Diacylglycerols Containing Omega 3 and Omega 6 Fatty Acids Bind to RasGRP and Modulate MAP Kinase Activation

We elucidated the effects of different diacylglycerols (DAGs), i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG), on [3H]PDBu binding to RasGRP. The competition studies with these DAGs on [3H]PDBu binding to RasGRP revealed different Ki values for these DAG molecular species. Furthermore, we transfected human Jurkat T cells by a plasmid containing RasGRP and assessed the implication of endogenous DAGs on activation of MAP kinases ERK1/ERK2, induced by phorbol-12-myristate-13-acetate (PMA). In control cells, GF109203X, a protein kinase C inhibitor, inhibited ERK1/ERK2 activation. However, this agent curtailed but failed to completely diminish ERK1/ERK2 phosphorylation in RasGRP-overexpressing cells, though calphostin C, a DAG binding inhibitor, suppressed the phosphorylation of MAP kinases in these cells. In cells incubated with arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), PMA induced the production of endogenous DAGs containing these fatty acids, respectively: DAG-AA, DAG-DHA, and DAG-EPA. The inhibition of production of DAG-AA and DAG-DHA significantly inhibited MAP kinase activation in RasGRP overexpressing, but not in control, cells. Our study demonstrates that three DAG molecular species bind to RasGRP, but only DAG-AA and DAG-DHA participate in the modulation of RasGRP-mediated activation of MAP kinases in Jurkat T cells. We elucidated the effects of different diacylglycerols (DAGs), i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG), on [3H]PDBu binding to RasGRP. The competition studies with these DAGs on [3H]PDBu binding to RasGRP revealed different Ki values for these DAG molecular species. Furthermore, we transfected human Jurkat T cells by a plasmid containing RasGRP and assessed the implication of endogenous DAGs on activation of MAP kinases ERK1/ERK2, induced by phorbol-12-myristate-13-acetate (PMA). In control cells, GF109203X, a protein kinase C inhibitor, inhibited ERK1/ERK2 activation. However, this agent curtailed but failed to completely diminish ERK1/ERK2 phosphorylation in RasGRP-overexpressing cells, though calphostin C, a DAG binding inhibitor, suppressed the phosphorylation of MAP kinases in these cells. In cells incubated with arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), PMA induced the production of endogenous DAGs containing these fatty acids, respectively: DAG-AA, DAG-DHA, and DAG-EPA. The inhibition of production of DAG-AA and DAG-DHA significantly inhibited MAP kinase activation in RasGRP overexpressing, but not in control, cells. Our study demonstrates that three DAG molecular species bind to RasGRP, but only DAG-AA and DAG-DHA participate in the modulation of RasGRP-mediated activation of MAP kinases in Jurkat T cells. The Ras family of small GTPases is comprised of the classical Ras GTPases (H-Ras, N-Ras, and K-Ras) as well as a more divergent group of Ras-related GTPase (TC21, R-Ras, R-Ras3, Rals, and Raps) (1.Bokoch G.M. Der C.J. FASEB J. 1993; 7: 750-759Crossref PubMed Scopus (184) Google Scholar). These proteins cycle between an inactive form bound to GDP and an active GTP-bound state. To reach the active GTP-bound state, Ras proteins must first release bound GDP. This rate-limiting step in GTP binding is catalyzed by guanine-nucleotide exchange factors (GEFs). 1The abbreviations used are: GEF, guanine-nucleotide exchange factors; AA, arachidonic acid; DAG, diacylglycerol; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; SAG, 1-stearoyl-2-arachidonoyl-sn-glycerol; SDG, 1-stearoyl-2-docosahexaenoyl-sn-glycerol; SEG, 1-stearoyl-2-eicosapentaenoyl-sn-glycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PLD, phospholipase D; PCPLD, PC-specific PLD; PDBu, phorbol 12,13-dibutyrate; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5 biphosphate; PLC, phospholipase C; PI-PLC, PI-specific-PLC; PS, phosphatidylserine; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PUFAs, polyunsaturated fatty acids; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin; ANOVA, analysis of variance. Several mammalian GEFs have been identified so far, including Sos (2.Chardin P. Camonis J.H. Gale N.W. van Aelst L. Schlessinger J. Wigler M.H. Bar-Sagi D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (659) Google Scholar), RasGRF (3.Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar), and RalGDS (4.Albright C.F. Giddings B.W. Liu J. Vito M. Weinberg R.A. EMBO J. 1993; 12: 339-347Crossref PubMed Scopus (158) Google Scholar). A new class of GEFs, expressed mainly in brain and T cells (5.Pierret P. Dunn R.J. Djordjevic B. Stone J.C. Richardson P.M. J. Neurocytol. 2000; 29: 485-497Crossref PubMed Scopus (12) Google Scholar, 6.Pierret P. Vallee A. Mechawar N. Dower N.A. Stone J.C. Richardson P.M. Dunn R.J. Neuroscience. 2001; 108: 381-390Crossref PubMed Scopus (10) Google Scholar), is composed of at least four members: 1) RasGRP, the first member characterized as a GEF for Ras (7.Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (552) Google Scholar); 2) CalDAGI or RasGRP2, which possesses GEF activity for N-Ras, K-Ras, and Rap1 (8.Rebhun J.F. Castro A.F. Quilliam L.A. J. Biol. Chem. 2000; 275: 34901-34908Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar); 3) CalDAGIII or Ras-GRP3, which can activate both Ras and Rap 1 (9.Rebhun J.F. Chen H. Quilliam L.A. J. Biol. Chem. 2000; 275: 13406-13410Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar); 4) RasGRP4, recently discovered, has been shown to activate H-Ras in a cation-dependent manner (10.Yang Y. Li L. Wong G.W. Krilis S.A. Madhusudhan M.S. Sali A. Stevens R.L. J. Biol. Chem. 2002; 277: 25756-25774Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 11.Reuther G.W. Lambert Q.T. Rebhun J.F. Caligiuri M.A. Quilliam L.A. Der C.J. J. Biol. Chem. 2002; 277: 30508-30514Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). All these GRP members have a pair of atypical EF-hands (a calcium-binding motif), and the C1 domain, which represents a signature motif that is involved in the recognition of phorbol ester and diacylglycerol (DAG) (12.Lorenzo P.S. Kung J.W. Bottorff D.A. Garfield S.H. Stone J.C. Blumberg P.M. Cancer Res. 2001; 61: 943-949PubMed Google Scholar, 13.Tognon C.E. Kirk H. Passmore L.A. Whitehead I.P. Der C.J. Kay R.J. Mol. Cell. Biol. 1998; 18: 6995-7008Crossref PubMed Scopus (205) Google Scholar, 14.Rong S.B. Enyedy I.J. Qiao L. Zhao L. Ma D. Pearce L.L. Lorenzo P.S. Stone J.C. Blumberg P.M. Wang S. Kozikowski A.P. J. Med. Chem. 2002; 45: 853-860Crossref PubMed Scopus (21) Google Scholar). Although calmodulin-like EF-hands are present in RasGRP, [3H]phorbol 12,13-dibutyrate (PDBu) binding is independent of calcium (12.Lorenzo P.S. Kung J.W. Bottorff D.A. Garfield S.H. Stone J.C. Blumberg P.M. Cancer Res. 2001; 61: 943-949PubMed Google Scholar). This is in marked contrast to the calcium-dependence observed for conventional protein kinase C (PKC) (15.Lorenzo P.S. Beheshti M. Pettit G.R. Stone J.C. Blumberg P.M. Mol. Pharmacol. 2000; 57: 840-846PubMed Google Scholar). For many years, DAG was believed to act solely through the PKC family of isoenzymes. Now, RasGRP provides a direct link between DAG generation and Ras activation (12.Lorenzo P.S. Kung J.W. Bottorff D.A. Garfield S.H. Stone J.C. Blumberg P.M. Cancer Res. 2001; 61: 943-949PubMed Google Scholar, 14.Rong S.B. Enyedy I.J. Qiao L. Zhao L. Ma D. Pearce L.L. Lorenzo P.S. Stone J.C. Blumberg P.M. Wang S. Kozikowski A.P. J. Med. Chem. 2002; 45: 853-860Crossref PubMed Scopus (21) Google Scholar). Ebinu et al. (7.Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (552) Google Scholar) monitored the subcellular fractionation of RasGRP in Rat2 cells, and they observed that PMA binding increased the recruitment of RasGRP to plasma membrane where it interacted efficiently with ras. Activated RasGRP and PKC, in turn, stimulate the mitogen activated protein (MAP) kinase-signaling pathway (12.Lorenzo P.S. Kung J.W. Bottorff D.A. Garfield S.H. Stone J.C. Blumberg P.M. Cancer Res. 2001; 61: 943-949PubMed Google Scholar, 16.Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (402) Google Scholar). Propagation of these signaling cascades promotes the transcription of numerous genes including those encoding cytokines critical for T cell development, activation, and proliferation. In fact, deletion of C1 domain of the RasGRP not only abolishes phorbol ester binding but also MAP kinase activation (7.Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (552) Google Scholar, 13.Tognon C.E. Kirk H. Passmore L.A. Whitehead I.P. Der C.J. Kay R.J. Mol. Cell. Biol. 1998; 18: 6995-7008Crossref PubMed Scopus (205) Google Scholar). Numerous reports have a of polyunsaturated fatty as docosahexaenoic acid and eicosapentaenoic on of human and has been shown to T cell and an in T 1998; 57: PubMed Scopus Google Scholar). the fatty cell cell signaling DAG production In T cells, DAG is in a N.A. A. in in Scholar). The production of DAG is by the of phosphatidylinositol catalyzed by phospholipase C The of DAG generation is with an in the activation of phospholipase phosphatidic acid which can to DAG by the of N.A. A. J. PubMed Scopus Google Scholar, R.J. R.J. J. 1992; PubMed Scopus Google Scholar). In of has been shown to cell the production of DAG, containing this fatty acid A. S. 1992; Google Scholar). the DAG has been to with H. Res. 1998; Scopus Google Scholar). In the we have recently that different of conventional and and and in to DAGs containing or in a S. A. A. J. N.A. FASEB J. 2001; PubMed Scopus Google Scholar). as the modulation of RasGRP by DAG is to a studies are Lorenzo et al. (15.Lorenzo P.S. Beheshti M. Pettit G.R. Stone J.C. Blumberg P.M. Mol. Pharmacol. 2000; 57: 840-846PubMed Google Scholar) have shown that was more for binding to C1 RasGRP with These also the of in to in cells (12.Lorenzo P.S. Kung J.W. Bottorff D.A. Garfield S.H. Stone J.C. Blumberg P.M. Cancer Res. 2001; 61: 943-949PubMed Google Scholar). that induced to the and to a to the plasma study is on the of RasGRP to bind DAGs containing or it was to 1) the effects of different DAGs molecular species containing arachidonic acid (AA), an and and EPA, PUFAs, on binding of phorbol 12,13-dibutyrate to RasGRP, and 2) the implication of DAGs containing these fatty in the of activation in human Jurkat T cells. and and was bovine PMA, AA, DHA, and [3H]PDBu and and MAP kinase calphostin C, and The MAP kinase was and of RasGRP in for RasGRP was brain (7.Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (552) Google Scholar). RasGRP in was used for the with the induced for at The was for at at and the was in 1 and was by of The was at for and the was used as the for the The protein was an to the on the of protein used for the binding of SAG, SDG, and SDG, and by the of phospholipase C on and as S. A. A. J. N.A. FASEB J. 2001; PubMed Scopus Google Scholar). DAGs on a and with The DAGs identified by with those of of DAGs as and by by The by phospholipase C, and DAGs as in this DAGs at for by as acid with by in a and with a and a with The analysis as at and at was used as with a of of fatty acid was with to the by of of PS, DAG, [3H]PDBu and in or a of in a and in by and at for of of [3H]PDBu was as by and Blumberg N.A. Blumberg P.M. Cancer Res. 45: Google Scholar). The 1 RasGRP protein and the or for at binding was an of and binding was in at For competition on [3H]PDBu binding to RasGRP by different DAG the but a of [3H]PDBu and of the In atypical competition different of the values the competition and the Ki for the was the by the Ki where is the of [3H]PDBu used and is the of for [3H]PDBu for RasGRP. The was to the as a of which a where a of the and is to is the of the and competition studies the by which for the was used for and To a for mammalian the of RasGRP was and and the effects of DAGs on the of RasGRP and MAP kinase Jurkat T cells, which a RasGRP activity J.H. R. C.J. R. P. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), to the RasGRP The Jurkat T cells in with and serum at in a containing and and assessed for by in and of Jurkat cells with was cells RasGRP control and cells used for To in cells transfected with containing and was monitored a as by et al. J.H. R. C.J. R. P. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of RasGRP by protein and cells. was by and to and of RasGRP was by a at the of RasGRP of This with both and human RasGRP. the membrane with activity was with of AA, DHA, and at in of the to fatty bovine serum in was to a fatty acid of The incubated for at of DAG the of and PKC in DAG we used of and was used for inhibition of for and for control and Jurkat T cells incubated for in incubated for at in in the or not of or the cells three in incubated for with or for at in the or of by the of and DAGs three with and to as by et al. J. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the was in a and incubated with DAG kinase in the of The was and by The to was and the was by liquid to the or DAG containing fatty with to the activity of these fatty incubated in the of cells. MAP to both control and Jurkat T cells incubated for in incubated for at in in the or not of at AA, DHA, or cells three in incubated for with or calphostin C and incubated for at in the or of PMA the cells and for 1 The protein of cell with a protein was by and to and was by ERK1/ERK2 the membrane with activity was with MAP kinase activity was to the with the The activity was assessed by the of a of containing the which is a more for MAP kinases the by cell by with was by liquid are shown as analysis of was The of the between values was by of [3H]PDBu to RasGRP and by SAG, SDG, and shown in [3H]PDBu bound to RasGRP with high the To the for recognition by RasGRP, we competition binding studies different DAGs in which or fatty The SAG, an and and SEG, and The of [3H]PDBu by DAG molecular species revealed values of and for SAG, SDG, and SEG, The competition of the of [3H]PDBu by at with an of In the of SDG, the is and this DAG to the binding of [3H]PDBu The of [3H]PDBu by at 1 and provides an of The Ki values for SDG, and and These a high of RasGRP for and and for of [3H]PDBu binding to RasGRP by different DAG molecular species. with different DAGs SDG, or and the of [3H]PDBu binding was as a of of DAG as are the the of [3H]PDBu binding to RasGRP by different DAG molecular species binding studies as are inhibition the The values of inhibition have been the Ki where is the of and the of the in a new AA, DHA, and to the of with of AA, DHA, or in a containing of fatty for to a of membrane The of three fatty in cell was with a of of the present in the with and not acid was and for AA, DHA, and EPA, of including and not the the three fatty acids, i.e. DHA, and EPA, at a in PC, and and of not In the AA, DHA, and was and of including and To the production of DAG by PMA, the cells incubated for different and in the of We observed that PMA of cells for effects on DAG production as shown in DAG production was not different between control and transfected cells or not cells incubated with We that in the of AA, DHA, or EPA, cells DAGs with these fatty DAG containing AA, DHA, and and of endogenous DAG, The of was with or and cells, We that DHA, but not or EPA, significantly increased by DAG production which is to the in DAG-DHA PMA activation significantly increased DAG in cells In cells, PMA induced a DAG production with cells, and PMA increased DAG production by and for and DHA, PMA also the of DAGs containing and in both control and cells those containing of the cell PMA DAG by and the of C and in DAG we used an of PI-PLC, and an of that GF109203X, a PKC inhibitor, DAG production both in control and transfected cells, that PMA increased DAG production PKC activation. In the of PMA, and on DAG production not the these significantly DAG Furthermore, and effects on the inhibition of DAG in both control and RasGRP Jurkat T cells and was to the of cells an of inhibitor, completely failed to DAG production in of cells not These that both and are activated by PMA in Jurkat T cells, and PKC the of and a of DAG RasGRP to ERK1/ERK2 of RasGRP by a of Ras GRP in Jurkat T cells. the Ras GRP protein is expressed in cells shown in and PMA induced ERK1/ERK2 RasGRP in Jurkat T cells is by GF109203X, a PKC inhibitor, and calphostin C, a of binding of DAG and phorbol ester to C1 domain present on PKC and RasGRP (15.Lorenzo P.S. Beheshti M. Pettit G.R. Stone J.C. Blumberg P.M. Mol. Pharmacol. 