Casein kinase 1α regulates an MDMX intramolecular interaction to stimulate p53 binding

Abstract

MDMX is an important regulator of p53 during embryonic development and malignant transformation. Previous studies showed that casein kinase 1α (CK1α) stably associates with MDMX, stimulates MDMX-p53 binding, and cooperates with MDMX to inactivate p53. However, the mechanism by which CK1α stimulates MDMX-p53 interaction remains unknown. Here, we present evidence that p53 binding by the MDMX N-terminal domain is inhibited by the central acidic region through an intramolecular interaction that competes for the p53 binding pocket. CK1α binding to the MDMX central domain and phosphorylation of S289 disrupts the intramolecular interaction, allowing the N terminus to bind p53 with increased affinity. After DNA damage, the MDMX-CK1α complex is disrupted by Chk2-mediated phosphorylation of MDMX at S367, leading to reduced MDMX-p53 binding. Therefore, CK1α is an important functional partner of MDMX. DNA damage activates p53 in part by disrupting CK1α-MDMX interaction and reducing MDMX-p53 binding affinity.

Fig 1

CK1α promotes MDMX-p53 binding in vivo. (a) H1299 cells transfected as indicated were immunoprecipitated with MDMX antibody, and coprecipitated p53 and CK1α were detected by Western blotting (WB). (b) H1299 cells were transfected with MDMX and CK1α. MDMX was immunoprecipitated and probed with anti-phospho-S289 antibody. Phosphatase (CIP) treatment was performed on immunoprecipitated MDMX prior to pS289 Western blotting. (c) H1299 cells were transfected as indicated, and p21 expression was detected by Western blotting. (d) CK1α was knocked down by siRNAs in HCT116 cells, and p53-MDMX binding was determined by p53 IP and Western blotting for MDMX. (e) The ability of different CK1 isoforms to regulate MDMX-p53 binding was compared using the assay described for panel a. WCE, whole-cell extract.

Fig 2

CK1α promotes MDMX-p53 binding in vitro. (a) ELISA plates coated with His₆-p53 were incubated with H1299 lysate expressing the MDMX wild type and C306S and S289A mutants in the presence or absence of CK1α. The amount of captured MDMX was detected using MDMX antibody. (b) Western blot confirmation of equalized MDMX levels in lysate used for ELISA. (c) Gal4–p53-1-82 was cotransfected with MDMX and CK1α into H1299, and the binding between MDMX and Gal4–p53-1-82 was detected by MDMX IP and p53 Western blotting. Control IP contained MDMX antibody but no cell extract. The antibody background band is marked by an asterisk.

Fig 3

CK1α stimulates p53 binding by MDM2-MDMX chimera. (a) H1299 cells transfected with p53, FLAG-tagged MDM2-MDMX hybrid constructs, and CK1α were immunoprecipitated with M2 beads followed by Western blotting for coprecipitated p53 and CK1α. (b) Diagram of MDM2-MDMX chimeric constructs.

Fig 4

CK1α disrupts MDMX N-terminal-central domain interaction. (a) PONDR analysis of MDMX amino acid sequence. Segments with low PONDR scores (below 0.5) are predicted to be stably folded, and high scores (near 1) indicate disorder. (b) For the top panel, glutathione-agarose beads loaded with the indicated GST-MDMX fusion proteins were incubated with Myc-tagged MDMX-100-361 expressed in H1299. The captured MDMX-100-361 was detected by Myc Western blotting. The lower panel shows verification of purified GST fusion proteins by Coomassie blue staining. (c) Glutathione beads loaded with GST-MDMX or GST-MDM2 N terminus were incubated with H1299 lysate transfected with Myc–MDMX-100-361 and CK1α. The captured MDMX-100-361 was detected by Myc Western blotting. (d) GST–MDMX-1-120 pulldown of MDMX-100-361 was inhibited by wild-type CK1α but not kinase-deficient CK1α-K46D mutant. (e) Introduction of S289A mutation into MDMX-100-361 abrogates the response to CK1α in the GST–MDMX-1-120 pulldown assay.

