GSK3 beta N-terminus binding to p53 promotes its acetylation

Abstract

The prevalence in human cancers of mutations in p53 exemplifies its crucial role as a tumor suppressor transcription factor. Previous studies have shown that the constitutively active serine/threonine kinase glycogen synthase kinase-3beta (GSK3 beta) associates with the C-terminal basic domain of p53 and regulates its actions. In this study we identified the GSK3 beta N-terminal amino acids 78-92 as necessary for its association with p53. Inhibitors of GSK3 impaired the acetylation of p53 at Lys373 and Lys382 near the GSK3 beta binding region in p53, indicating that GSK3 beta facilitates p53 acetylation. We also found that acetylation of p53 reduced its association with GSK3 beta, as well as with GSK3alpha. These results indicate that the N-terminal region of GSK3 beta binds p53, this association promotes the acetylation of p53, and subsequently acetylated p53 dissociates from GSK3.

Figure 1

Nuclear localization of expressed GSK3β constructs. p53-null human lung carcinoma H1299 cells that express inducible wild-type HA-tagged p53 were transiently transfected with wild-type GSK3β-NLS-myc (1–420) or the indicated mutants of GSK3β-NLS-myc. Constructs generated by ligating rat GSK3β cDNA into the pShooter vector pCMV/myc/nuc (Invitrogen) were expressed using FuGENE 6 (Roche). NLS-myc vector (myc) was used as a negative control. After 24 hr, expression was examined by immunoblotting and immunostaining with anti-myc-tag (Cell Signaling) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). Nuclei were labeled with 1 μg/ml bisbenzimide (Bis; blue). The expression level of each mutant construct was similar to wild-type GSK3β-NLS-myc and all GSK3β-NLS-myc constructs (green) were expressed in the nucleus. 100× magnification.

Figure 2

Identification of the GSK3β domain required for association with p53. Schematic diagrams (left) depict GSK3β constructs (A) sequentially truncated from the C-terminal, (B) sequentially truncated from the N-terminal, and (C) deletion constructs. GSK3β constructs were transiently transfected into H1299 cells and HA-p53 was inducibly expressed by removing doxycycline from the medium for 24 hr [9]. Cells were subjected to lysis, immunoprecipitation using sheep anti-mouse IgG Dynabeads (Dynal Biotech) and 1 μg anti-HA (Covance) and immunoblotting as indicated. The heavy chain (HC) and light chain (LC) of IgG are indicated, and β-actin was used as loading control.

Figure 3

Diagram of the GSK3β region required for association with p53. (A) The sequence of GSK3β shows the region required for association with p53, consisting of amino acids 78–92, that is located close to the N-terminus of GSK3β. The molecular rendering of a monomer of GSK3β was generated using the Cn3D software from the NCBI based on the protein data bank file 1O9U , and the p53 binding domain of GSK3β was depicted as yellow. (B) H1299 cells were transiently transfected to express wild-type GSK3β-NLS-myc (1–420 amino acids; WT) or kinase-dead GSK3β-NLS-myc (K85A/K86A), and HA-p53 was inducibly expressed, for 24 hr. Cells were subjected to lysis and anti-HA was used to immunoprecipitate p53, followed by immunoblotting with anti-myc antibody to detect GSK3β-NLS-myc. The levels of expressed HA-p53, NLS-myc-GSK3β, and β-actin were determined by immunoblotting.

Figure 4

GSK3 regulates p53 acetylation which regulates association with GSK3β. (A) SH-SY5Y cells were preincubated with 1 μM TSA and 5 mM nicotinamide (T/N) for 2 hr and treated with 1 μM camptothecin (CA) for 3 hr. Total p53 was immunoprecipitated followed by immunoblotting with antibodies specific for acetylated Lys373 (Ac-K373-p53), Lys382 (Ac-K382-p53), Lys320 (Ac-K320-p53) (Trevigen), total p53 (Santa Cruz), and β-actin (Sigma). (B) Quantitative analysis of the percentage of acetylated p53. SH-SY5Y cells were preincubated with 1 μM TSA and 5 mM nicotinamide for 2 hr, with GSK3 inhibitors, 20 mM lithium (Li), 10 μM SB216763 (SB), or 10 μM CHIR99021 (CHIR), for 30 min, and treated with 1 μM camptothecin for 3 hr. Quantitative values are means ± S.E.; n = 3. *p < 0.05, ANOVA with Dunnett's post hoc multiple comparisons test. (C) SH-SY5Y cells were preincubated with 1 μM TSA and 5 mM nicotinamide (T/N) for 2 hr and treated with 1 μM camptothecin (CA) for 3 hr. The association of GSK3α/β with p53 was assessed by immunoprecipitation of GSK3α or GSK3β followed by immunoblotting total p53. The endogenous total p53, total GSK3α/β, acetylated p53 (Ac-K373-p53, Ac-K382-p53, Ac-K320-p53), and β-actin levels were determined by immunoblotting.