Optimal Lysophosphatidic Acid-induced DNA Synthesis and Cell Migration but Not Survival Require Intact Autophosphorylation Sites of the Epidermal Growth Factor Receptor

Abstract

Lysophosphatidic acid (LPA)-elicited transphosphorylation of receptor tyrosine kinases has been implicated in mediating extracellular signal-regulated kinase (ERK) 1/2 activation, which is necessary for LPA-induced cell proliferation, migration, and survival. B82L cells lack epidermal growth factor receptor (EGFR) but express LPA1–3, platelet-derived growth factor (PDGF), ErbB2, and insulin-like growth factor receptor transcripts, yet LPA caused no detectable transphosphorylation of these receptor tyrosine kinases. LPA equally protected B82L cells, or transfectants expressing EGFR, the kinase dead EGFRK721A, EGFRY5F receptor mutant, which lacks five autophosphorylation sites, or EGFRY845F, which lacks the Src phosphorylation site from tumor necrosis factor-α-induced apoptosis. In contrast, LPA-elicited DNA synthesis and migration were augmented in cells expressing EGFR, EGFRK721A, or EGFRY845F, but not EGFRY5F, although the PDGF responses were indistinguishable. LPA-induced transphosphorylation of the EGFR, ErbB2, or PDGF receptor was not required for its antiapoptotic effect. EGFR with or without intrinsic kinase activity or without the Src-phosphorylation site augmented, but was not required for, LPA-elicited cell proliferation or migration. In B82L cells, augmentation of these two LPA responses required intact autophosphorylation sites because among the four EGFR mutants, only cells expressing the EGFRY5F mutant showed no enhancement. In EGFRY5F-expressing cells, LPA failed to elicit tyrosine phosphorylation of Src homologous and collagen protein (SHC) and caused only a modest increase in ERK1/2 phosphorylation similar to that in wild-type B82L cells. The present data pinpoint the lack of importance of the intrinsic kinase activity in contrast to the importance of autophosphorylation sites of the EGFR for SHC phosphorylation in the enhancement of select ERK1/2-dependent LPA responses. Lysophosphatidic acid (LPA)-elicited transphosphorylation of receptor tyrosine kinases has been implicated in mediating extracellular signal-regulated kinase (ERK) 1/2 activation, which is necessary for LPA-induced cell proliferation, migration, and survival. B82L cells lack epidermal growth factor receptor (EGFR) but express LPA1–3, platelet-derived growth factor (PDGF), ErbB2, and insulin-like growth factor receptor transcripts, yet LPA caused no detectable transphosphorylation of these receptor tyrosine kinases. LPA equally protected B82L cells, or transfectants expressing EGFR, the kinase dead EGFRK721A, EGFRY5F receptor mutant, which lacks five autophosphorylation sites, or EGFRY845F, which lacks the Src phosphorylation site from tumor necrosis factor-α-induced apoptosis. In contrast, LPA-elicited DNA synthesis and migration were augmented in cells expressing EGFR, EGFRK721A, or EGFRY845F, but not EGFRY5F, although the PDGF responses were indistinguishable. LPA-induced transphosphorylation of the EGFR, ErbB2, or PDGF receptor was not required for its antiapoptotic effect. EGFR with or without intrinsic kinase activity or without the Src-phosphorylation site augmented, but was not required for, LPA-elicited cell proliferation or migration. In B82L cells, augmentation of these two LPA responses required intact autophosphorylation sites because among the four EGFR mutants, only cells expressing the EGFRY5F mutant showed no enhancement. In EGFRY5F-expressing cells, LPA failed to elicit tyrosine phosphorylation of Src homologous and collagen protein (SHC) and caused only a modest increase in ERK1/2 phosphorylation similar to that in wild-type B82L cells. The present data pinpoint the lack of importance of the intrinsic kinase activity in contrast to the importance of autophosphorylation sites of the EGFR for SHC phosphorylation in the enhancement of select ERK1/2-dependent