Ephrin-B1 Reverse Signaling Activates JNK through a Novel Mechanism That Is Independent of Tyrosine Phosphorylation

Abstract

Eph receptors and their cognate ligand ephrins play important roles in various biological processes such as cell migration, axon guidance, and synaptic plasticity. One characteristic feature of the Eph-ephrin signal transduction is that, upon interaction with the receptor, the transmembrane B-class ephrins become tyrosine-phosphorylated and transduce intracellular signals that lead to reorganization of the cytoskeleton. Although in vitro and genetic studies have demonstrated unequivocally the significance of this reverse signaling, the underlying mechanism remains unclear. We report here that transfection of ephrin-B1 into 293 cells resulted in robust increase in JNK activity, whereas expression of truncated ephrin-B1 lacking the cytoplasmic domain had a negligible effect, indicating that the induction of JNK activity was attributed mainly to the reverse signaling. The ephrin-B1-mediated JNK activation was reduced significantly by dominant-negative TAK1, MKK4, or MKK7. Ephrin-B1 over-expressing 293 cells became rounded in morphology. Surprisingly, ephrin-B1 that lacked all six intracellular tyrosine residues still triggered JNK activation and rounding morphology of the transfected cells. Consistent with these observations, activation of JNK and the resulting morphological changes mediated by ephrin-B1 could be abolished by the JNK inhibitor SP600125 but not the Src inhibitor PP2. Taken together, our findings have identified a novel reverse signaling pathway transduced by ephrin-B1, which is independent of tyrosine phosphorylation but involves the activation of JNK through TAK1 and MKK4/MKK7 and leads to changes in cell morphology. Eph receptors and their cognate ligand ephrins play important roles in various biological processes such as cell migration, axon guidance, and synaptic plasticity. One characteristic feature of the Eph-ephrin signal transduction is that, upon interaction with the receptor, the transmembrane B-class ephrins become tyrosine-phosphorylated and transduce intracellular signals that lead to reorganization of the cytoskeleton. Although in vitro and genetic studies have demonstrated unequivocally the significance of this reverse signaling, the underlying mechanism remains unclear. We report here that transfection of ephrin-B1 into 293 cells resulted in robust increase in JNK activity, whereas expression of truncated ephrin-B1 lacking the cytoplasmic domain had a negligible effect, indicating that the induction of JNK activity was attributed mainly to the reverse signaling. The ephrin-B1-mediated JNK activation was reduced significantly by dominant-negative TAK1, MKK4, or MKK7. Ephrin-B1 over-expressing 293 cells became rounded in morphology. Surprisingly, ephrin-B1 that lacked all six intracellular tyrosine residues still triggered JNK activation and rounding morphology of the transfected cells. Consistent with these observations, activation of JNK and the resulting morphological changes mediated by ephrin-B1 could be abolished by the JNK inhibitor SP600125 but not the Src inhibitor PP2. Taken together, our findings have identified a novel reverse signaling pathway transduced by ephrin-B1, which is independent of tyrosine phosphorylation but involves the activation of JNK through TAK1 and MKK4/MKK7 and leads to changes in cell morphology. Eph receptors, the largest family of receptor tyrosine kinases comprising 14 members in mammals, play critical roles in diverse biological processes during development as well as in the mature animal (1Flanagan J.G. Vanderhaeghen P. Annu. Rev. Neurosci. 