Research ArticleBiochemistry

Mutant and wild-type p53 form complexes with p73 upon phosphorylation by the kinase JNK

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Science Signaling  03 Apr 2018:
Vol. 11, Issue 524, eaao4170
DOI: 10.1126/scisignal.aao4170
  • Fig. 1 Phosphorylation of Thr81 of p53 in response to JNK activation.

    (A) Western blot of phosphorylated c-Jun N-terminal kinase (JNK), phosphorylated Thr81 of p53, and actin in cellular extracts from TMD231, MDA-MB-468, and MDA-MB-157 cells treated with either camptothecin or anisomycin for the indicated times. Blots are representative of three independent experiments. (B) Western blot of phosphorylated JNK or Thr81 of p53 and actin from cellular extracts from 3T3, SF767, HME-1, BJ, and U87 treated with anisomycin or camptothecin for the indicated times. Blots are representative of three independent experiments (quantified in fig. S1). (C) U87 or SF767 cellular extracts from DMSO, SP600125, anisomycin, or pretreatment with SP600125 and then anisomycin and Western-blotted for p53–up-regulated modulator of apoptosis (PUMA) and actin. Blots are representative of two independent experiments.

  • Fig. 2 Predicted structure for a Thr81 phosphomimic reveals a possible mode of binding to p73.

    (A and B) Structures (A) and amino acid coverage (B) of domains in wild-type and mutant p53. Whole protein represents amino acids 1 to 393 of p53. Transactivation domains 1 and 2 (TAD1/TAD2) represent amino acids 1 to 40 and 43 to 54. The proline-rich domain (PRD) represents amino acids 63 to 97. The tetramerization and regulatory domains (TET/REG) represent amino acids 323 to 393. T81, Thr81; E, glutamic acid in the T81E phosphomimic mutant domain. Arrows in the TET/REG domain structures indicate the region of a conformational change that is permissive to binding.

  • Fig. 3 Formation and activity of a p53-p73 complex.

    (A) Immunoprecipitation of p53 or control immunoglobulin G (IgG) from TMD231 cells treated with DMSO or SP600125 and Western-blotted for p73 and p53. Blots are representative of four independent experiments. (B) Transient transfection of H1299 cells with p53 and HA-p73 and treated with either DMSO or anisomycin. Western blot of p53 and HA from p53 immunoprecipitated from cellular extracts. Blots are representative of two independent experiments. (C) Immunoprecipitation of p53 or control IgG and Western blot of p53 and p73 from H1299 cells transfected with wild-type p53 or T81Ep53. WCL is the whole-cell lysate from H1299 cells. Blots are representative of two independent experiments. (D) Immunoprecipitation of endogenous p73 from U87 cells treated with SP600125, anisomycin, or both and Western blot of phospho-Thr81, p53, and p73. Blots are representative of two independent experiments. (E) Far-Western analysis of p73 binding to p53. Lane 1 corresponds to p53 and the phosphomimics of p53; lane 2 is MDM2 and MDMX. Right, blotting for p73 to detect binding to p53, MDM2, and MDMX. Left, the relative amount of p53 on the membrane. Blots are representative of two independent experiments.

  • Fig. 4 Colocalization of p53 with p73.

    (A to C) Immunofluorescence assessing nuclear colocalization of p53 (green) and p73 (red) in MDA157 (A) and HME cells (B) treated with anisomycin (Aniso) or DMSO and in TMD231 cells (C) treated with SP600125 or DMSO. Arrows indicate the cell magnified in the inset (bottom right) of each image. Scale bars, 25 μm (MDA157 and TMD231) or 50 μm (HME). Data are mean ± SE from three independent experiments; P is determined by two-tailed unpaired t test.

  • Fig. 5 Downstream effects of the p53-p73 pathway.

    (A) Western blot of BAX, PUMA, and p53 in cellular extracts from BJ cells transfected with empty vector control (E.V.) or p53–short hairpin RNA (shp53) and treated with anisomycin for the indicated time. Blots are representative of three independent experiments; E.V. and shp53 samples were developed on the same membrane. (B) Methylene blue staining cell viability assay in E.V.- and shp53-transfected BJ cells treated with DMSO or anisomycin for 9 hours. Data are representative of three independent experiments. (C) Relative green fluorescent protein (GFP) fluorescence was measured in GFP shp53 and LVTHM empty vector BJ cells. Cells were treated with DMSO or anisomycin for 48 hours. Relative fluorescence was derived from DMSO controls. Data are representative of four independent experiments. Significance was determined by a two-tailed unpaired t test (***P < 0.005). (D) Green fluorescence was measured in H1299 cells transfected with GFP alone or GFP and either wild-type or T81A p53 and then treated with DMSO or anisomycin. Fold-change survival was calculated as the change in survival of anisomycin-treated cells relative to DMSO-treated cells and normalized to the GFP-alone control. Data are means ± SE from three independent experiments.

  • Fig. 6 Cell fate is dictated by the formation of a p53-p73 complex.

    Schematic of our model. Apoptotic stimuli (for example, DNA damage) results in the phosphorylation and activation of JNK. JNK phosphorylates wild-type p53 (green hexagons) at Thr81, causing a structural change in p53 and the subsequent binding of p73 (red diamonds). This complex drives the transcription of PUMA and BAX, leading to apoptosis. Mutant p53 (blue hexagons) does not require phosphorylation by JNK to form a complex with p73. Phosphorylation of mutant p53 instead serves to enhance p53 binding with p73, hence increasing total complex formation. However, because of the dysfunctional DNA binding domain in mutant p53, as well as its sequestering of p73 by complex formation, PUMA and BAX are not transcribed, thus decreasing the apoptotic signal and promoting tumor cell survival.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/524/eaao4170/DC1

    Fig. S1. Phosphorylation of Thr81 in cell lines with wild-type p53.

    Fig. S2. U87 cell death.

    Fig. S3. JNK activation or inhibition in HME, MDA157, and TMD231 cell lines.

    Table S1. Manders’ coefficients for microscopy colocalization analysis.

    Data file S1. Wild-type p53 structure [Protein Data Bank (PDB)].

    Data file S2. p53 R248W structure (PDB).

    Data file S3. p53 T81E structure (PDB).

    Data file S4. p53 S46D structure (PDB).

    Data file S5. p53 R248W/T81E structure (PDB).

  • Supplementary Materials for:

    Mutant and wild-type p53 form complexes with p73 upon phosphorylation by the kinase JNK

    Eric R. Wolf, Ciarán P. McAtarsney, Kristin E. Bredhold, Amber M. Kline, Lindsey D. Mayo*

    *Corresponding author. Email: ldmayo{at}iu.edu

    This PDF file includes:

    • Fig. S1. Phosphorylation of Thr81 in cell lines with wild-type p53.
    • Fig. S2. U87 cell death.
    • Fig. S3. JNK activation or inhibition in HME, MDA157, and TMD231 cell lines.
    • Table S1. Manders' coefficients for microscopy colocalization analysis.
    • Legends for data files S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (.pdb format). Wild-type p53 structure [Protein Data Bank (PDB)].
    • Data file S2 (.pdb format). p53 R248W structure (PDB).
    • Data file S3 (.pdb format). p53 T81E structure (PDB).
    • Data file S4 (.pdb format). p53 S46D structure (PDB).
    • Data file S5 (.pdb format). p53 R248W/T81E structure (PDB).

    [Download Data files S1 to S5]


    © 2018 American Association for the Advancement of Science

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