LPA responses. The phospholipid growth factor, lysophosphatidic acid (1-acyl-2-hydroxy-sn-glycero-3-phosphate, LPA) 1The abbreviations used are: LPA, lysophosphatidic acid 18:1; GPCR, G protein-coupled receptor; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; PDGF, platelet-derived growth factor BB; IGF, insulin-like growth factor; PTX, pertussis toxin; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; IGFR, insulin-like growth factor receptor; ERK, extracellular signal-regulated kinase; SHC, Src homologous and collagen protein; TNF, tumor necrosis factor; C4, IGFR-positive F32 VIII C4 mouse hybridoma cells., a normal component of blood plasma, is a confirmed ligand of the LPA1, LPA2, LPA3 and putative agonist of the GPR23 G protein-coupled receptors (GPCR) (1Tigyi G. Parrill A.L. Prog. Lipid Res. 2003; 42: 498-526Crossref PubMed Scopus (157) Google Scholar, 2Tigyi G. Prostaglandins Other Lipid Mediat. 2001; 64: 47-62Crossref PubMed Scopus (92) Google Scholar). Cell survival, proliferation, and migration are responses elicited by LPA GPCR and also by activation of polypeptide growth factor receptors tyrosine kinase (RTK) for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF). RTK activation results in ligand-induced autophosphorylation of the receptor, which recruits adapter and docking proteins to initiate the assembly and activation of a multimolecular signaling complex (3Carpenter G. J. Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar, 4Gschwind A. Zwick E. Prenzel N. Leserer M. Ullrich A. Oncogene. 2001; 20: 1594-1600Crossref PubMed Scopus (414) Google Scholar, 5Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar, 6Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar, 7Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar, 8Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PubMed Scopus Google Scholar, E. P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: Full Text Full Text PubMed Scopus Google Scholar). is that of the G protein by pertussis the LPA-elicited and (1Tigyi G. Parrill A.L. Prog. Lipid Res. 2003; 42: 498-526Crossref PubMed Scopus (157) Google Scholar, 2Tigyi G. Prostaglandins Other Lipid Mediat. 2001; 64: 47-62Crossref PubMed Scopus (92) Google Scholar, Sci. A. PubMed Scopus Google Scholar, J. Biol. Full Text PubMed Google Scholar, A. A. M.J. Mol. Cell. Biol. 20: PubMed Scopus Google Scholar). In but not cell Daaka Y. Lefkowitz R.J. L.M. J. Biol. 1999; Full Text Full Text PubMed Scopus Google Scholar, J. M. J. J. Biol. 2001; Full Text Full Text PubMed Scopus Google activation of LPA GPCR is by phosphorylation of the epidermal growth factor receptor (EGFR) L.M. Lefkowitz R.J. J. Biol. Full Text Full Text PubMed Scopus Google Scholar, L.M. Lefkowitz R.J. J. Biol. Full Text Full Text PubMed Scopus Google Scholar, L.M. Lefkowitz R.J. J. Biol. Full Text Full Text PubMed Scopus Google Scholar, Y. L.M. Lefkowitz R.J. J. Biol. Full Text Full Text PubMed Scopus Google Scholar, Ullrich A. PubMed Scopus Google Scholar, A. A. Ullrich A. J. PubMed Scopus Google the platelet-derived growth factor receptor Y. A. A. J. Biol. 2003; Full Text Full Text PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar, A. A. Ullrich A. G. Sci. A. PubMed Scopus Google or insulin-like growth factor Y. A. A. J. Biol. 2003; Full Text Full Text PubMed Scopus Google Scholar). a the LPA GPCR and RTK in which ligand activation of the GPCR the of RTK by kinases of the Src L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar, L.M. Lefkowitz R.J. J. Biol. Full Text Full Text PubMed Scopus Google the of to a of the cell ligand-induced activation of the EGFR A. Zwick E. Prenzel N. Leserer M. Ullrich A. Oncogene. 