Fig 5

MDMX central domain inhibits interaction with p53. (a and b) Glutathione-agarose beads loaded with GST–p53-1-82 were incubated with similar amounts of MDMX truncation mutants (a) and internal deletion mutants (b) expressed in H1299, and the captured MDMX was detected by Western blotting. The position of each mutant MDMX protein of interest is marked by an arrow head. (c) Diagram of MDMX mutants and summary of p53 binding activity. (d) The ability of MDMX internal deletion mutants to inhibit p53 transactivation function was analyzed by a reporter assay after cotransfection with p53 and p53-responsive luciferase reporter BP100-luc into p53/MDMX double null mouse embryo fibroblasts (41.4 cells).

Fig 6

MDMX central domain inhibits N-terminal interaction with p53. (a) MDMX-1-120 and MDMX-1-361 fragments were analyzed for binding to p53 peptide using isothermal titration calorimetry (ITC). Dissociation constants (K_d), stoichiometry (N), and changes in enthalpy (ΔH) and entropy (ΔS) for peptide binding are summarized in the table. The asterisk indicates that due to the weak binding of MDMX-1-361, the best fit for K_d determination was achieved by fixing the N value to 1.0. (b) Glutathione beads loaded with GST–MDMX-1-120 were incubated with H1299 lysate expressing Myc-tagged MDMX-100-361 in the presence of pDI or pDI-3A peptides. The captured MDMX-100-361 protein was detected by Western blotting. (c) MDMX and Myc-CK1α were transfected separately into H1299 cells. MDMX lysate was preincubated with 5 μM pDI and pDI-3A peptides, followed by coincubation with Myc-CK1α lysate. In vitro MDMX-CK1α complex formation was analyzed by MDMX IP-Myc Western blotting.

Fig 7

Phosphorylation of S367 by Chk2 regulates MDMX-CK1α interaction. (a) Wild-type and Chk2-null HCT116 cells were treated with 10 Gy IR in the presence of MG132 (to normalize protein levels). MDMX-CK1α interaction was determined by MDMX IP followed by Western blotting for CK1α. The input showed equal expression levels of each protein. con, control. (b) HCT116 cells were treated with 10 Gy IR in the presence of MG132, and MDMX immunoprecipitated by MDMX antibody or CK1α antibody (loading was adjusted to obtain similar levels of total MDMX) was probed using S367 phosphorylation-specific antibody (pS367) or MDMX polyclonal antibody. (c) U2OS cells stably expressing MDMX and MDMX-S367A were treated with 10 Gy IR and cell extracts were prepared after 2 h and mixed with H1299 extract expressing Myc-CK1α. De novo formation of MDMX-CK1α complex was detected by MDMX IP and Myc Western blotting.

Fig 8

S289 is required to mediate the effect of S367 phosphorylation. (a) U2OS cells stably expressing wild-type MDMX or the MDMX-S367A mutant were treated with 10 Gy IR and analyzed for MDMX-CK1α coprecipitation and S289 phosphorylation after 4 h. (b) H1299 cells were transiently transfected with MDMX mutants, CK1α, and p53. MDMX-p53 binding was determined by IP-Western blotting. (c) U2OS cells stably expressing wild-type MDMX, the S367A mutant, or the S289A/S367A double mutant were treated with 10 Gy IR and analyzed for p21 induction after 4 h.

Fig 9

DNA damage inhibits MDMX-p53 binding. (a) HCT116 cells were treated with 10 Gy IR in the presence of MG132 (to normalize protein levels). MDMX-p53 binding was determined by p53 IP using Pab1801 and MDMX Western blotting using a polyclonal antibody. Western blots of whole-cell extract (WCE) show the expression levels of each protein. (b and c) Glutathione-agarose beads loaded with GST-p53 or GST-MDM2 were incubated with extract of control and irradiated HCT116 cells (treated with MG132). The captured MDMX was analyzed by gradient gel SDS-PAGE and MDMX Western blotting. GST-p53 selectively captured nonphosphorylated MDMX (b), and GST-MDM2 specifically captured phosphorylated MDMX (c). (d) A model of MDMX intramolecular autoinhibition and regulation by DNA damage. The central acidic region of MDMX interacts with the N-terminal domain through p53 mimicry, thus reducing p53 binding affinity. CK1α interaction with the central region and phosphorylation of S289 disrupts the intramolecular interaction, allowing the N-terminal domain to bind p53 and inhibit p53 transcriptional function. After DNA damage, phosphorylation of S367 by Chk2 inhibits CK1α-MDMX interaction, thus releasing p53 to activate transcription.