1998; 21: 309-345Google Scholar, 2Kullander K. Klein R. Nat. Rev. Mol. Cell. Biol. 2002; 3: 475-486Google Scholar). They are activated by membrane-bound ligands called ephrins, which are classified into two subclasses based on their modes of membrane anchorage. The ephrin-A ligands (ephrin-A1–A5) are glycosylphosphatidylinositol-linked and prefer to bind to EphA receptors (EphA1–A8). The ephrin-B ligands (ephrin-B1–B3), which possess a transmembrane moiety and a short cytoplasmic domain, bind to EphB receptors (EphB1–B6). The interactions between ephrins and Eph receptors are generally promiscuous within each subclass. There is little cross-reactivity between members from different subclasses, with the notable exception of EphA4 (3Gale N.W. Holland S.J. Valenzuela D.M. Flenniken A. Pan L. Ryan T.E. Henkemeyer M. Strebhardt K. Hirai H. Wilkinson D.G. Pawson T. Davis S. Yancopoulos G.D. Neuron. 1996; 17: 9-19Google Scholar). Based on the prominent expression of ephrins and Eph receptors in the brain, early studies focused on their significance in the nervous system. Elegant in vitro and genetic studies have demonstrated that ephrins act as a repulsive axon guidance cue, which are essential in the formation of topographic mapping in various regions of the central nervous system. Moreover, interactions between Eph receptors and ephrins also govern neural crest cell migration and segmental patterning in the brain. Besides the fact that Eph receptors and ephrins perform important functions during development, more recent studies have unraveled their significance in the mature nervous system, especially in the aspect of synapse formation and functioning. Eph receptors and ephrins are localized at the neuronal synapses as well as the neuromuscular junction (4Torres R. Firestein B.L. Dong H. Staudinger J. Olson E.N. Huganir R.L. Bredt D.S. Gale N.W. Yancopoulos G.D. Neuron. 1998; 21: 1453-1463Google Scholar, 5Lai K.O. Ip F.C.F. Cheung J. Fu A.K.Y. Ip N.Y. Mol. Cell. Neurosci. 2001; 17: 1034-1047Google Scholar), and ephrin-B ligands are involved in the formation of neuronal synapses and long-term potentiation in the hippocampus (6Dalva M.B. Takasu M.A. Lin M.Z. Shamah S.M. Hu L. Gale N.W. Greenberg M.E. Cell. 2000; 103: 945-956Google Scholar, 7Grunwald I.C. Korte M. Wolfer D. Wilkinson G.A. Unsicker K. Lipp H.P. Bonhoeffer T. Klein R. Neuron. 2001; 32: 1027-1040Google Scholar, 8Henderson J.T. Georgiou J. Jia Z. Robertson J. Elowe S. Roder J.C. Pawson T. Neuron. 2001; 32: 1041-1056Google Scholar). Recent studies have also demonstrated the regulation of cell adhesion by Eph receptors, which is central to their participation in vascular development and tumorigenesis outside of the nervous system (9Adams R.H. Klein R. Trends Cardiovasc. Med. 2000; 10: 183-188Google Scholar, 10Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). One unusual feature of Eph-ephrin interaction is that signals can be transduced bidirectionally in both the ligand- and receptor-expressing cells (11Cowan C.A. Henkemeyer M. Trends. Cell Biol. 2002; 12: 339-346Google Scholar). The glycosylphosphatidylinositol-linked A-class ephrins lack a cytoplasmic domain and intrinsic kinase activity, requiring the recruitment of other signaling molecules to trigger the reverse signaling. One possible candidate is the non-receptor tyrosine kinase Fyn, which was found necessary for the reverse signaling mediated by ephrin-A5 (12Davy A. Gale N.W. Murray E.W. Klinghoffer R.A. Soriano P. Feuerstein C. Robbins S.M. Genes Dev. 1999; 13: 3125-3135Google Scholar). The reverse signaling of ephrin-A5 leads to modulation of integrin functions and in turn to the regulation of cell adhesion (13Davy A. Robbins S.M. EMBO J. 2000; 19: 5396-5405Google Scholar, 14Huai J. Drescher U. J. Biol. Chem. 2001; 276: 6689-6694Google Scholar). Interactions between the glycosylphosphatidylinositol-linked ephrins and VAB-1, the Caenorhabditis elegans homolog of EphA receptors, regulate