2001; 20: 1594-1600Crossref PubMed Scopus (414) Google Scholar, E. P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: Full Text Full Text PubMed Scopus Google Scholar, N. Zwick E. Leserer M. Ullrich A. 1999; PubMed Scopus Google Scholar). of these that LPA-induced activation of ERK1/2 and was by of the EGFR tyrosine and A. A. Ullrich A. J. PubMed Scopus Google Scholar, A. A. Ullrich A. G. Sci. A. PubMed Scopus Google Scholar, N. Zwick E. Leserer M. Ullrich A. 1999; PubMed Scopus Google Scholar, J. J. J. Biol. Full Text Full Text PubMed Scopus Google and the activation of these kinases for the of EGFR in the responses to the that LPA was of or of its or signaling responses in cells in which and PDGF not elicit responses migration J. Biol. 1999; Full Text Full Text PubMed Scopus Google or in cells that EGFR J. M. J. J. Biol. 2001; Full Text Full Text PubMed Scopus Google Scholar, A. A. Ullrich A. G. Sci. A. PubMed Scopus Google Scholar, J. J. J. Biol. Full Text Full Text PubMed Scopus Google or in cells from and J. M. J. J. Biol. 2001; Full Text Full Text PubMed Scopus Google and J. M. J. J. Biol. 2001; Full Text Full Text PubMed Scopus Google that although Src was necessary for ERK1/2 activation in to LPA, a activation of EGFR and that the antiapoptotic of LPA ERK1/2 activation Y. J. J. 2001; Scopus Google Scholar, Y. J. R.J. G. J. PubMed Scopus Google Scholar, G. Full Text Full Text PubMed Scopus Google Scholar, E. G. J. 2003; PubMed Scopus Google that is to and to the EGFR kinase but not to G. Full Text Full Text PubMed Scopus Google in cells. In these cells, LPA-elicited phosphorylation of the EGFR was by but not by PTX, the of EGFR transphosphorylation is required for ERK1/2 the phosphorylation of RTK, the of in the of responses elicited by LPA J. 2003; PubMed Scopus Google for a signaling LPA receptors to EGFR and ERK1/2 cells, in to component but not EGFR, of which were required for the activation of The activation of was by the because SHC phosphorylation was not by PTX, has been by A. A. Ullrich A. J. PubMed Scopus Google Scholar). The mediating the increase in but the factor that is by the Cell 1999; 11: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. J. 2003; PubMed Google Scholar). In tyrosine phosphorylation of SHC to a the of the is by LPA or In the present the of EGFR in ERK1/2-dependent responses migration, and elicited by LPA a receptor in B82L cells that lack The of EGFR was by wild-type EGFR J. J. Biol. 1999; Full Text Full Text PubMed Scopus Google a mutant J. J. Biol. Full Text Full Text PubMed Scopus Google the EGFRY5F receptor mutant, in which the sites of tyrosine autophosphorylation been to N. J. Oncogene. Google and with its Src phosphorylation site J.S. A.L. Sci. A. 1999; PubMed Scopus Google Scholar). data that in B82L cells, LPA GPCR are of the responses of the of PDGFR, and LPA-elicited and but not the antiapoptotic are by EGFR a that not EGFR tyrosine kinase activity or a Src phosphorylation site but intact autophosphorylation sites to tyrosine phosphorylation of SHC for activation of was from and was to cells with was from and were from PTX, and were from and were from protein kinase and mouse were from Cell and were from receptor was from was from The was from used for was from mouse used for was from and were from Cell were in with in a B82L expressing the wild-type EGFR or were by of and in the J. J. Biol. 1999; Full Text Full Text PubMed Scopus Google Scholar). B82L cells were with by or a from of and by the with The cells were used without The was cells were and by without the of was from cells with the and with and with the The from of was with for for and for were used to EGFR and and and and were by and LPA by was from cells and with of of from was with was the with the of The and were with and were LPA1, LPA2, and and and for and was with and were of and of was J. PubMed Scopus Google and the of LPA receptor was to was and the results are of for the was with the of of by DNA B82L cells were with for DNA was by the with the Cell from G. Full Text Full Text PubMed Scopus Google Scholar, E. G. J. 2003; PubMed Scopus Google Scholar). DNA was of protein and of cells were with in protein with and and of EGFR, PDGFR, or IGFR, cells were in a and and The protein was the 1/2 and its were by for the