ATP stimulates MDM2-mediated inhibition of the DNA-binding function of E2F1: ATP stimulated inhibition of E2F1 by MDM2

Murine double minute 2 (MDM2) protein exhibits many diverse biochemical functions on the tumour suppressor protein p53, including transcriptional suppression and E3 ubiquitin ligase activity. However, more recent data have shown that MDM2 can exhibit ATP‐dependent molecular chaperone activity and directly mediate folding of the p53 tetramer. Analysing the ATP‐dependent function of MDM2 will provide novel insights into the evolution and function of the protein. We have established a system to analyse the molecular chaperone function of MDM2 on another of its target proteins, the transcription factor E2F1. In the absence of ATP, MDM2 was able to catalyse inhibition of the DNA‐binding function of E2F1. However, the inhibition of E2F1 by MDM2 was stimulated by ATP, and mutation of the ATP‐binding domain of MDM2 (K454A) prevented the ATP‐stimulated inhibition of E2F1. Further, ATP stabilized the binding of E2F1 to MDM2 using conditions under which ATP destabilized the MDM2:p53 complex. However, the ATP‐binding mutant of MDM2 was as active as an E3 ubiquitin ligase on E2F1 and p53, highlighting a specific function for the ATP‐binding domain of MDM2 in altering substrate protein folding. Antibodies to three distinct domains of MDM2 neutralized its activity, showing that inhibition of E2F1 is MDM2‐dependent and that multiple domains of MDM2 are involved in E2F1 inhibition. Dimethylsulfoxide, which reduces protein unfolding, also prevented E2F1 inhibition by MDM2. These data support a role for the ATP‐binding domain in altering the protein–protein interaction function of MDM2

Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

<div><p>Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes.</p> </div

MDM2 and HSP70/HSPA family members change the localization and aggregation state of EYFP-p53 R175H in MEFs.

<p>(<b>A</b>) MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) were transfected with various combinations of plasmids encoding EYFP-p53 R175H, MDM2, HSC70, WT HSP70 and HSP70 K71S, as labelled above. Cells shown in 9–16 were treated with MG132 (2,5 µM) for 16 hours before imaging. Detailed description in text. Scale bar 10 µm. (<b>B</b>) MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) were transfected with plasmid encoding EYFP-p53 R175H together with HSP70 and optionally MDM2 (lower panel). 24 hours post-transfection cells were treated with MG132 and scanned using Olympus ScanR Station widefield fluorescent microscope (time-lapse imaging) for additional 16 hours. 0 h represents 8 hours post-transfection, subsequent frames were acquired every 1 hour. The obtained images were analysed by ScanR Analysis software.</p

MDM2 and HSP70 colocalize with p53R175H in an aggressome.

<p>(<b>A</b>) Immunostaining of MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) transfected with plasmids encoding p53 R175H, HSP70 and optionally MDM2 (lower panel) shows that only in the presence of MDM2, A11 antibodies (labelling amyloid-oligomers) recognize aggregates composed of mutant p53 and HSP70. (<b>B</b>) Immunostaining of MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) transfected with plasmids encoding p53R175H, MDM2 and HSP70 revealed recruitment of both <b>(1)</b> the E3 ubiquitin ligase and <b>(2)</b> the chaperone to the large inclusion body. (<b>3)</b> The inclusion is formed at the centrosome marked by the presence of gamma-tubulin, suggesting that the large inclusion body containing p53 R175H is an aggresome. (<b>4</b>) Disruption of microtubules with nocodazole treatment impairs protein transport and results in the disappearance of the aggresome; smaller, scattered cytoplasmic p53-containing aggregates form instead. Scale bar 10 µm.</p

Endogenous HSP70 protein levels in H1299 cells are sufficient for nuclear aggregation of p53R175H.

<p>(<b>A</b>) SK-BR-3, H1299 and MEF cells were seeded 24 hours prior to harvesting. Immunoblotting with HSP70-specific antibody revealed high levels of HSP70 in the cancer-derived SKBR3 and H1299 cell lines when compared to double knockout MEF (<i>Trp53^−/−, Mdm2^−/−</i>) cells. (<b>B</b>) H1299 cells were treated (or not) with a combination of two compounds –17-AAG (1 µM, HSP90 inhibitor) and VER155008 (10 µM, HSP70 inhibitor) – for 16 hours. Immunoblotting of cell lysates shows the accumulation of HSP70 protein upon treatment with 17-AAG and the decrease in p53 R175H protein level as a result of HSP70 inhibition by VER155008. (<b>C</b>) H1299 cells were transfected with plasmids encoding p53 R175H and optionally low or high amount of HSP70 and then treated with MG132 (2 µM) for 16 hours prior to fixation. Immunostaining with p53-specific antibody revealed that the endogenous HSP70 protein level is sufficient for induction of nuclear p53 R175H aggregate formation (left panel) and that an increase in HSP70 level results in appearance of cytoplasmic p53-containing speckles (middle and right panel). (<b>D</b>) Immunofluorescence of H1299 cells treated with 17-AAG further supports the notion that endogenous HSP70 shifts the equilibrium between various oligomeric states of mutated p53. The lower level of HSP70 (6 hours after 17-AAG treatment, middle panel) triggers p53 R175H nuclear aggregation, whereas the higher level of HSP70 (16 hours after 17-AAG treatment, right panel) causes the dissociation of the nuclear aggregates into smaller speckles and their relocalization to the cytoplasm. Scale bar 10 µm.</p

Nuclear diffusion of p53 R175H is limited.

<p>H1299 cells were transfected with plasmids encoding for indicated proteins and 24 hours post-transfection subjected to the FRAP analysis. Mobility of tandem EYFP protein and EYFP fused to indicated p53 mutant was measured in the nuclei. Recovery curves were normalized and each set of replicates was fitted to one or two phase exponential association equation with an R² of no less than 0.95. The differences between all curves shown are statistically significant (larger than 95% confidence intervals, not shown for chart clarity).</p

HSP70 inhibition prevents the formation of cytoplasmic p53 R175H speckles in MEFs.

<p>MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) were transfected with plasmids encoding p53 R175H and HSP70 and treated (or not) with HSP70 inhibitor (VER155008, Tocris Bioscience) and subsequently labelled for p53 and HSP70. In non-treated cells cytoplasmic spots containing p53 and HSP70 were observed. Small folding intermediates were visible when HSP70 inhibitor was added 6h post-transfection and no spots were detected in the case of HSP70 inhibition induced in parallel with transfection. Scale bar 10 µm.</p