Protein L-isoaspartyl methyltransferase regulates p53 activity

Abstract

Protein methylation plays important roles in most, if not all, cellular processes. Lysine and arginine methyltransferases are known to regulate the function of histones and non-histone proteins through the methylation of specific sites. However, the role of the carboxyl-methyltransferase protein L-isoaspartyl methyltransferase (PIMT) in the regulation of protein functions is relatively less understood. Here we show that PIMT negatively regulates the tumour suppressor protein p53 by reducing p53 protein levels, thereby suppressing the p53-mediated transcription of target genes. In addition, PIMT depletion upregulates the proapoptotic and checkpoint activation functions of p53. Moreover, PIMT destabilizes p53 by enhancing the p53-HDM2 interaction. These PIMT effects on p53 stability and activity are attributed to the PIMT-mediated methylation of p53 at isoaspartate residues 29 and 30. Our study provides new insight into the molecular mechanisms by which PIMT suppresses the p53 activity through carboxyl methylation, and suggests a therapeutic target for cancers.

Figure 1. PIMT regulates p53 protein levels.

(a) A significant association of PIMT expression in human lung (n=442) and breast cancer (n=404) was found with the overall survival (OS) or recurrence-free survival (RFS). The data are represented as the mean±s.e.m. (b) Immunoblot analysis of cell lysates from seven different cell lines (BJ: foreskin fibroblast; U2OS: osteosarcoma; SH-SY5Y: neuroblastoma; NTERA2: embryonal carcinoma; HCT116: colorectal carcinoma; HeLa: cervical adenocarcinoma; NCCIT: embryonal carcinoma). (c) Immunoblot analysis of cell lysates from U2OS cells transfected with PIMT siRNA or control siRNA for the indicated times. (d) Immunoblot analysis of cell lysates from U2OS cells transfected with three different PIMT shRNA constructs (H2, H3 and H4) or scramble shRNA (SC). (e) Immunoblot analysis of cell lysates from several cancer cell lines transfected with PIMT siRNA or control siRNA. (f) Immunoblot analysis of cell lysates from U2OS cells transfected with the indicated amounts of the Flag-PIMT expression vector. (g) Immunoblot analysis of cell lysates from U2OS cells transfected with PIMT siRNA targeting a non-CDS sequence of the PIMT mRNA (line 2), followed by transfection with the Flag-PIMT expression vector (line 3). The relative protein levels were measured by Image Gauge software (version 3.12) and described as histograms to the right of each blot.

Figure 2. PIMT inhibits the activity of p53.

(a) Immunoblot analysis of cell lysates from U2OS cells transfected with PIMT siRNA or control siRNA. (b) Quantitative real-time PCR was used to measure the relative mRNA levels of HDM2, p21, PUMA, TSP1 and CD44 in U2OS cells transfected with control siRNA or PIMT siRNA. The data are represented as the mean±s.d., n=3. (c) Immunoblot analysis of cell lysates from U2OS cells transfected with PIMT siRNA, p53 siRNA or both. (d) Chromatin immunoprecipitation assay to assess the recruitment of p53 to the promoters of the p53-responsive element of the p21 or HDM2 gene in U2OS cells transfected with control siRNA or PIMT siRNA. The data are represented as the mean±s.d., n=3. (e) Immunoblot analysis of PIMT knockdown or control stable U2OS cell lines. (f) The growth rate of the PIMT knockdown or control stable U2OS cell lines. The data are represented as the mean±s.d., n=4. (g) Soft agar colony-forming assay using the PIMT knockdown or control stable MCF7 cell lines. The colonies were counted and visualized using nitroblue tetrazolium (NBT) staining. The data are represented as the mean±s.d., n=3. Scale bar, 250 μM (upper panel) or 100 μM (lower panel). (h) Flow cytometry analysis of U2OS cells transfected with PIMT siRNA or control siRNA, followed by adriamycin treatment. The data are represented as the mean±s.d., n=4. (i) Luciferase assay of cell lysates from H1299 cells transfected with expression vectors for either wild-type PIMT (WT) or mutant-type PIMT (G88A), together with p21-luc, as indicated. The data are represented as the mean±s.d., n=4. (j) Quantitative real-time PCR or immunoblot analysis of cell lysates from U2OS cells transfected with expression vectors for either wild-type PIMT (WT) or mutant-type PIMT (G88A), as indicated. Statistical significance was analyzed using the Student's t-test and expressed as a P-value. *P<0.05; **P<0.01; ***P<0.005; NS, not significant.

Figure 3. PIMT interacts with p53.

(a) Immunoblot analysis of Flag immunoprecipitates (IP line) and cell lysates (Input line) from H1299 cells transfected with the expression vector for Flag-PIMT and HA-p53. (b) Immunoblot analysis of p53 immunoprecipitates (IP line) and cell lysates (Input line) from H1299 cells transfected with the expression vector for Flag-PIMT and HA-p53. (c) Immunoblot analysis of PIMT immunoprecipitates (upper panel) and p53 immunoprecipitates (lower panel) from MCF7 cells. (d) Immunoblot analysis of p53 immunoprecipitates (upper panel) and PIMT immunoprecipitates (lower panel) from U2OS cells treated with adriamycin.