of of protein was with of by for by in and to were with in and for and with the for with were with in for by four and with the and EGFR, IGFR, ErbB2, and were also and by In of of cell was by of protein for and for the was with with by of protein The were by with and in and to and DNA synthesis was by B82L or cells were in the cells were and and to LPA or of cells were with for and the was by acid migration were A. M.J. Y. G. Full Text Full Text PubMed Scopus Google Scholar). B82L cells were to to and The cell was with a were and to LPA to or and the of migration was with the of Cell migration was by cells was in and two the migration results the of data are or was was used for the and the of was used to of the EGFR a the migration and proliferation of the and for the of LPA-induced enhancement of migration and proliferation the cell of was of LPA and RTK in B82L that B82L cells for the LPA receptors of the The of LPA receptor by was in wild-type B82L cells and in the B82L expressing wild-type or mutant EGFR In with A. A. Ullrich A. G. Sci. A. PubMed Scopus Google Scholar, J. J. Biol. 1999; Full Text Full Text PubMed Scopus Google B82L cells not express EGFR and protein and In B82L cells with wild-type EGFR, the of the receptor protein in the cell and were in B82L cells also the of and protein in B82L cells only of protein with although the used the mouse in F32 VIII C4 mouse hybridoma cells used a increase in tyrosine phosphorylation of the in C4 cells, failed to increase in in B82L cells a results the a of LPA transphosphorylation in B82L cells. the and LPA in B82L is a factor in cell Y. J. J. 2001; Scopus Google Scholar, Y. J. R.J. G. J. PubMed Scopus Google Scholar, G. Full Text Full Text PubMed Scopus Google Scholar, E. G. 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The receptor by to tyrosine phosphorylation of the receptor to LPA for failed to elicit a detectable phosphorylation of the results the that EGFR or activation is not necessary and not LPA-induced from apoptosis. PDGF not elicit a in B82L cells or the of LPA LPA has been to elicit the transphosphorylation of the in cell cells, cells, and cells Y. A. A. J. Biol. 2003; Full Text Full Text PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar, A. A. Ullrich A. G. Sci. A. PubMed Scopus Google Scholar). the that in the of EGFR, in mediating LPA by and A. A. Ullrich A. G. Sci. A. PubMed Scopus Google in a of B82L cells. in B82L cells that express PDGF receptor and of the that of B82L cells to LPA for not increase phosphorylation the the receptor LPA DNA in B82L cells, of the cells with kinase showed antiapoptotic activity the LPA-induced antiapoptotic results the that is a of LPA-induced transphosphorylation and that the of LPA in B82L cells. and ERK1/2 for the LPA-elicited in B82L of ERK1/2 is required in the antiapoptotic of LPA in a of cell Y. J. J. 2001; Scopus Google Scholar, Y. J. R.J. G. J. PubMed Scopus Google Scholar, G. Full Text Full Text PubMed Scopus Google Scholar, E. G. 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EGFR LPA-induced in B82L The receptor tyrosine kinase and been used to and to for EGFR results with B82L cells that the antiapoptotic to LPA not the of the B82L cells to the by EGFR kinase and of LPA to wild-type B82L cells DNA by with or a of LPA-induced DNA no was in of the LPA-elicited apoptosis. the activation of ERK1/2 was required for the antiapoptotic and signaling with the of DNA of B82L cells with in of the LPA elicited phosphorylation of ERK1/2 was with not results the of of these two EGFR tyrosine kinase in of only because ERK1/2 DNA results in B82L cells that EGFR was not required for LPA-induced which the of EGFR in the to In B82L cells, with LPA DNA synthesis in a the of EGFR LPA-induced the of LPA, EGF, and PDGF DNA synthesis in B82L and cells with LPA a increase of DNA synthesis in cells with that in B82L cells DNA synthesis was by in the two cell with DNA synthesis only in cells and no in the wild-type B82L cells, which