation to membranes.Fig. 8 depicts the proposed role of insulin-induced generation of PA. According to this model, PA has the following two functions: 1) the recruitment of Raf-1 to the plasma membrane where it is activated by factors which reside on the plasma membrane, such as activated Ras and PKC, and 2) facilitation of endocytic vesicle formation. These two effects, acting in conjunction, may result in the recruitment of many important components of signal transduction to the plasma membrane and to the membranes of endocytic vesicles. Among these components are receptor tyrosine kinases, Raf-1 through its association with PA, and proteins that associate with PIP2 through pleckstrin homology domains. Our work and that of Daaka et al. (47Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar) further suggest that the internalization of these signaling components is necessary for full activation of the MAPK signaling cascade, probably by bringing Raf-1 in contact with downstream targets such as MEK. In this paper, we demonstrate that this phenomenon is mediated by phosphatidic acid. 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 Text Full Text PDF PubMed Scopus (140) Google Scholar). Activation of PLD occurs through interaction with the small G-proteins of the ADP-ribosylation factor (ARF) (5Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (820) Google Scholar, 6Hammond S.M. Altshuller Y.M. Sung T.-C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar) and Rac/Rho families (7Malcolm K.C. Ross A.H. Qui R.-G. Symons M. Exton J.H. J. Biol. Chem. 1994; 269: 25951-25954Abstract Full Text PDF PubMed Google Scholar) as well as with protein kinase C (PKC) (8Frohman M.A. Morris A.J. Curr. Biol. 1996; 6: 945-947Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 9Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (347) Google Scholar). The relative contribution of these factors to the activation of PLD is highly dependent on the cell type and signaling model examined. For example, stimulation of Rat-1 fibroblasts overexpressing the human insulin receptor (HIRcB cells) with insulin activates PLD exclusively through the ARF pathway (10Shome K. Vasudevan C. Romero G. Curr. Biol. 1997; 7: 387-396Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), whereas the activation of PLD by insulin in adipocytes appears to be primarily Rho-mediated (11Karnam P. Standaert M.L. Galloway L. Farese R.V. J. Biol. Chem. 1997; 272: 6136-6140Abstract Full Text Full Text PDF PubMed Scopus (83)