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Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β.

Jiang J, Wang ZH, Qu M, Gao D, Liu XP, Zhu LQ, Wang JZ - Sci Rep (2015)

Bottom Line: EphB2 activation inhibits glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase, and activates phosphatidylinositol-3-kinase (PI3K)/Akt both in vitro and in vivo, whereas simultaneous inhibition of PI3K or upregulation of GSK-3β abolishes the EphB2 stimulation-induced tau dephosphorylation.Finally, we confirm that ephrinB1/Fc treatment induces tyrosine phosphorylation (activation) of EphB2, while deletion of the tyrosine kinase domain (VM) of EphB2 eliminates the receptor stimulation-induced GSK-3β inhibition and tau dephosphorylation.Our data provide a novel membranous target to antagonize AD-like tau pathology.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Key Laboratory of Neurological Diseases of Education Ministry of China, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China [2] Department of Oncology, The Central Hospital of Wuhan, 430014, Wuhan, China.

ABSTRACT
Abnormal tau hyperphosphorylation is an early pathological marker of Alzheimer's disease (AD), however, the upstream factors that regulate tau phosphorylation are not illustrated and there is no efficient strategy to arrest tau hyperphosphorylation. Here, we find that activation of endogenous EphB2 receptor by ligand stimulation (ephrinB1/Fc) or by ectopic expression of EphB2 plus the ligand stimulation induces a remarkable tau dephosphorylation at multiple AD-associated sites in SK-N-SH cells and human embryonic kidney cells that stably express human tau (HEK293-tau). In cultured hippocampal neurons and the hippocampus of human tau transgenic mice, dephosphorylation of tau proteins was also detected by stimulation of EphB2 receptor. EphB2 activation inhibits glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase, and activates phosphatidylinositol-3-kinase (PI3K)/Akt both in vitro and in vivo, whereas simultaneous inhibition of PI3K or upregulation of GSK-3β abolishes the EphB2 stimulation-induced tau dephosphorylation. Finally, we confirm that ephrinB1/Fc treatment induces tyrosine phosphorylation (activation) of EphB2, while deletion of the tyrosine kinase domain (VM) of EphB2 eliminates the receptor stimulation-induced GSK-3β inhibition and tau dephosphorylation. We conclude that activation of EphB2 receptor kinase arrests tau hyperphosphorylation through PI3K-/Akt-mediated GSK-3β inhibition. Our data provide a novel membranous target to antagonize AD-like tau pathology.

No MeSH data available.


Related in: MedlinePlus

EphrinB1/Fc stimulates tyrosine phosphorylation of EphB2 and deletion of the kinase domain (VM) but not the PDZ binding domain eliminates EphB2-induced tau dephosphorylation.HEK293-tau cells were treated with ephrinB1/Fc for 45 min, and then the phosphorylation level of EphB2 was measured by immunoprecipitation with anti-EphB2 antibody and Western blotting probed by PY99 and anti-EphB2 antibody (a, b). HEK293-tau cells were transfected with deletion of PDZ (c, d) or VM (e, f) domain of EphB2 or the control Vector for 24 h and then treated with ephrinB1/Fc for 45 min. The phosphorylation levels of tau at Thr231 and Ser396, or GSK-3β at Ser9, or PI3K at Tyr458/Tyr199 were measured by Western blotting. The phosphorylation levels of tau and the kinases were normalized against the total levels. Data were representative of at least three independent experiments from 9 ~ 12 culture wells for each group and presented as the mean ± SD. *P < 0.05, **P <0.01 versus Vector.
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f6: EphrinB1/Fc stimulates tyrosine phosphorylation of EphB2 and deletion of the kinase domain (VM) but not the PDZ binding domain eliminates EphB2-induced tau dephosphorylation.HEK293-tau cells were treated with ephrinB1/Fc for 45 min, and then the phosphorylation level of EphB2 was measured by immunoprecipitation with anti-EphB2 antibody and Western blotting probed by PY99 and anti-EphB2 antibody (a, b). HEK293-tau cells were transfected with deletion of PDZ (c, d) or VM (e, f) domain of EphB2 or the control Vector for 24 h and then treated with ephrinB1/Fc for 45 min. The phosphorylation levels of tau at Thr231 and Ser396, or GSK-3β at Ser9, or PI3K at Tyr458/Tyr199 were measured by Western blotting. The phosphorylation levels of tau and the kinases were normalized against the total levels. Data were representative of at least three independent experiments from 9 ~ 12 culture wells for each group and presented as the mean ± SD. *P < 0.05, **P <0.01 versus Vector.