is in with a J. J. J. Biol. Full Text Full Text PubMed Scopus Google Scholar). that of the EGFR a in LPA-induced DNA LPA-induced DNA synthesis in cells and in B82L cells of protein kinase kinase activation by the LPA in B82L and cells. The increase in DNA synthesis in cells was not by but was by the protein kinase kinase results that of the EGFR in B82L cells to DNA synthesis and that EGFR also the of of the Cell elicited only a modest increase in DNA synthesis that was by the of the a LPA and LPA-induced cell migration in wild-type B82L and cells a In B82L cells, LPA a increase in migration activation by LPA was by or not In cells, LPA a increase in migration that was that in B82L cells. In cells, the LPA-induced migration was with or failed to a in wild-type B82L or cells, which is in with a J. J. Biol. 1999; Full Text Full Text PubMed Scopus Google Scholar). of cells with not LPA migration and no the of cell migration. In contrast to EGF, PDGF elicited a increase in migration although that elicited by The in wild-type B82L cells and cells was by no the of LPA-induced cell migration of EGFR LPA-induced in B82L were by the that although DNA synthesis in cells, failed to increase cell migration, yet the LPA-induced cell migration was The B82L cells and cells used in been not to or J. J. Biol. 1999; Full Text Full Text PubMed Scopus Google Scholar). of LPA GPCR and EGFR activation of the intrinsic kinase activity the transphosphorylation of the or transfectants of B82L cells expressing mutant, EGFRY5F that lacks the five autophosphorylation sites, and that lacks the Src phosphorylation and the responses to LPA, EGF, and no in LPA-induced of DNA among wild-type or cells expressing receptor and of or PDGF to of the five cell failed DNA and DNA synthesis was not in of the five cell of which EGFR in and cells, LPA elicited in wild-type B82L cells elicited DNA synthesis in and cells The augmented responses in LPA-induced DNA synthesis were not and cells. In cell and DNA only the LPA-induced increase in In cells, LPA-induced was from that in wild-type B82L cells, that EGFR mutant failed to LPA-induced DNA these results that the tyrosine kinase activity of the EGFR is not necessary for the augmentation of LPA-induced DNA of the five autophosphorylation sites but not the Src phosphorylation site the augmentation of LPA-induced in cells. also LPA-elicited cell migration in the cell In cells, LPA elicited a increase in the of cell migration that was to that in cells, although the of cell migration was also in of cells failed to a in cell migration in cell with or without EGFR In contrast, PDGF elicited a in cell that was by the in the DNA synthesis and that the tyrosine kinase activity of the EGFR is not required for to LPA-induced cell migration. LPA-induced transphosphorylation of the EGFR implicated Src or tyrosine kinases in transphosphorylation L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. 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In contrast, LPA only a increase in cells that was to that in wild-type B82L cells the and of migration was not in of the B82L cells with EGFR of the EGFRY5F mutant B82L cells failed to enhancement in LPA-induced of cell migration. these data that the of the EGFR receptor cell migration and DNA synthesis elicited by intrinsic tyrosine kinase activity and the Src phosphorylation site are not necessary but the tyrosine autophosphorylation sites are necessary for the cells to LPA-induced DNA synthesis and J. 2003; PubMed Scopus Google that activation of by LPA is signaling and that EGFR a that tyrosine phosphorylation of the adapter protein SHC is a of the LPA-elicited SHC phosphorylation in cells expressing the four mutant EGFR LPA elicited a increase in SHC with the of wild-type B82L and cells the LPA-elicited increase in was to the of SHC in the cells, cells showed the and was no and cells. the also LPA-elicited in in the five cell and that the increase in activation was in wild-type B82L