Figure 4. PIMT methylates p53 at N29 and N30.

(a) Fluorography results of the methylation assay using recombinant GST-PIMT on GST-p53. Asterisk indicates methylated GST-p53. (b) Immunoblot (IB) analysis of purified recombinant GST-PIMT and GST-p53 peptides 1–82, 83–289 and 290–393 (left panel). Fluorography results of the methylation assay using recombinant GST-PIMT and GST-p53 peptides 1–82, 83–289 or 290–393 (right panel). NS means nonspecific. Asterisk indicates methylated GST-p53 (1–82). (c) Fluorography results of the methylation assay using recombinant GST-PIMT (wild-type) or GST-PIMT (G88A) and GST-p53 peptides 1–82. (d) Fluorography results of the methylation assay using recombinant GST-PIMT or His-PIMT and GST-p53 peptides 1–82, followed by NaOH treatment, as indicated. NS means nonspecific. Asterisk indicates methylated GST-p53 (1–82). (e) Fluorography results of the methylation assay using recombinant GST-PIMT and GST-p53 peptides 1–82 or GST-p53 peptides 1–82 bearing D7A, D21A, N29A, N30A, D41A, D42A, D48A or D49A replacements (upper panel). A liquid scintillation counter (LSC) was used to measure the relative c.p.m. (counts per minute) of each sample (lower panel). The data are represented as the mean±range, n=2. (f) Fluorography of the methylation assay with recombinant GST-PIMT and p53 peptides, as indicated. (g) Fluorography of the methylation assay with recombinant GST-PIMT and the full-length p53 mutant (N29,30A). Asterisks indicate methylated GST-p53 wild-type. Asterisk indicates methylated GST-p53. (h) Fluorography of methylated GST-p53 peptide incubated in Tris buffer under several pH conditions. LSC was used to measure the relative c.p.m. of each sample. The data are represented as the mean±range, n=2. (i) Fluorography of the methylated p53 peptides incubated for different time intervals at 37 °C or 4 °C in Tris buffer.

Figure 5. Deamidation of p53 at N29 and N30.

(a) Schematic outline of the experiment. (b) Fluorography of the methylation assay with recombinant GST-PIMT and p53 peptides 22–40 (upper panel). Liquid scintillation counting (LSC) was used to measure the c.p.m. (counts per minute) of each sample (lower panel). The data are represented as the mean±range, n=2. (c) Nano-LC-ESI-MS/MS analysis of the p53 peptide incubated for 1 h in Tris buffer. CID mode is used for MS/MS. Ion score is 65 and charge value of selected parent peptide is +2.

Figure 6. PIMT influences the stability of p53.

(a) Locations of the methylated asparagine residues in p53. (b) Immunoblot (IB) analysis and quantitative real-time PCR of cell lysates from U2OS cells transfected with control siRNA or PIMT siRNA. The data are represented as the mean±s.e.m., n=3. (c) IB analysis of cell lysates from U2OS cells transfected with PIMT siRNA or control siRNA, followed by treatment with cycloheximide (CHX, 20 μg ml⁻¹) for the indicated times. The relative protein levels of p53 were measured using the ImageGauge software and analyzed with GraphPad Prism 5. (d) IB analysis of p53 immunoprecipitation (IP) in U2OS cells transfected with control or PIMT siRNA, followed by treatment with MG132 (40 μM). (e) IB analysis of p53 IP in H1299 cells transfected with the expression vector for HA-p53 and PIMT siRNA, followed by MG132 treatment (40 μM). (f) IB analysis of the p53 IP in H1299 cells transfected with the expression vector for Flag-PIMT and HA-p53, followed by MG132 treatment (40 μM). (g) IB analysis of p53 IP in U2OS cells transfected with control or PIMT siRNA. (h) IB analysis of p53 IP in H1299 cells transfected with expression vectors for HA-p53 or Flag-PIMT, as indicated. (i) IB analysis of cell lysates from H1299 cells transfected with expression vectors for wild-type HA-p53 (WT) or mutant HA-p53 (N29A, N30A). (j) IB analysis of HA IP (IP panel) and cell lysates (Input panel) in H1299 cells transfected with expression vectors for either HA-p53 (WT) or mutant HA-p53 (N29A or N30A) together with HDM2, as indicated. (k) IB analysis of cell lysates from H1299 cells transfected with expression vectors for wild-type HA-p53 (WT) or mutant HA-p53 (N29E, N30E, N29,30E, N29Q, N30Q, N29,30Q). (l) IB analysis of HA IP (IP panel) and cell lysates (Input panel) in H1299 cells transfected with the expression vectors for either HA-p53 (WT) or mutant HA-p53 (N29Q, N30Q or N29,30Q) together with HDM2, as indicated. Statistical significance was analyzed using the Student's t-test and expressed as a P-value. **P<0.01; NS, not significant.