Mentions: The activity of tyrosine kinases is stimulated by autophosphorylation within the kinase domain3233. To confirm the receptor activation, we measured the tyrosine phosphorylation by EphB2 immunoprecipitation and P-Tyr-99 blotting in SK-N-SH cells after treatment of ephrinB1/Fc for 45 min. The results showed that ephrinB1/Fc treatment increased remarkably the phosphorylation level of EphB2 (Fig. 6a,b), indicating the kinase activation. Both the kinase domain (VM) and PDZ binding domain are important functional regions of EphB2 receptor. To further confirm the role of EphB2 kinase domain and possible involvement of PDZ in regulating PI3K/ GSK-3β pathway and tau dephosphorylation, we transfected EphB2 VM or PDZ deletion mutant plasmids into the HEK293-tau cells and treated the cells with ephrinB1/Fc for 45 min. We found that deletion of PDZ domain did not affect the EphB2 stimulation-induced phosphorylation levels of PI3K/GSK-3β and tau proteins (Fig. 6c,d), whereas deletion of VM kinase domain almost abolished the EphB2 activation-induced alterations of PI3K/GSK-3β and tau phosphorylation (Fig. 6e,f). These data suggest that the tyrosine phosphorylation and the kinase domain (VM) of EphB2 play a key role in coupling PI3K/GSK-3β pathway with the reduced tau phosphorylation.


Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β.

Jiang J, Wang ZH, Qu M, Gao D, Liu XP, Zhu LQ, Wang JZ - Sci Rep (2015)

EphrinB1/Fc stimulates tyrosine phosphorylation of EphB2 and deletion of the kinase domain (VM) but not the PDZ binding domain eliminates EphB2-induced tau dephosphorylation.HEK293-tau cells were treated with ephrinB1/Fc for 45 min, and then the phosphorylation level of EphB2 was measured by immunoprecipitation with anti-EphB2 antibody and Western blotting probed by PY99 and anti-EphB2 antibody (a, b). HEK293-tau cells were transfected with deletion of PDZ (c, d) or VM (e, f) domain of EphB2 or the control Vector for 24 h and then treated with ephrinB1/Fc for 45 min. The phosphorylation levels of tau at Thr231 and Ser396, or GSK-3β at Ser9, or PI3K at Tyr458/Tyr199 were measured by Western blotting. The phosphorylation levels of tau and the kinases were normalized against the total levels. Data were representative of at least three independent experiments from 9 ~ 12 culture wells for each group and presented as the mean ± SD. *P < 0.05, **P <0.01 versus Vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: EphrinB1/Fc stimulates tyrosine phosphorylation of EphB2 and deletion of the kinase domain (VM) but not the PDZ binding domain eliminates EphB2-induced tau dephosphorylation.HEK293-tau cells were treated with ephrinB1/Fc for 45 min, and then the phosphorylation level of EphB2 was measured by immunoprecipitation with anti-EphB2 antibody and Western blotting probed by PY99 and anti-EphB2 antibody (a, b). HEK293-tau cells were transfected with deletion of PDZ (c, d) or VM (e, f) domain of EphB2 or the control Vector for 24 h and then treated with ephrinB1/Fc for 45 min. The phosphorylation levels of tau at Thr231 and Ser396, or GSK-3β at Ser9, or PI3K at Tyr458/Tyr199 were measured by Western blotting. The phosphorylation levels of tau and the kinases were normalized against the total levels. Data were representative of at least three independent experiments from 9 ~ 12 culture wells for each group and presented as the mean ± SD. *P < 0.05, **P <0.01 versus Vector.
Mentions: The activity of tyrosine kinases is stimulated by autophosphorylation within the kinase domain3233. To confirm the receptor activation, we measured the tyrosine phosphorylation by EphB2 immunoprecipitation and P-Tyr-99 blotting in SK-N-SH cells after treatment of ephrinB1/Fc for 45 min. The results showed that ephrinB1/Fc treatment increased remarkably the phosphorylation level of EphB2 (Fig. 6a,b), indicating the kinase activation. Both the kinase domain (VM) and PDZ binding domain are important functional regions of EphB2 receptor. To further confirm the role of EphB2 kinase domain and possible involvement of PDZ in regulating PI3K/ GSK-3β pathway and tau dephosphorylation, we transfected EphB2 VM or PDZ deletion mutant plasmids into the HEK293-tau cells and treated the cells with ephrinB1/Fc for 45 min. We found that deletion of PDZ domain did not affect the EphB2 stimulation-induced phosphorylation levels of PI3K/GSK-3β and tau proteins (Fig. 6c,d), whereas deletion of VM kinase domain almost abolished the EphB2 activation-induced alterations of PI3K/GSK-3β and tau phosphorylation (Fig. 6e,f). These data suggest that the tyrosine phosphorylation and the kinase domain (VM) of EphB2 play a key role in coupling PI3K/GSK-3β pathway with the reduced tau phosphorylation.