and cells. In contrast, and cells showed a activation of ERK1/2 phosphorylation and results that enhancement of LPA-induced SHC and ERK1/2 activation or of the five of the The of RTK by GPCR is tyrosine phosphorylation of RTK to ligand activation of the the RTK phosphorylation by the polypeptide growth factor or GPCR and its ERK1/2 activation been the of the of receptor not been in the mediating the responses to LPA, a GPCR ligand that RTK phosphorylation in of cells, has to the of EGFR in the of LPA-induced antiapoptotic and migration a receptor in B82L cells that lack B82L cells express of LPA receptors in to ErbB2, PDGFR, and in B82L cells J. J. Biol. 1999; Full Text Full Text PubMed Scopus Google LPA failed to elicit a detectable increase in tyrosine phosphorylation of these RTK or EGFR, although receptor was in to its LPA-induced cell migration and DNA synthesis but not antiapoptotic was augmented in cells of EGFR, of or dead tyrosine kinase or the Src phosphorylation was is to that of RTK by GPCR cell Daaka Y. Lefkowitz R.J. L.M. J. Biol. 1999; Full Text Full Text PubMed Scopus Google Scholar, A. A. Ullrich A. J. PubMed Scopus Google not only the EGFR but also of the Y. A. A. J. Biol. 2003; Full Text Full Text PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar, A. A. Ullrich A. G. Sci. A. PubMed Scopus Google Scholar, L.M. J. Biol. Full Text Full Text PubMed Scopus Google Scholar). ERK1/2 activity was required for LPA-induced antiapoptotic and migration and and data not In wild-type B82L cells, LPA elicited ERK1/2 activation although in cells was to cells from and that LPA-elicited cell migration and DNA synthesis with LPA-induced SHC phosphorylation and a activation of ERK1/2 to a of the and of LPA-elicited with the of the signaling to in of the and of its no LPA-induced and The B82L cell and its and been J. J. Biol. 1999; Full Text Full Text PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar, J. J. Biol. Full Text Full Text PubMed Scopus Google Scholar, Y. J. Biol. 1999; Full Text Full Text PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). cells, the used by A. A. Ullrich A. G. Sci. A. PubMed Scopus Google no LPA-induced transphosphorylation of the no for the of the LPA responses by the although the PDGF and the autophosphorylation of and to a the cell is that J. J. Biol. 1999; Full Text Full Text PubMed Scopus Google that not increase the migration in cells but migration in to or in the of EGF, which is with a of EGFR in the mediating migration. In also failed to migration in cells, yet LPA-induced migration was augmented in and but and these data that G proteins of the and ERK1/2 activation are for LPA effect. that RTK GPCR responses that not elicit in a cell in B82L cells are not without because in mouse cells, the of and PDGF to migration, LPA has been to migration J. Biol. 1999; Full Text Full Text PubMed Scopus Google Scholar). The LPA and in was to and the of G proteins and results are similar to of and J. 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PubMed Scopus Google and with the receptor has been implicated in mediating the activation of M.J. PubMed Scopus Google Scholar). autophosphorylation site mutant been to N. J. Oncogene. Google Scholar). data that these are not by LPA in the of the EGFR The lack of for EGFR tyrosine kinase activity in responses is in the Ullrich A. J. J. Biol. Full Text PubMed Google Scholar, M.J. PubMed Scopus Google Scholar). is that the the EGFR required for the of LPA responses that not only the intrinsic tyrosine kinase of the EGFR but also tyrosine kinases. was to EGFR but although was not necessary for ERK1/2 activation by LPA J. M. J. J. Biol. 2001; Full Text Full Text PubMed Scopus Google Scholar). The although in ERK1/2 phosphorylation among the that migration and DNA synthesis a activation of the protein kinase in which of the EGFR, with its to activation by J. 2003; PubMed Scopus Google a necessary The of the sites and the LPA docking to these EGFR of