Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event

Abstract

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.

Regulation and Function of Glycogen Synthase Kinase-3 Isoforms in Neuronal Survival

“…Moreover, tyrosine phosphorylation of GSK-3␣ and GSK-3␤ in cortical neurons was decreased after treatment with either GSK-3␤ inhibitor I or VII for 1 day or longer. Our data are supported by a previous study demonstrating that GSK-3␤ is capable of catalyzing the autophosphorylation of Tyr 216 in vitro (26) and suggest that the phosphorylation of Tyr 279 / 216 in cells is also catalyzed by GSK-3. However, our results are in variance with other previous reports that GSK-3␤ Tyr 216 phosphorylation is not autoregulated following GSK-3 inhibition (21,25).…”

“…Although some evidence supports active regulation of tyrosine phosphorylation in the brain (21), the vast majority of research on GSK-3 has been focused on the aspect of inhibitory serine phosphorylation, and little is known about possible kinases involved in GSK-3 activation through tyrosine phosphorylation. In addition, previous studies have led to conflicting conclusions as to whether the tyrosine phosphorylation of GSK-3 is catalyzed by GSK-3 itself or by a distinct tyrosine kinase (25)(26)(27)(28)(29)(30).…”

Blockade of Tau Hyperphosphorylation and Aβ1–42 Generation by the Aminotetrahydrofuran Derivative ANAVEX2-73, a Mixed Muscarinic and σ1 Receptor Agonist, in a Nontransgenic Mouse Model of Alzheimer’s Disease

“…GSK-3b is the main isoform involved in AD pathology. It must be outlined that inhibition of GSK-3b has more influence on activity than activation, as the enzyme is constitutively active and activation sites are Blockade of Tau hyperphosphorylation and Ab 1-42 by ANAVEX2-73 V Lahmy et al subjected to autophosphorylation (Cole et al, 2004). One of the most characterized regulation pathways for GSK-3b is through protein kinase B (Akt) activation.…”

An evolutionary perspective on the kinome of malaria parasites

“…The strongest evidence for tyrosine phosphorylation of P. falciparum proteins was in the activation loop of two protein kinases: PfGSK3 (PFC0525c) and PfCLK3 (PF11_0156) [19]. Phosphorylation of PfGSK3 occurred at Y229, analogous to Y279 and Y216 on mammalian GSK3a/b, the activation loop auto-phosphorylation sites necessary for enzymatic activity [59,60]. The tyrosine phosphorylation of the CDK-like kinase PfCLK3 appears to follow similar lines: this kinase is a serine/threonine kinase related to the pre-mRNA-processing kinase hPRP4 found in higher Figure 3.…”