Glycogen Synthase Kinase-3β Induces Neuronal Cell Death via Direct Phosphorylation of Mixed Lineage Kinase 3

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3β has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3β during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3β, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3β-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3β activation, indicating that induction of GSK-3β lies upstream of MLK3. In GSK-3β-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3β. The activation of MLK3 by GSK-3β occurred via phosphorylation of MLK3 on two amino acid residues, Ser789 and Ser793, that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3β was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3β. Taken together, our data provide a direct link between GSK-3β and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3β. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation. Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3β has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3β during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3β, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3β-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3β activation, indicating that induction of GSK-3β lies upstream of MLK3. In GSK-3β-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3β. The activation of MLK3 by GSK-3β occurred via phosphorylation of MLK3 on two amino acid residues, Ser789 and Ser793, that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3β was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3β. Taken together, our data provide a direct link between GSK-3β and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3β. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation. The decision by cells to either undergo cell death or to allow survival is governed by a myriad of extracellular cues such as growth factor availability, heat shock, and cytotoxic insults. The extracellular changes are recognized by specific cellular receptors and transmitted to intracellular targets leading to prevalence of death or survival. Activation of family members of the c-Jun N-terminal kinases (JNKs) 3The abbreviations used are: JNK, c-Jun N-terminal kinase; HEK, human embryonic kidney; MAP3K, mitogen-activated protein kinase kinase kinase; MAP2K, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; DAPI-4′, 6-diamidino-2-phenylindole; MLK, mixed lineage kinase; GSK, glycogen synthase kinase; NGF, nerve growth factor; MEF, mouse embryonic fibroblast; MEKK, MAPK/extracellular signal-regulated kinase kinase kinase; GST, glutathione S-transferase; HA, hemagglutinin; WT, wild type; AKT, AKT/protein kinase B. by their upstream kinases has been implicated in cell death responses (1Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2897) Google Scholar, 2Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar, 3Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar, 4Shaulian E. Karin M. Nat. Cell Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2251) Google Scholar). JNK is stimulated by pro-inflammatory cytokines, oxidative stress, growth factor deprivation, heat shock, UV, γ-radiation, and other cellular stress (1Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2897) Google Scholar, 2Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). The signals in the stress-activated JNK pathway are transmitted via three core protein kinase modules: MAP3K, MAP2K, and MAPK. In the JNK pathway, the activating stimuli activate the MAP3K members, such as members of mixed lineage kinases (MLKs) or MEKKs, which, in turn, phosphorylate MAP2K members such as SEK1/MKK4 and MKK7 (5Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 6Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 663-672Crossref PubMed Scopus (458) Google Scholar). The activated MAP2Ks then phosphorylate JNK at a tyrosine and threonine residue leading to activation and phosphorylation of transcription factors, such as, c-Jun, ELK1, and ATF2. The phosphorylated transcription factors bind to their cognate DNA targets and modulate gene transcription and cellular fate. MLKs are members of a significant MAP3K family and have been implicated in neuronal cell death pathways (7Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gessner G. Xu Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. J. 2001; PubMed Google Scholar, Z. A.C. P. Greene L.A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, Johnson Rev. PubMed Scopus Google Scholar). the MLK family members, MLK3 has been in K.A. Johnson G.