2000; 57: 840-846PubMed Google Scholar). We observed that the ERK1/ERK2 phosphorylation in control cells, it but not completely the phosphorylation of in cells, that PMA induced MAP kinase activation PKC in control cells, in transfected cells, the activation of MAP kinase is both a and DAG-AA and DAG-DHA, but in ERK1/ERK2 we have observed in that of Jurkat T cells in the of DHA, or can to these fatty acids, we the effects of of on activation. that ERK1/ERK2 activity was in transfected cells with control cells or not they incubated in the of and is that Jurkat T cells with and activation those cells fatty acid The effects of an of PI-PLC, and an of on activation in transfected cells in control cells, which incubated with and The of these phospholipase This inhibition is more in cells, that DAGs are involved in kinase activation an of inhibitor, failed to diminish activation not Furthermore, in the effects of and was observed in cells in of the group of cells RasGRP has been shown to a DAG and phorbol which possesses to or C1 domain, of PKC (7.Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (552) Google Scholar, P.S. Beheshti M. Pettit G.R. Stone J.C. Blumberg P.M. Mol. Pharmacol. 2000; 57: 840-846PubMed Google Scholar, J.H. R. C.J. R. P. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In the present we the of RasGRP as a ester We observed that [3H]PDBu bound to RasGRP with high with a of These well with the of Lorenzo et al. (12.Lorenzo P.S. Kung J.W. Bottorff D.A. Garfield S.H. Stone J.C. Blumberg P.M. Cancer Res. 2001; 61: 943-949PubMed Google Scholar) have human a to To study is on the of binding of different DAG molecular species to RasGRP. In the present for the first the in the of RasGRP to bind AA, EPA, and The of [3H]PDBu by different DAG molecular species a high binding of RasGRP for SAG, SEG, and to and The between and the in binding to RasGRP with these to the that AA, DHA, and cell through DAG and through a of and on RasGRP on MAP kinase the these DAGs by cells, and effects on MAP kinase activation. To this human Jurkat T cells that present a RasGRP activity J.O. Stang S.L. C. Bottorff D.A. J. Blumberg P.M. M. Stone J.C. 2000; PubMed Google Scholar) to the RasGRP We observed that cells expressed a high of RasGRP proteins with control Jurkat T cells. The of RasGRP in Jurkat T cells was by and calphostin C. calphostin C, of DAG, and phorbol domain inhibited ERK1/ERK2 phosphorylation in both of cells, GF109203X, a PKC inhibitor, curtailed but not completely the of PMA on ERK1/ERK2 phosphorylation in RasGRP Jurkat T cells with control cells. These that PMA RasGRP promotes but signaling in transfected Jurkat T cells. In the present both control and RasGRP Jurkat T cells fatty DHA, and of not of fatty has been in a of cell R.J. J. Google Scholar, S. M. M. A.F. J. Res. 1998; Full Text Full Text PDF PubMed Google Scholar). to the that of Jurkat T cells can to the production of DAGs activation of and N.A. A. J. PubMed Scopus Google Scholar, R.J. R.J. J. 1992; PubMed Scopus Google Scholar, S. M. M. A.F. J. Res. 1998; Full Text Full Text PDF PubMed Google Scholar), this phorbol ester was used to activate these in We observed that cells high production of DAG-DHA or not with This is in with the of et al. S. M. M. A.F. J. Res. 1998; Full Text Full Text PDF PubMed Google Scholar) have also high production of DAG-DHA in cells. These have shown that DHA, with or EPA, both phospholipase and which an increased DAG production at the of it is also that a high of DAG-DHA form the of this DAG to to phosphatidic DAG and to is on the of DAG kinase on We also observed that the of DAG-DHA was as with The of was not significantly in both control and