Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: MDM2 associates with sHSPs in the nucleus. A and B , recombinant MDM2 protein (1 μg) was incubated with 0.125, 0.25, 0.5, 1, 2, or 4 μg of αBC ( A ) and HSP27 ( B ) proteins. MDM2 was pulled down, and the association with αBC and HSP27 was analyzed using anti-αBC and anti-HSP27 antibodies. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , Co-IP of endogenous MDM2 from MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. IB analyzed the MDM2, αBC, HSP27, and GAPDH IPed by MDM2. Veh, vehicle; Cis, cisplatin-treated. D and E , Confocal images of IF staining against αBC ( D ) and HSP27 ( E ) along with MDM2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear levels of MDM2, αBC, and HSP27 were normalized to vehicle-treated cells, and their nuclear co-localization was determined by immunofluorescence. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. F and G , PLA of αBC–MDM2 ( F ) and HSP27-MDM2 ( G ) in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear PLA foci of αBC–MDM2 and HSP27-MDM2 were quantified. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment.

Article Snippet: Monoclonal antibodies against PIP 2 (clone 2C11, #Z-P045, Echelon Biosciences), PIP 2 (clone KT10, #MSBS2283, MilliporeSigma), p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), HSP27 (clone F-4, #SC-13132, Santa Cruz Biotechnology), αBC (D6S9E, #45844, Cell Signaling), PIPKIIα (PIP4K2A, clone D83C1, #5527, Cell Signaling), GAPDH (clone 0411, #SC-47724, Santa Cruz Biotechnology), and polyclonal antibodies against MDM2 (clone D1V2Z, #86934, Cell Signaling), MDM2 (#AF1244, R&D Systems), αBC (#ab13497, Abcam), PIPKIα (PIP5K1A, #9693, Cell Signaling), PIPKIγ (PIP5K1C, #3296, Cell Signaling), PIPKIIβ (PIP4K2B, #9694, Cell Signaling), FOXO3a (clone 75D8, #2497, Cell Signaling), Histone H2A (#2578, Cell Signaling), Histone H2B (clone D2H6, #12364, Cell Signaling), Ubiquitin (P37, #58395, Cell Signaling) were utilized in this study.

Techniques: Recombinant, Incubation, Co-Immunoprecipitation Assay, Staining, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 regulates the interaction of MDM2, sHSPs and p53 and controls MDM2 stability. A and B , 0.1 μg of MDM2 recombinant protein and 0.1 μg of αBC ( A ) or HSP27 ( B ) protein were incubated with 0, 0.5, 1, 2, or 5 μM PIP 2 . MDM2 was pulled down, and the associated αBC or HSP27 was analyzed with an anti-αBC or an anti-HSP27 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-468 cells were transfected with siRNAs for αBC or HSP27 for 48 h. An empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB MDM2 IBs were quantified, and the graph is shown as mean ± s.d. of n = 4 independent experiments. KD, knockdown. D , MDA-MB-468 cells were transfected with siRNAs for αBC for 48 h. Before harvesting, cells were treated with 10 μM of MG132 for 4 h. Empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB, and MDM2 IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. E , recombinant MDM2 protein (0.1 μg) and GST-tagged p53 WT or PIP2-binding-defective p53 6Q mutant (p53 6Q, 0.1 μg) were incubated with 0, 1, 5, 10, or 50 μM PIP 2 . MDM2 was pulled down, and the associated p53 was analyzed with an anti-p53 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. p53 WT, wild type p53; p53 6Q, p53 6Q mutant.

Article Snippet: Monoclonal antibodies against PIP 2 (clone 2C11, #Z-P045, Echelon Biosciences), PIP 2 (clone KT10, #MSBS2283, MilliporeSigma), p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), HSP27 (clone F-4, #SC-13132, Santa Cruz Biotechnology), αBC (D6S9E, #45844, Cell Signaling), PIPKIIα (PIP4K2A, clone D83C1, #5527, Cell Signaling), GAPDH (clone 0411, #SC-47724, Santa Cruz Biotechnology), and polyclonal antibodies against MDM2 (clone D1V2Z, #86934, Cell Signaling), MDM2 (#AF1244, R&D Systems), αBC (#ab13497, Abcam), PIPKIα (PIP5K1A, #9693, Cell Signaling), PIPKIγ (PIP5K1C, #3296, Cell Signaling), PIPKIIβ (PIP4K2B, #9694, Cell Signaling), FOXO3a (clone 75D8, #2497, Cell Signaling), Histone H2A (#2578, Cell Signaling), Histone H2B (clone D2H6, #12364, Cell Signaling), Ubiquitin (P37, #58395, Cell Signaling) were utilized in this study.