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 663-672Crossref PubMed Scopus (458) Google Scholar). has been shown that ectopic expression of MLK3 or NGF neuronal cell death, which is blocked by a of (7Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gessner G. Xu Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Z. A.C. P. Greene L.A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). Similarly, CEP-11004, of has also been shown to have in NGF withdrawal-induced cell death M. Gessner G. Angeles Chem. 2002; PubMed Scopus Google Scholar). we that cell death induced by MLK3 activation is by via pathway activation P. B. A. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The cell survival protein in the C-terminal regulatory domain of MLK3. The phosphorylation of MLK3 by MLK3 kinase and its cell death induction P. B. A. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). has been implicated in neuronal during S. J. Google Scholar). Inhibition of MLKs by neuronal cell death, by Xu Z. A.C. Connors T.J. Greene L.A. J. 2001; PubMed Google Scholar, P. S. Vaught J.L. Scott R.W. J. 2001; PubMed Scopus Google Scholar). a significant and a direct of MLKs in a cell death pathway. However, the by which modulate MLK3 and cell death is to GSK-3β activation also neuronal cell death M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Johnson Full Text Full Text PDF PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). of GSK-3β in PC-12 cells M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google or its expression in of the in GSK-3β neuronal cell death P. J. J. 2001; PubMed Scopus Google Scholar). has been shown that NGF withdrawal-induced cell death in PC-12 cells by the GSK-3 inhibitor, 2001; PubMed Scopus Google Scholar, J. Scott S. A. 2000; PubMed Scopus Google Scholar). GSK-3β has also been shown to activate the JNK pathway activation M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to such as deprivation, to or GSK-3β activation, by induction of M. Z. J. 2000; PubMed Google Scholar). to also activates GSK-3β kinase and cell death A. T. K. G. M. K. PubMed Scopus Google Scholar, A. K. G. M. M. K. K. 1996; PubMed Scopus Google is by M. M. S. A. K. K. S. A. PubMed Scopus Google Scholar). that GSK-3β, which is for and also in cell death However, the by which GSK-3β activation neuronal cell death and the functional between GSK-3β and MLK3 in cell death pathway to In we that MLK3 is a direct of GSK-3β, and their functional interaction is by stimuli NGF in PC-12 cells. regulatory that is induced by GSK-3β has on MLK3 downstream activation of JNK and apoptosis. data also that MLK3 is a cellular target of GSK-3β and the that such as neuronal by MLK3 kinase Cell cells were in and as Z. A.C. P. Greene L.A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). PC-12 cells were for in and of embryonic GSK-3β-deficient and mouse embryonic were in as J. Nature. 2000; PubMed Scopus Google Scholar). and and were the expression as and were for the the NGF was induced by the cells and was by the of and and were in as (5Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The cell were by at for and the protein were and glutathione were either by specific or The were and either for or kinase protein of cell or the protein were The were on and and as The used JNK and GSK-3β for JNK was by Avruch MLK3, the C-terminal of the protein was in the as P. B. A. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. A. G. A. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase was as M. E. 1996; PubMed Scopus Google as the MLK3 kinase was as the as (5Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). in kinase were and as (5Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The GSK-3β was cells were to and were for 3 as P. B. A. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and and GSK-3β were used as J. Nature. 2000; PubMed Scopus Google Scholar, L. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar). MLK3 were in the by and of human MLK3 was a The MLK3 and were the the wild MLK3 is by a The were used as a to and were the of was in expression between and The wild was used as a for The used for were as and of the in the its protein was and used as the for in GSK-3β kinase Cell NGF withdrawal-induced cell death in PC-12 cells was by the and in a cell death and as J. Scott S. A. 2000; PubMed Scopus Google Scholar, S. J. Scott J. 2002; 4: PubMed Scopus Google Scholar). The cell in for and Cell death was also in PC-12 cells PC-12 cells were the the as Cell death was in the cells the cells were in and by The in cells were for by The of cells was as the of cells by the of cells. NGF GSK-3β, MLK3, and JNK and Cell in PC-12 used of neuronal is trophic factor withdrawal-induced cell death in neuronal cells. the of trophic factor on the protein kinase activities of GSK-3β, MLK3, and JNK and its on neuronal cell death, NGF was at PC-12 cell NGF was by to the cell and the cells were at to The kinase activities of JNK, GSK-3β, and MLK3 were specific for was a activation of JNK which at of NGF NGF also activated GSK-3β and MLK3 kinase activities in a GSK-3β was activated at of NGF withdrawal, and MLK3 kinase also at of the activation of JNK, GSK-3β, and MLK3 NGF withdrawal-induced cell death in PC-12 we the of PC-12 cell NGF withdrawal-induced cell death in PC-12 cells was as as withdrawal, which was the and to a in cell death the cells. a between induction of cell death and activation of GSK-3β, JNK, and MLK3 in also that the of cell death is by the activation of is via downstream of cell death NGF Cell by MLK3 and GSK-3 NGF withdrawal-induced GSK-3β and MLK3 activation in PC-12 cell death, the cells were either M. K. S. M. L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. J. G.L. P. L. J. 2000; PubMed Scopus Google a GSK-3 inhibitor, or M. Gessner G. Angeles Chem. 2002; PubMed Scopus Google a MLK3 inhibitor, NGF The of either GSK-3 or MLK3 NGF withdrawal-induced cell death in PC-12 that protein kinases a significant in trophic factor neuronal cell Furthermore, NGF withdrawal-induced JNK activation was also by and also NGF withdrawal-induced MLK3 activation that was in the of of the although was to inhibit MLK3 activation the MLK3 failed to NGF withdrawal-induced GSK-3β activation that GSK-3β upstream of MLK3. GSK-3β of are two of and GSK-3β. GSK-3β a direct in MLK3 activation, we of GSK-3β-deficient J. Nature. 2000; PubMed Scopus Google Scholar). the expression of GSK-3β and MLK3 were by GSK-3β was in in GSK-3β-deficient MLK3 expression in GSK-3β-deficient was the has been shown that activates GSK-3β kinase M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. Xu J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). activation of GSK-3β by MLK3 kinase activity, GSK-3β-deficient and were to of The MLK3 in was activated by However, in MLK3 was activated upon MLK3 activation was induced by in GSK-3β-deficient the and MLK3 kinase activities were the MLK3 kinase in was that of GSK-3β-deficient the for GSK-3β for MLK3 activation, the GSK-3β-deficient were either or GSK-3β expression The GSK-3β restored MLK3 activation that GSK-3β is for MLK3 GSK-3β and GSK-3 phosphorylation are in the C-terminal of human MLK3 NGF activated MLK3 and GSK-3 we that the activation of MLK3 mediated GSK-3β phosphorylation of MLK3. we for between MLK3 and GSK-3β in PC-12 cells NGF GSK-3β MLK3 in to NGF was blocked by the GSK-3 inhibitor, that GSK-3β MLK3 in a and MLK3. interaction between MLK3 and GSK-3β was in PC-12 between or phosphorylation of of MLK3 was in cells. NGF interaction between MLK3 and GSK-3β in PC-12 cells. The PC-12 cells were or for to NGF MLK3 was and was by The cell were also for MLK3 and GSK-3β. GSK-3β the C-terminal regulatory domain of MLK3. as were either WT, or