Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event.
Phosphorylation of the endogenous GSK3alpha (glycogen synthase kinase-3alpha) at Tyr279 and GSK3beta at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3beta transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3beta expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216 in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3beta was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3beta lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.
MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, Scotland, UK.
Adam
A
Cole
Sheelagh
S
Frame
Philip
P
Cohen
D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D009154:Mutation; D020932:Nerve Growth Factor / Q000502:physiology; D010766:Phosphorylation; D019311:Staurosporine / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D004791:Enzyme Inhibitors; D014443:Tyrosine; D020932:Nerve Growth Factor; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D019311:Staurosporine