the organization of epidermal cells independently of the kinase activity of VAB-1, thereby providing genetic evidence for the significance of ephrin-A reverse signaling. Upon interaction with EphB receptors, the transmembrane B-class ephrins also transduce intracellular signal. Ample evidence from different in vivo studies supports the biological significance of ephrin-B reverse signaling in axon guidance, hind brain segmentation, and neuronal plasticity. Axons of the anterior commissure, which express the ephrin-B ligands rather than EphB receptors, migrate aberrantly in EphB2 null mice but not in transgenic mice, in which the kinase domain of EphB2 is deleted (15Henkemeyerk M. Orioli D. Henderson J.T. Saxton T.M. Roder J. Pawson T. Klein R. Cell. 1996; 86: 35-46Google Scholar). Similarly, axon pathfinding of retinal ganglion cells within the retina depends on EphB2 in a kinase-independent manner (16Birgbauer E. Oster S.F. Severin C.G. Sretavan D.W. Development. 2001; 128: 3041-3048Google Scholar). During segmentation of the hind brain, EphB-ephrin-B interactions at the boundary of adjacent rhombomeres are crucial to prevent the intermingling of distinct cell populations, and bidirectional signaling of both EphB receptors and ephrin-B ligands is required (17Mellitzer G. Xu Q. Wilkinson D.G. Nature. 1999; 400: 77-81Google Scholar, 18Xu Q. Mellitzer G. Robinson V. Wilkinson D.G. Nature. 1999; 399: 267-271Google Scholar). Mice with targeted deletion of EphB2 display defects in neuronal plasticity, which can be rescued by EphB2 truncated at the carboxyl terminus (7Grunwald I.C. Korte M. Wolfer D. Wilkinson G.A. Unsicker K. Lipp H.P. Bonhoeffer T. Klein R. Neuron. 2001; 32: 1027-1040Google Scholar). The signaling pathways following ephrin-B activation have begun to be elucidated; they may be classified into the phosphotyrosine-dependent pathway and PDZ 1The abbreviations used are: PDZ, PSD95/Dlg/ZO-1; SFK, Src family kinases; MAP, mitogen-activated protein; MAP3K, MAP kinase kinase kinase; MKK, MAP kinase kinase; PAK, p21-activated kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; GFP, green fluorescence protein; PTP, protein tyrosine phosphatase; SH2 and SH3, Src homology 2 and 3; RGS, regulator of heterotrimeric G protein signaling; HA, hemagglutinin; GST, glutathione S-transferase. -dependent pathway (2Kullander K. Klein R. Nat. Rev. Mol. Cell. Biol. 2002; 3: 475-486Google Scholar). In the former, ephrin-B is tyrosine-phosphorylated upon interaction with EphB receptors by the Src family kinases (SFK) (19Holland S.J. Gale N.W. Mbamalu G. Yancopoulos G.D. Henkemeyer M. Pawson T. Nature. 1996; 383: 722-725Google Scholar, 20Bruckner K. Pasquale E.B. Klein R. Science. 1997; 275: 1640-1643Google Scholar, 21Palmer A. Zimmer M. Erdmann K.S. Eulenburg V. Porthin A. Heumann R. Deutsch U. Klein R. Mol. Cell. 2002; 9: 725-737Google Scholar). Sprouting angiogenesis of endothelial cells induced by EphB4-Fc is abolished by the Src inhibitor PP2, that tyrosine phosphorylation by is essential for ephrin-B reverse signaling A. Zimmer M. Erdmann K.S. Eulenburg V. Porthin A. Heumann R. Deutsch U. Klein R. Mol. Cell. 2002; 9: 725-737Google Scholar). the other a protein tyrosine is to the activated ephrin-B1 in a interaction between the PDZ domain of and the carboxyl of ephrin-B1, which ephrin-B1 is and the phosphotyrosine-dependent reverse signaling is A. Zimmer M. Erdmann K.S. Eulenburg V. Porthin A. Heumann R. Deutsch U. Klein R. Mol. Cell. 2002; 9: 725-737Google Scholar). The tyrosine phosphorylation of ephrin-B1 in the of the protein interaction with SH2 The in turn various signaling molecules such as adhesion kinase and protein to the ephrin-B signaling to of and rounding cell morphology C.A. Henkemeyer M. Nature. 