GSK-3β. MLK3 were and GSK-3β protein was The cell were also and to the expression of GSK-3β and MLK3, GSK-3β the C-terminal regulatory domain of MLK3. MLK3 as were and as In kinase were in the of kinase GSK-3β, either in the or of as The kinase activities of GSK-3β were protein as the The kinase was and then GSK-3β MLK3 in cells were in either or kinase GSK-3β. the cells were and the MLK3 were and to of MLK3 was by The cell were also and to the expression of MLK3 and GSK-3β MLK3 is phosphorylated by NGF withdrawal-induced GSK-3β. PC-12 cells were and either or as for The cells were and NGF was for as in the to The MLK3 was and by The data in of three and the data in and of two the potential GSK-3β phosphorylation on MLK3 are in the C-terminal of the we the interaction between MLK3 and GSK-3β is mediated of MLK3. The N-terminal or the C-terminal of MLK3 were a GSK-3β expression cells and for The MLK3 and C-terminal of MLK3 were to wild GSK-3β, the N-terminal of MLK3 failed to GSK-3β our interaction data indicating that the interaction between C-terminal regulatory domain of MLK3 and GSK-3β is dependent upon the activation of GSK-3β. we GSK-3β the C-terminal of MLK3 The C-terminal MLK3 protein was and in kinase were in of GSK-3β The GSK-3β phosphorylated the C-terminal the N-terminal of the MLK3 which was in of data that GSK-3β has the to phosphorylate MLK3 in occurred in we in cells. that GSK-3β was of MLK3 in phosphorylation of MLK3 by GSK-3β was that MLK3 is in of GSK-3β is also at the cellular by GSK-3β. our in and in phosphorylation data and that GSK-3β is a kinase of MLK3, we the phosphorylation of MLK3 in PC-12 cells NGF The cells were either to inhibit GSK-3β or as a cells were and NGF was for The phosphorylation of MLK3 was The NGF induced increased phosphorylation of MLK3, which was blocked by GSK-3β as a kinase of MLK3 growth factor GSK-3β on C-terminal of three GSK-3β phosphorylation in the C-terminal of MLK3 are and are by GSK-3β, we residues to residue in MLK3 and the of MLK3. were as residue or in as in we their interaction GSK-3β to the interaction GSK-3β. The wild and MLK3 GSK-3β, the between GSK-3β and MLK3 were other MLK3 and are by GSK-3β, the C-terminal of MLK3 or were in The were used in in kinase GSK-3β The C-terminal of wild and MLK3 were phosphorylated by GSK-3β. However, the other MLK3 of phosphorylation wild or C-terminal MLK3 in GSK-3β phosphorylation on MLK3 are also in cells were GSK-3β MLK3, GSK-3β phosphorylation as in In of cells that GSK-3β was to phosphorylate MLK3, phosphorylation of or the of MLK3, was the phosphorylation of MLK3 was other two and that the in GSK-3β phosphorylation on MLK3. GSK-3β JNK Activation via MLK3 has been that GSK-3β activation of JNK and that to M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. T. M.J. Mol. Cell. Biol. PubMed Scopus Google Scholar). and have that MLK3 is a of JNK (5Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, L.A. J. J. 1996; PubMed Scopus Google Scholar). we have that the activation of JNK in to or factor is mediated via MLK3 P. A. G. A. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation of MLK3 by GSK-3β to JNK activation, the GSK-3β phosphorylation of MLK3 or wild MLK3 were wild GSK-3β cells The JNK protein was and in kinase were protein as the of wild MLK3 or GSK-3β activated JNK which was in the of MLK3 and GSK-3β The activation of JNK by GSK-3β was to by or of MLK3 the JNK activation was blocked by the MLK3 two GSK-3β phosphorylation MLK3 to that the of MLK3 by GSK-3β to the activation of The MLK3 also blocked JNK our data and that by GSK-3β. The to and thus is that is by other GSK-3β. the phosphorylation of MLK3 by GSK-3β the activation of MLK3, we also the protein kinase activities of MLK3 The activation of the MLK3 by GSK-3β was wild MLK3 Cell via MLK3 of MLK3 by GSK-3β to neuronal cell death, PC-12 cells were either GSK-3β or MLK3 by we that MLK3 is a downstream of GSK-3β PC-12 cells were either or to cell of MLK3 or GSK-3β induced cell death, and was blocked by that activation of MLK3 by GSK-3β to cell the phosphorylation of MLK3 by NGF withdrawal-induced GSK-3β