Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation

Abstract

Glycogen synthase kinase 3beta (GSK3beta) is involved in metabolism, neurodegeneration, and cancer. Inhibition of GSK3beta activity is the primary mechanism that regulates this widely expressed active kinase. Although the protein kinase Akt inhibits GSK3beta by phosphorylation at the N terminus, preventing Akt-mediated phosphorylation does not affect the cell-survival pathway activated through the GSK3beta substrate beta-catenin. Here, we show that p38 mitogen-activated protein kinase (MAPK) also inactivates GSK3beta by direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation of beta-catenin. p38 MAPK-mediated phosphorylation of GSK3beta occurs primarily in the brain and thymocytes. Activation of beta-catenin-mediated signaling through GSK3beta inhibition provides a potential mechanism for p38 MAPK-mediated survival in specific tissues.

Fig. 1

Regulation of the β-catenin pathway by p38 MAPK. (A) Western blot showing c-Myc and Lef in whole-cell extracts from Rag^-/- thymocytes (Rag^-/-) and MKK6 thymocytes (MKK6). Actin was examined as a control. (B) Western blot showing c-Myc and Lef in thymocytes from Rag^-/-, MKK6, and Rag^-/-/MKK6 mice. (C) Western blot showing β-catenin in nuclear extracts from Rag^-/- and MKK6 thymocytes. (D) Western blot showing β-catenin and p38 MAPK in whole-cell extracts from 293T cells transfected with GSK3β (-) or GSK3β with p38 MAPK and MKK6 (p38/MKK6).

Fig. 2

Direct phosphorylation of GSK3β by p38 MAPK. (A) Western blot showing phospho-Ser⁹ GSK3β (P-S⁹) and total GSK3β in Rag^-/- and MKK6 thymocytes. (B) Western blot showing P-Ser⁹ GSK3β, GSK3β, phospho-p38 MAPK (P-p38), and p38 MAPK in 293T cells transfected with an empty vector (Con), MKK6 alone, or MKK6 and p38 MAPK (MKK6/p38). (C) In vitro p38 MAPK assay with inactive recombinant GSK3β as the substrate and p38 MAPK immunoprecipitated from MKK6-thymocytes (MKK6 Thy) or MKK6-transfected 293T cells (293T). In vitro reactions were incubated in the presence (SB) or absence (-) of the specific p38 MAPK inhibitor SB203580. Total GSK3β was visualized by PonceauS staining, and phosphorylated GSK3β was detected by autoradiography. (D) In vitro p38 MAPK kinase assay as described in (C), using total or Akt-depleted extracts (Akt-dep) from MKK6 thymocytes (MKK6 Thy) or MKK6-transfected 293T cells (293T). (E) In vitro kinase assay as described in (C), with recombinant active p38 MAPK kinase. (F) Western blot showing GSK3β and p38 MAPK in the GSK3β and p21 (Con) immunoprecipitates (IP) and whole-cell extracts from MKK6 thymocytes (Input). (G) In vitro kinase assay for recombinant active p38 MAPK kinase using catalytically inactive GSK3α as a substrate. Phosphorylation of activating transcription factor 2 (ATF2) was examined as a positive control.

Fig. 3

Inhibition of GSK3β by p38 MAPK is mediated by phosphorylation at Thr³⁹⁰. (A) In vitro kinase assays for recombinant p38 MAPK with catalytically inactive GSK3β and GSK3β-T⁴³A mutantas substrates. (B) In vitro kinase assay for recombinant p38 MAPK with kinase-inactive GSK3β (WT), GSK3β-T⁴³A, GSK3β-T³⁹⁰A, and GSK3β-T⁴³A/T³⁹⁰A mutants as substrates. (C) In vitro kinase assay for recombinant active ERK with catalytically inactive GSK3β, GSK3β-T⁴³A, GSK3β-T³⁹⁰A, and GSK3β-T⁴³A/T³⁹⁰A mutants as substrates. (D) In vitro kinase assay for active GSK3β, GSK3β-T⁴³A, and GSK3β-T³⁹⁰A mutants before (Con) or after incubation with activated Akt or activated p38 MAPK. GSK3β activity relative to the activity without Akt or p38 MAPK (Con) is shown. Error bars represent SD (n =3 replicates). (E) In vitro GSK3β kinase reactions alone (-) or in the presence of unphosphorylated-Thr³⁹⁰ (Thr³⁹⁰), phospho-Ser⁹ (P-Ser⁹), or phospho-Thr³⁹⁰ (P-Thr³⁹⁰) peptides, as described in (D). Error bars represent SD. (F) GSK3β in vitro kinase assays as in (D), using various concentrations of phospho-Thr³⁹⁰, phospho-Ser⁹, and unphosphorylated-Thr³⁹⁰ peptides. Each point is the average of two measurements. GSM, modified glycogen synthase peptide.

Fig. 4

Phosphorylation of endogenous GSK3β by p38 MAPK. (A) Western blot showing the presence of endogenous phospho-Ser³⁸⁹ GSK3β (P-Ser³⁸⁹) in WT, GSK3α^-/-, and GSK3β^-/- ES cells. Total GSK3α, GSK3β, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined as controls. (B) Western blot showing P-Ser³⁸⁹ in GSK3β^-/- ES cells transfected with WT GSK3β or GSK3β-S³⁸⁹A mutant alone or with p38 MAPK and MKK6 (p38/MKK6). (C) Western blot showing P-Ser³⁸⁹, Flag-tagged mouse GSK3β, and β-catenin in nontransfected 293T cells (-) or cells transfected with mouse GSK3β alone or in combination with p38 MAPK and MKK6 (p38/MKK6). (D) P-Ser³⁸⁹, total GSK3β, and β-catenin in 293T cells transfected with GSK3β, p38, and MKK6 in the absence (-) or presence of SB203580. (E) P-Ser³⁸⁹ and total GSK3β in MEF or total GSK3α and GSK3β in ES cells nontreated (-) or treated with SB203580. (F) P-Ser³⁸⁹, P-Ser⁹, total GSK3β, P-p38, total p38, and β-catenin in WT and MKK3^-/-MKK6^-/- (3/6^-/-) MEF. (G) The tissue distribution of P-Ser³⁸⁹, P-Ser⁹, and total GSK3β. Quantification of the levels of P-Ser³⁸⁹ relative to P-Ser⁹ in each tissue is also shown (lower panel). Thy, thymocytes; Spl, spleen cells. (H) P-Ser³⁸⁹ and total GSK3β in thymocytes and brain from WT mice treated in vivo with vehicle (-) or SB203580 (SB). (I) P-S³⁸⁹ and total GSK3β in thymocytes from Rag^-/- and MKK6 transgenic mice.