2001; Scholar). In to the pathway in the reverse signaling that the of ephrin-B1 Besides a of PDZ such as and have identified as with the carboxyl of ephrin-B1 (4Torres R. Firestein B.L. Dong H. Staudinger J. Olson E.N. Huganir R.L. Bredt D.S. Gale N.W. Yancopoulos G.D. Neuron. 1998; 21: 1453-1463Google Scholar, K. J. P. A. Klein R. Neuron. 1999; Scholar, D. G.D. Z. Pawson T. J. Biol. Chem. 1999; their significance with to the of reverse signaling is not a novel called was found to with ephrin-B1 PDZ a regulator of heterotrimeric G protein signaling domain, which the G protein and the signaling transduced by G was found that interaction between ephrin-B1 and was essential in the ephrin-B1-mediated cell in Q. Klein J.G. Cell. 2001; Scholar). the activation of ephrin-B1 reverse signaling of cells induced by the and the depends on between the ephrin-B1 reverse signaling and the G receptor signaling Although is not activation of ephrin-B1 the activity of is that the between ephrin-B1 and is and not on activation by Q. Klein J.G. Cell. 2001; Scholar). in recent of the reverse signaling of the ephrins unclear. One candidate protein that identified to with the domain of is C.A. Henkemeyer M. Nature. 2001; Scholar). is a of the protein family and as a regulator of signaling A. R. Annu. Rev. Cell Dev. Biol. 1998; Scholar). In was demonstrated to the MAP kinase c-Jun NH2-terminal kinase J. Lin J. Biol. Chem. 1999; Scholar). In of this and the regulation of MAP kinase activity by the signaling of Eph receptors E. U. J. Biol. Chem. 1998; Scholar, H. D.M. Q. T. E. Nat. Cell Biol. 2001; 3: Scholar, S. Holland S.J. S. Pawson T. Mol. Cell. Biol. 2001; 21: Scholar, J. Elowe S. P. Pawson T. J. Biol. Chem. Scholar), the of JNK in ephrin-B1 reverse signaling report a novel reverse signaling pathway mediated by ephrin-B1 in 293 which involves activation of JNK TAK1 and MKK4/MKK7 and leads to a morphological in the transfected cells. this pathway not tyrosine phosphorylation of of ephrin-B1 was from two and The for was from a and was ephrin-B1 and into the expression of ephrin-B1 and by in vitro the dominant-negative as M. R. Mol. Cell. Biol. Scholar). by In to resulting from the to by for and JNK as as J. Lin J. Biol. Chem. 1999; Scholar). was a from J. and cells in with of and 2 in transfection to the The of transfected was to with the at transfection and as J. Lin J. Biol. Chem. 1999; Scholar). was and the JNK activity was as of as by to the phosphorylation of c-Jun J. Lin J. Biol. Chem. 1999; Scholar). activation of the kinase was by and to their expression are as kinase activation with that in with the the from at cells by as C.A. Henkemeyer M. Nature. 2001; Scholar). and transfected cells in in for and at for at Ephrin-B1 from the cell with and protein and with The on and to with in phosphate-buffered with for the with The and with by of 293 by and cells by or of as K.O. Ip F.C.F. Ip N.Y. 1999; Scholar). the cells with in for by the cells by for 2 by with at for the cells with with for The cells by and with the inhibitor in for the of the and of 293 cells in with of and of or various ephrin-B1 by The cells in on the and the cell morphology was fluorescence the of the cells in on the transfection and with the JNK inhibitor SP600125 the Src inhibitor or or for of the cell morphology. the the of and rounded cells that was in different from each The from independent of JNK by Ephrin-B1 TAK1, MKK4, and ephrin-B1 reverse signaling could JNK, cells with ephrin-B1 and The kinase activity of JNK was by phosphorylation of following Although of activity in cells with expression of ephrin-B1 resulted in a robust increase in JNK activity JNK activity was not in the of One possible for the lack of a for is that cells express EphB receptors, which with the ephrin-B1, to the activation of 293 cells could be by indicating expression of EphB receptors ephrin-B1 may and transduce reverse signaling. MAP kinase kinase kinases have to the JNK pathway Cell. 2000; 103: Scholar, G. Robinson T. Xu M. K. Rev. 2001; Scholar, R. Science. 