transfected Jurkat T cells. Furthermore, PMA significantly increased the of DAG-AA and to a DAG-DHA in both control and cells those containing of the cell In cells, we observed that the of in DAG was and for and DHA, PMA These that PMA not stimulate as is the of cells. PMA activate PKC, which in activate DAG This the production of DAG-DHA in cells. The of DAG molecular species is to and as and the of and significantly curtailed the DAG-AA and DAG-DHA in control and RasGRP Jurkat T cells. we a cell in and ERK1/ERK2 activation RasGRP. we the ERK1/ERK2 activity in of phosphorylation state, and this to the that the phosphorylation of activity that on by different factors kinases and 1993; Google Scholar). the small in activation in can not so as we can in we to that we have shown that of Jurkat T cells with also significantly the ERK1/ERK2 activation A. A. N.A. J. Res. 2001; Full Text Full Text PDF PubMed Google Scholar), in the present study was to the of PMA In fact, the are in the of cells with was for BSA, in the present study the cells are incubated for with in to and PMA is to activate the the production of DAG-DHA was that of DAG-AA, the of activation not the of in cells incubated with and the of activation in cells with and is that DAG-AA, in small can high DAG-AA to more DAG-DHA to bind to RasGRP as is revealed by Ki values SAG, This that the is of and involved in MAP kinases activation. We have also that is a more of PKC in a cell S. A. A. J. N.A. FASEB J. 2001; PubMed Scopus Google Scholar). as is it not to in activation as was and Jurkat T cells not in PMA induced activation in cells with was to the cells which fatty acid in both the group of cells and These the of et al. R.J. Res. PubMed Scopus (65) Google Scholar) have also shown that and are more for PKC activation. Furthermore, the inhibition of the production of DAG-AA and DAG-DHA by and significantly but not completely the ERK1/ERK2 activation in but not in control, Jurkat T cells. This by the of RasGRP on DAG for activation, with In the Lorenzo et al. (15.Lorenzo P.S. Beheshti M. Pettit G.R. Stone J.C. Blumberg P.M. Mol. Pharmacol. 2000; 57: 840-846PubMed Google Scholar) have shown that DAG was more for binding to C1 RasGRP with of both and completely curtailed DAG production in transfected and control cells and inhibited MAP kinase activity in transfected cells. This inhibition not reach MAP kinase activity to the of control cells, and this is to the direct PMA activation of RasGRP and activation was in the of phospholipase these that i.e. but and are in activation in these cells. inhibitor, to DAG also J.H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) and can that an in activation. the implication of in inhibition of activation can as et al. PubMed Scopus (21) Google Scholar) have shown that in Jurkat T cells, failed to ERK1/ERK2 In the present it is also that DAG-DHA on PKC and the In fact, is S. M. M. A.F. J. Res. 1998; Full Text Full Text PDF PubMed Google Scholar). et al. M.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) have that DAG not participate in the activation of PKC in et al. A. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have that DAG by PKC, that by is not to activate this However, we the of of endogenous DAG on the that endogenous DAGs participate in 1) of and significantly activation as with transfected and fatty acid cells and 2) of this the that was in cells that not with and the of activation was in these cells with PMA, and of as with control cells with and this is to of this phorbol ester on PKC and RasGRP in transfected Jurkat T cells. We that RasGRP, in a bound phorbol ester and DAG with a high the of the DAG binding to on the of DHA, and present at of RasGRP Jurkat T cells with these PUFAs, through phospholipase and to the production of DAG containing these These those containing and AA, to effects on MAP kinase activation Our study is of as in and by with cell We C. Stone of of for the plasmid containing RasGRP. We also for

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