Membrane depolarization induces the undulating phosphorylation/dephosphorylation of glycogen synthase kinase 3beta, and this dephosphorylation involves protein phosphatases 2A and 2B in SH-SY5Y human neuroblastoma cells.
Changes in plasma membrane electrical potential evoke signals that regulate the expressions of various genes in the nervous system. However, the role of glycogen synthase kinase 3beta (GSK-3beta) in this process has not been elucidated. Thus, this study was performed to examine whether membrane depolarization can regulate the phosphorylation of GSK-3beta and to identify the molecular mechanisms involved in this regulation. The depolarization by treating with 100 mm KCl for 5 min resulted in the undulating phosphorylation of GSK-3beta at Ser-9 in SH-SY5Y human neuroblastoma cells, in H19 -7/IGF-IR rat embryonic hippocampal cells, and in PC12 rat pheochromocytoma cells, but not in A172 human glioblastoma cells. Cellular beta-catenin contents showed a temporal pattern similar to that of the Ser-9 phosphorylation of GSK-3beta. Treatment with wortmannin or calphostin C or the expression of dominant negative Akt inhibited phosphorylation of GSK-3beta at Ser-9 following the KCl-induced depolarization of SH-SY5Y cells. Moreover, pretreatment with okadaic acid or cyclosporin A blocked the dephosphorylation of GSK-3beta at Ser-9 at 0, 15, and 30 min after KCl-induced depolarization, and the activity of protein phosphatases (PP) 2A and 2B increased at these times. Treatment with nifedipine or calcium-free medium inhibited GSK-3beta dephosphorylation following membrane depolarization, and the amounts of co-immunoprecipitated GSK-3beta and PP2A changed in parallel with GSK-3beta dephosphorylation. Our study demonstrated that KCl-induced depolarization caused undulating GSK-3beta phosphorylation/dephosphorylation, which was regulated for the most part by phosphatidylinositol 3-kinase and Akt (phosphorylation) and PP2A and PP2B (dephosphorylation), respectively.
Lee
Yun-Il
YI
Department of Biochemistry and Department of Psychiatry, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea.
Seo
MiRan
M
Kim
Yeni
Y
Kim
So-Young
SY
Kang
Ung Gu
UG
Kim
Yong-Sik
YS
Juhnn
Yong-Sung
YS
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D019703:Calcineurin / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005909:Glioblastoma / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D009281:Naphthalenes / Q000494:pharmacology; D016716:PC12 Cells; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D013912:Threonine; D012694:Serine; D011189:Potassium Chloride; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; C058819:calphostin C; D002118:Calcium; D000077191:Wortmannin