2002; Scholar). and which of the was for ephrin-B1-mediated JNK dominant-negative of different MAP3K, as well as the p21-activated kinase with ephrin-B1 in cells. of the dominant-negative not the JNK activity and of dominant-negative or also not JNK activation of dominant-negative TAK1 significantly reduced JNK activity in a manner and JNK is and activated by and two MAP kinase kinases of Cell. 2000; 103: Scholar, G. Robinson T. Xu M. K. Rev. 2001; Scholar, R. Science. 2002; Scholar). their possible in JNK dominant-negative and with ephrin-B1 in cells. dominant-negative and could the JNK activation that expression of ephrin-B1 in cells leads to activation of JNK TAK1 and of JNK by Ephrin-B1 on but of Ephrin-B1 of the expression of EphB receptors in the increase in JNK activity upon of ephrin-B1 could be attributed to both and reverse signaling. their the JNK activity was between of ephrin-B1 and a truncated ephrin-B1 in which the transmembrane domain was but the cytoplasmic domain of was deleted The expression of could not be by with the the carboxyl terminus of with that both ephrin-B1 and and targeted to the membrane The reverse ephrin-B1 the to JNK indicating that JNK activation was for the to the reverse signaling. The are the ephrin-B and these the tyrosine residues and the (19Holland S.J. Gale N.W. Mbamalu G. Yancopoulos G.D. Henkemeyer M. Pawson T. Nature. 1996; 383: 722-725Google Scholar). the significance of the a truncated ephrin-B1 that lacked the carboxyl terminus was transfected into cells. of induced JNK activity with or the activation was than that induced by ephrin-B1 with that was and targeted to the to the ephrin-B1 that truncated ephrin-B1 that the intracellular tyrosine residues and the can still transduce reverse signaling to residues and of ephrin-B1 identified as the phosphorylation Pasquale E.B. J. Biol. Chem. 2001; 276: Scholar). the of ephrin-B1 tyrosine phosphorylation in JNK the six intracellular tyrosine residues of ephrin-B1 or by to of these into cells induced robust JNK activation and In ephrin-B1 in which all six tyrosine residues could JNK to to that of is SH2 domain that identified as with the activated ephrin-B1 that to reorganization C.A. Henkemeyer M. Nature. 2001; Scholar). The of in JNK activation in cells was of not increase the JNK activity, and the JNK activity was not by of Similarly, of different dominant-negative which or of or by could not the ephrin-B1 induced JNK activity that the recruitment of is not involved in the JNK which is with the findings that of can JNK Ephrin-B1 by Src upon in that expression of ephrin-B1 in cells could turn on the reverse signaling and JNK activity independently of tyrosine with the pathway in which ephrin-B1 was tyrosine-phosphorylated by in to on the reverse signaling A. Zimmer M. Erdmann K.S. Eulenburg V. Porthin A. Heumann R. Deutsch U. Klein R. Mol. Cell. 2002; 9: 725-737Google Scholar, C.A. Henkemeyer M. Nature. 2001; Scholar). The for the is unclear. is possible that the mechanism of ephrin-B1 reverse signaling is cell such that the activation of ephrin-B1 in cells not tyrosine phosphorylation by as in other cell tyrosine phosphorylation of ephrin-B1 by upon activation in cells but is not involved in JNK between the two tyrosine phosphorylation of ephrin-B1 was in transfected cells. ephrin-B1, but not in which tyrosine residues became tyrosine-phosphorylated in cells the of Moreover, tyrosine phosphorylation of ephrin-B1 was abolished the transfected cells with the Src kinase inhibitor that ephrin-B1, in tyrosine-phosphorylated by but that this phosphorylation pathway is not essential for the JNK of Ephrin-B1 to of 293 on JNK but Ephrin-B1 biological significance of the JNK pathway in was 293 cells with ephrin-B1 and GFP, the cells became rounded and whereas the cells and not In 293 cells with and or both the and cells and The induction of cell rounding by ephrin-B1 was with the the changes of cells in to C.A. Henkemeyer M. Nature. 