Techniques: Recombinant, Incubation, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 and sHSPs regulate the MDM2-p53 interaction. A and B , 0.1 μg of MDM2 and GST-p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC ( A ) or HSP27 ( B ) in the presence or absence of 10 μM of PIP 2 . After pulling down with MDM2 antibody-conjugated beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. sup, supernatant; pel, pellet. C , 0.5 μg Bacteria (His-tagged) and mammalian-expressed (FLAG-tagged) MDM2 ( A ) and p53 ( B ) proteins were analyzed by fluorescence IB. Fluorescence IB detects purified proteins and PIP 2 stably associated to proteins. The MDM2 and PIP 2 association is shown in the merged image. D , recombinant MDM2 protein (0.1 μg) and FLAG-, His-p53, or GST-p53 6Q mutant proteins were incubated with 0, 1, 5, or 10 μM PIP 2 . MDM2 was pulled down, and the associated different p53 was analyzed with an anti-p53 antibody. The graphs are shown bound p53 normalized to the strongest signal (His-p53 with 10 μM PIP 2 ) as mean ± s.d. of n = 3 independent experiments. p53 6Q, p53 6Q mutant. E , 0.1 μg of FLAG-MDM2 and p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC or HSP27. After pulling down with MDM2 beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. FLAG-tagged proteins; mammalian-derived proteins.

Article Snippet: Monoclonal antibodies against PIP 2 (clone 2C11, #Z-P045, Echelon Biosciences), PIP 2 (clone KT10, #MSBS2283, MilliporeSigma), p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), HSP27 (clone F-4, #SC-13132, Santa Cruz Biotechnology), αBC (D6S9E, #45844, Cell Signaling), PIPKIIα (PIP4K2A, clone D83C1, #5527, Cell Signaling), GAPDH (clone 0411, #SC-47724, Santa Cruz Biotechnology), and polyclonal antibodies against MDM2 (clone D1V2Z, #86934, Cell Signaling), MDM2 (#AF1244, R&D Systems), αBC (#ab13497, Abcam), PIPKIα (PIP5K1A, #9693, Cell Signaling), PIPKIγ (PIP5K1C, #3296, Cell Signaling), PIPKIIβ (PIP4K2B, #9694, Cell Signaling), FOXO3a (clone 75D8, #2497, Cell Signaling), Histone H2A (#2578, Cell Signaling), Histone H2B (clone D2H6, #12364, Cell Signaling), Ubiquitin (P37, #58395, Cell Signaling) were utilized in this study.

Techniques: Recombinant, Incubation, Bacteria, Fluorescence, Purification, Stable Transfection, Mutagenesis, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 and sHSPs regulate the ubiquitination function of MDM2. A , for MDM2 autoubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with different concentrations of αBC, HSP27 (0.9, 1.8, or 2.7 μM), or PIP 2 (10, 20, or 30 μM) for 1 h. Ubiquitin, MDM2, αBC and HSP27 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. B , for in vitro ubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of His- and FLAG-tagged MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with 1 μM of His- and FLAG-tagged p53 for 1 h. For IP, samples were incubated with anti-p53-conjugated agarose overnight. Ubiquitin, MDM2, and p53 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-231 cells were transfected with siRNAs for PIPKIα and treated with vehicle or 30 μM cisplatin for 24 h. Empty vector (Mock) was used as a negative control. After cells were treated with 10 μM of MG132 for 4 h, cells were harvested for IP of MDM2 and p53. Expression of the indicated proteins was analyzed by IB, and ubiquitin IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown; UT, untreated; Cis, cisplatin treated; Cis/MG132, Cisplatin/MG132 treated. D , MDA-MB-468 cells were transfected with siRNAs for αBC and HSP27 for 48 h in absence or presence of MG132. Empty vector (Mock) was used as a negative control. Cells were processed for IP of MDM2. IB was used to analyze indicated proteins, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown. E , a model of PIP 2 regulation of the interaction between MDM2 and sHSPs, which differentially control the stability of MDM2 and function of of MDM2. In the presence of PIPKIα, PIP 2 is linked to MDM2, which recruits αBC to MDM2 to stabilize it and enhances p53 binding. Conversely, in the absence of PIPKIα and PIP 2 , HSP27 is recruited to MDM2, which increases the ubiquitin E3 ligase activity of MDM2.

Article Snippet: Monoclonal antibodies against PIP 2 (clone 2C11, #Z-P045, Echelon Biosciences), PIP 2 (clone KT10, #MSBS2283, MilliporeSigma), p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), HSP27 (clone F-4, #SC-13132, Santa Cruz Biotechnology), αBC (D6S9E, #45844, Cell Signaling), PIPKIIα (PIP4K2A, clone D83C1, #5527, Cell Signaling), GAPDH (clone 0411, #SC-47724, Santa Cruz Biotechnology), and polyclonal antibodies against MDM2 (clone D1V2Z, #86934, Cell Signaling), MDM2 (#AF1244, R&D Systems), αBC (#ab13497, Abcam), PIPKIα (PIP5K1A, #9693, Cell Signaling), PIPKIγ (PIP5K1C, #3296, Cell Signaling), PIPKIIβ (PIP4K2B, #9694, Cell Signaling), FOXO3a (clone 75D8, #2497, Cell Signaling), Histone H2A (#2578, Cell Signaling), Histone H2B (clone D2H6, #12364, Cell Signaling), Ubiquitin (P37, #58395, Cell Signaling) were utilized in this study.

Techniques: Ubiquitin Proteomics, Incubation, In Vitro, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Control, Binding Assay, Activity Assay