was for cell our data that on MLK3 the in GSK-3β phosphorylation and that the phosphorylation of MLK3 was phosphorylated by GSK-3β we or wild MLK3 PC-12 cells. The MLK3 NGF withdrawal-induced cell death, the phosphorylation MLK3 was wild MLK3 in NGF withdrawal-induced cell death in PC-12 cells Taken together, that of MLK3 by GSK-3β a significant in neuronal cell death GSK-3 activation has been to neuronal cell death during of 2001; PubMed Scopus Google Scholar, E. K. B. J. PubMed Scopus Google Scholar, K. T. J. Google Scholar). Activation of MLK3 has also been shown to a in neuronal cell in Johnson Rev. PubMed Scopus Google Scholar, A.C. Chem. 2002; PubMed Scopus Google Scholar). functional between GSK-3 and MLK3 in neuronal or cell death pathways has been In we that trophic factor activates GSK-3β, which in residues on the C-terminal regulatory domain of MLK3. phosphorylation of MLK3 to in trophic factor cell death in PC-12 cells. cells in to trophic factor deprivation, heat shock, and other stress NGF neuronal cell death in cell in trophic factor L. T. Rev. Google Scholar). the of death protein GSK-3β, MLK3, and JNK, in NGF withdrawal-induced cell death pathway in the PC-12 cell has been that the NGF neuronal cell death in PC-12 cells via GSK-3β on we the of GSK-3β, MLK3, and JNK activation in PC-12 in to NGF deprivation. The kinase activities of three protein kinases significant activation of NGF the that kinases are a pathway in to NGF a of the in of M. T. Vaught J.L. Angeles J.P. J. Chem. PubMed Scopus Google Scholar). was that the JNK of kinases at A.C. E. Finn J.P. Angeles T. J. PubMed Google Scholar, A.C. Finn J.P. Greene L.A. J. Google Scholar). was that targets the MLK of kinases that upstream in the JNK pathway (7Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gessner G. Xu Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google and neuronal in the of Scott R.W. J. 2000; PubMed Scopus Google Scholar). is also that and its also expression and activation in and MLK downstream JNK activation Johnson J. PubMed Scopus Google Scholar). in human was that was and significant PubMed Google Scholar). used CEP-11004, which is of to the of MLK3 in the cell death pathway. NGF withdrawal-induced cell death in PC-12 was blocked by or by the GSK-3 specific inhibitor, that GSK-3 and MLK3 were to neuronal cell PC-12 cells were the GSK-3 inhibitor, the kinase of MLK3 and cell death and were However, the MLK3 inhibitor, CEP-11004, although preventing cell death, was unable to GSK-3β kinase that MLK3 was downstream of GSK-3β in the trophic factor cell death pathway. was by GSK-3β-deficient UV, of GSK-3β, activate MLK3 in of GSK-3β of GSK-3β restored MLK3 activation our data that MLK3 was downstream of GSK-3β and that the kinases were activated in to NGF deprivation, we that GSK-3β activates MLK3 via of GSK-3 is its GSK-3 phosphorylation by the is amino the upstream as the phosphorylation for GSK-3 S. P. J. 2001; PubMed Scopus Google Scholar). of GSK-3 to phosphorylation for their by GSK-3 S. P. J. 2001; PubMed Scopus Google Scholar). of the MLK3 three GSK-3 phosphorylation in the C-terminal regulatory domain of MLK3 In light of we the MLK3 and GSK-3β in to NGF deprivation, which to the interaction was blocked by a GSK-3 inhibitor, indicating that GSK-3β MLK3 in a protein kinase dependent manner the C-terminal domain of MLK3 the GSK-3β phosphorylation we the C-terminal of MLK3 was a direct for GSK-3β. The GSK-3β phosphorylated the C-terminal of MLK3 are that as a regulatory on cellular as GSK-3 J. Cell PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar). have been to phosphorylated by transcription factors Johnson Full Text Full Text PDF PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar). However, of have the by and S. P. J. 2001; PubMed Scopus Google which are to that a protein is in of GSK-3β. data that MLK3 was a cellular of GSK-3β, the MLK3 was phosphorylated by GSK-3β, GSK-3β failed to phosphorylate MLK3 in were MLK3 was to phosphorylated by GSK-3 in PC-12 upon NGF at of the for MLK3 to as a of GSK-3β. to the that MLK3 is a of GSK-3β, the three and Ser793, were to either or in as in The in phosphorylation the of C-terminal of MLK3 that of three two Ser789 and were by GSK-3β However, the in the of MLK3 GSK-3β were that of the three GSK-3β was the target in and was by GSK-3β phosphorylation of MLK3 was wild MLK3 in is that the which to is a target of has also been as in MLK3 phosphorylation Gallo K.A. 2002; PubMed Scopus Google Scholar). However, the of the kinase for was data that GSK-3β in the phosphorylation of of the phosphorylation at or threonine located residues C-terminal to the of phosphorylation P. S. Nat. Rev. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). of our phosphorylation data also that is phosphorylated by to the Ser789 on MLK3. JNK activation to cell we and have that MLK3 as as other MLKs are of JNK (5Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 6Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 663-672Crossref PubMed Scopus (458) Google Scholar, L.A. J. J. 1996; PubMed Scopus Google Scholar). In GSK-3β also JNK activation M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. Xu J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we for the functional of MLK3 on JNK activation by GSK-3β, we that the phosphorylation of MLK3 by GSK-3β JNK activation, and GSK-3β phosphorylation were in the JNK activation, to the MLK3 was activated by GSK-3β at downstream JNK activation was blocked by is that GSK-3β other MLK family members to downstream JNK we the of other MLK family and and we that family members also either or and GSK-3β phosphorylation on other MLKs are also phosphorylated by GSK-3β is our data that GSK-3β JNK activation via MLK3 phosphorylation of MLK3 Z. A.C. P. Greene L.A. Mol. Cell. Biol. 2001; PubMed Scopus Google or GSK-3β M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google has been shown to neuronal cell we that of MLK3 also cell death in cells P. B. A. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on we phosphorylation of MLK3 by GSK-3β the cell death pathway in PC-12 cells. that of GSK-3β or MLK3 in PC-12 cells caused cell death, which was to cell death GSK-3β and MLK3 were The MLK cell death caused by of GSK-3β or MLK3 our indicating that GSK-3β was upstream of MLK3 in neuronal cell death pathway. we for functional of expression of MLK3 on NGF withdrawal-induced cell death in PC-12 we that the the growth factor withdrawal-induced cell death, the the of growth factor cell death the that GSK-3β neuronal cell death via MLK3 on our data and we that the pathway the cell death pathway via phosphorylation of on MLK3 by P. B. A. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we a of cell death and mediated MLK3 phosphorylation by and GSK-3β has been that NGF the pathway M. Z. J. 2000; PubMed Google Scholar). NGF the activation allow GSK-3β activation via of GSK-3β phosphorylation P. M. Nature. PubMed Scopus Google Scholar, M. P. PubMed Scopus Google Scholar). The and activated GSK-3β then phosphorylate MLK3 on Ser789 and Ser793, leading to activation of MLK3, which then JNK activation and cell In our we have shown GSK-3β activation cell death via JNK is that MLK3 activation cell death via JNK activation Z. A.C. P. Greene L.A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). we that GSK-3β, which is to activate JNK, cell death GSK-3β MLK3 JNK pathway. the growth factors growth factor and are the pathway P. B. A. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Full Text PDF PubMed Scopus Google Scholar, P. J. Z. PubMed Scopus Google Scholar, G. G. S. J. Cell Mol. PubMed Scopus Google Scholar). activated phosphorylate GSK-3β at at the also phosphorylate on MLK3. The direct phosphorylation of GSK-3β and MLK3 by to downstream JNK activation and thus cell survival. In for the we provide direct to the that GSK-3β is a upstream of MLK3 in the growth factor cell death pathway. also provide that MLK3 is a of GSK-3β, MLK3 was phosphorylated by GSK-3β in we have also that in GSK-3β-deficient UV, a of GSK-3β, to activate MLK3. is to that growth factor neurodegenerative diseases via the pathway. are to for

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