2001; Scholar). The fact that but not cells became rounded with ephrin-B1 that the cell rounding resulted mainly from the reverse signaling of ephrin-B1 rather than the activation of EphB receptors in 293 cells. was by the that was than ephrin-B1 in the rounding morphology of the transfected cells the other 293 cells that or a rounded indicating that activation of ephrin-B1 could cell rounding independently of tyrosine phosphorylation and PDZ domain was with the that of could JNK activation and The morphological for different ephrin-B1 a between the induction of JNK activation and the induction of cell the of JNK in the cell 293 cells transfected with ephrin-B1 or with the JNK inhibitor which JNK but not other MAP The of SP600125 for or is more than than that for JNK in the kinase Moreover, SP600125 to cells not the phosphorylation of and or the of of signaling B.L. Murray Xu J.C. A. S. D.W. U. S. A. 2001; Scholar). of transfected 293 cells with SP600125 abolished the phosphorylation of the expression of ephrin-B1 and the of SP600125 significantly the cell rounding of transfected 293 cells and thereby indicating the important of JNK in ephrin-B1 reverse signaling. induction of JNK activity and rounding morphology was over-expressing or in 293 could be that the ephrin-B1-mediated cell rounding was of tyrosine transfected 293 cells with the Src inhibitor could not the JNK activation cell rounding induced by over-expressing ephrin-B1 at a was demonstrated to be in the ephrin-B1 phosphorylation A. Zimmer M. Erdmann K.S. Eulenburg V. Porthin A. Heumann R. Deutsch U. Klein R. Mol. Cell. 2002; 9: 725-737Google Scholar), and a of to the of both transfected and cells not The of the ephrin-B1-mediated morphological changes by the JNK inhibitor but not the Src inhibitor evidence for the JNK pathway in ephrin-B1 reverse signaling. findings have identified the MAP kinase JNK as important of ephrin-B1 reverse signaling. of ephrin-B1 in cells the activity of We demonstrated that TAK1 was the of ephrin-B1-mediated JNK activation and MKK7. Moreover, that the reverse signaling of ephrin-B1 to rounding morphology of the transfected 293 which required the activity of evidence that this JNK activation and the morphological independent of tyrosine identified the kinase for the JNK activity induced by ephrin-B1 reverse signaling. of dominant-negative TAK1, MKK4, or could each the JNK activity, that ephrin-B1 reverse signaling in the activation of TAK1, which and to JNK JNK activity but not TAK1 J. Lin J. Biol. Chem. 1999; Scholar), the fact that dominant-negative could not the ephrin-B1-mediated JNK activation was with our that the JNK activation was not by dominant-negative not our also that which could with domain C.A. Henkemeyer M. Nature. 2001; Scholar), was not required in the JNK ephrin-B1 in 293 which EphB receptors, could both signaling from the EphB receptors and reverse signaling from the In a the phosphorylation of JNK during signaling of E. U. J. Biol. Chem. 1998; Scholar). is important to which pathway was for the JNK activation and induction of cell important by the reverse signaling. the activation of JNK upon over-expressing ephrin-B1 was than that of activation of JNK by over-expressing ephrin-B1 was in the of which could with the ephrin-B1 and act as the for signaling. of ephrin-B1 resulted in rounding morphology of the transfected whereas the which EphB receptors and in with the transfected and We that is the reverse signaling that JNK and the morphological a