Bottom Line: EphB2 activation inhibits glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase, and activates phosphatidylinositol-3-kinase (PI3K)/Akt both in vitro and in vivo, whereas simultaneous inhibition of PI3K or upregulation of GSK-3β abolishes the EphB2 stimulation-induced tau dephosphorylation.Finally, we confirm that ephrinB1/Fc treatment induces tyrosine phosphorylation (activation) of EphB2, while deletion of the tyrosine kinase domain (VM) of EphB2 eliminates the receptor stimulation-induced GSK-3β inhibition and tau dephosphorylation.Our data provide a novel membranous target to antagonize AD-like tau pathology.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Key Laboratory of Neurological Diseases of Education Ministry of China, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China [2] Department of Oncology, The Central Hospital of Wuhan, 430014, Wuhan, China.

ABSTRACT
Abnormal tau hyperphosphorylation is an early pathological marker of Alzheimer's disease (AD), however, the upstream factors that regulate tau phosphorylation are not illustrated and there is no efficient strategy to arrest tau hyperphosphorylation. Here, we find that activation of endogenous EphB2 receptor by ligand stimulation (ephrinB1/Fc) or by ectopic expression of EphB2 plus the ligand stimulation induces a remarkable tau dephosphorylation at multiple AD-associated sites in SK-N-SH cells and human embryonic kidney cells that stably express human tau (HEK293-tau). In cultured hippocampal neurons and the hippocampus of human tau transgenic mice, dephosphorylation of tau proteins was also detected by stimulation of EphB2 receptor. EphB2 activation inhibits glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase, and activates phosphatidylinositol-3-kinase (PI3K)/Akt both in vitro and in vivo, whereas simultaneous inhibition of PI3K or upregulation of GSK-3β abolishes the EphB2 stimulation-induced tau dephosphorylation. Finally, we confirm that ephrinB1/Fc treatment induces tyrosine phosphorylation (activation) of EphB2, while deletion of the tyrosine kinase domain (VM) of EphB2 eliminates the receptor stimulation-induced GSK-3β inhibition and tau dephosphorylation. We conclude that activation of EphB2 receptor kinase arrests tau hyperphosphorylation through PI3K-/Akt-mediated GSK-3β inhibition. Our data provide a novel membranous target to antagonize AD-like tau pathology.

No MeSH data available.


Related in: MedlinePlus