of signaling may be on ephrin-B1 is in 293 which for the of JNK activation and morphological in 293 cells transfected with signaling of Eph receptors in cell morphology and on the cell E. U. J. Biol. Chem. 1998; Scholar, U. E. H. EMBO J. 1999; Scholar, Pasquale E.B. E. U. S. A. 1999; Scholar, H. E. M. E. Nat. Cell Biol. 2000; Scholar, P. S.M. M. M. J. Cell 2002; Scholar, C. S. Mol. Cell. Biol. 2001; 21: Scholar). to the reverse signaling, in cell adhesion also (13Davy A. Robbins S.M. EMBO J. 2000; 19: 5396-5405Google Scholar, 14Huai J. Drescher U. J. Biol. Chem. 2001; 276: 6689-6694Google Scholar, C.A. Henkemeyer M. Nature. 2001; Scholar, J. Xu R.H. U. S. A. 1998; Scholar, U. C. H. J.T. M. J. J. Cell 2002; Scholar). the of cell adhesion mediated by ephrin-B reverse signaling. In our the that JNK activity is crucial for morphological studies have that JNK is the and reverse signaling pathways are on E. U. J. Biol. Chem. 1998; Scholar, U. C. H. J.T. M. J. J. Cell 2002; Scholar). not the significance of JNK in the as a of ephrin-B or EphB signaling. various ephrin-B1 have a between the activation of JNK and the induction of cell Moreover, the of JNK was by the of cell rounding in the of the JNK inhibitor SP600125 but not the Src kinase inhibitor PP2. ephrin-B1 JNK and cell rounding independently of tyrosine or of which intracellular tyrosine JNK activation and cell rounding to a as the was to be the critical that mediated the phosphotyrosine-dependent reverse signaling C.A. Henkemeyer M. Nature. 2001; Scholar), and JNK activation was not by various dominant-negative the of Src inhibitor abolished the tyrosine phosphorylation of ephrin-B1 but not the activation of JNK and cell from over-expressing that reverse signaling could be triggered interaction with PDZ domain from the findings the of tyrosine phosphorylation or in ephrin-B1 reverse signaling A. Zimmer M. Erdmann K.S. Eulenburg V. Porthin A. Heumann R. Deutsch U. Klein R. Mol. Cell. 2002; 9: 725-737Google Scholar, C.A. Henkemeyer M. Nature. 2001; Scholar, Q. Klein J.G. Cell. 2001; Scholar, U. C. H. J.T. M. J. J. Cell 2002; Scholar). is that, in ephrin-B1 was activated by the of to cells that ephrin-B ephrin-B1 was in 293 the ligands became activated the of of the in which Eph receptor was and triggered signaling in the of Pasquale E.B. E. U. S. A. 1999; Scholar, K. J. M. Pasquale E.B. J. Neurosci. 1997; Scholar, V.C. Pasquale E.B. Oncogene. 2001; Scholar, H. J. Biol. Chem. 2002; Scholar). 293 cells EphB receptors, the activation of ephrin-B1 interaction with EphB receptor, not the that ephrin-B1 became upon to the the receptor tyrosine kinase was K. J. M. Pasquale E.B. J. Neurosci. 1997; Scholar). of ephrin-B1 in this which be more with activation by could the of tyrosine phosphorylation and interaction and trigger reverse signaling. to the of reverse signaling, was that which the as in J. T. J. Dev. Cell. 2001; Scholar). was with our that ephrin-B1 in 293 which is independent of tyrosine phosphorylation not Moreover, ephrin-B1 was to with the protein independently of tyrosine phosphorylation or interaction with the PDZ domain protein M. T. S. H. EMBO J. Scholar). the of a ephrin-B1 that lacked of the tyrosine residues the into a induced a rounding morphology of the cells the to the cell ephrin-B1 was J. Xu R.H. U. S. A. 1998; Scholar). Taken together, the a novel signaling pathway transduced by ephrin-B1 that is independent of tyrosine phosphorylation and Moreover, of JNK as a critical of morphological to the that JNK activity is involved in studies have unraveled the significance of JNK in cell and M. M. Cell. 1998; Scholar, K. T. Dev. Cell. 2002; Scholar). findings have the that JNK may also play a in various processes that the participation of ephrins, such as axon guidance, segmentation, and We are to for in the