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PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells.

Davies L, Spiller D, White MR, Grierson I, Paraoan L - Cell Death Dis (2011)

Bottom Line: PERP (p53 apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest.Here, we identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53.These results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Eye and Vision Sciences, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.

ABSTRACT
The activation and regulation of target genes by the tumour-suppressor p53 dictates the fate of a cell, with cell cycle arrest or apoptosis being two distinct outcomes. PERP (p53 apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest. Downregulation of PERP is associated with the aggressive, monosomy 3-type of uveal melanoma (UM), the most common primary intraocular tumour in adults, and increased PERP expression has a pro-apoptotic effect in UM cells. Here, we identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53. Using fluorescent fusion proteins of PERP, p53 and MDM2, we demonstrate in single living UM cells that PERP expression significantly enhances p53 activity and its nuclear localization, increases p53-dependent transcription (including that of MDM2) while allowing oscillatory nucleo-cytoplasmic shuttling of p53/MDM2 complexes. Phosphorylation of p53 serine residues that interfere with the interaction between p53 and its negative regulator MDM2 and enhance pro-apoptotic gene transcription also occurs subsequent to PERP expression. These results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.

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Related in: MedlinePlus

p53 cellular localization is influenced by PERP expression and is subject to oscillatory regulation. (a) p53 localizes primarily in the nucleus following PERP expression. MEL202 cells transfected with p53–RFP or p53-RFP and GFP–PERP were monitored by confocal fluorescence microscopy and the analysis of the intracellular distribution of proteins of interest was undertaken at 20-h PT. The number of cells exhibiting a predominant nuclear localization (N>C) or a more even p53 distribution in the nucleus and cytoplasm (N≤C) were counted and are presented as the mean percentage of transfected cells from three independent transfections with S.D. p53–RFP was predominantly nuclear in a significantly higher proportion of cells when co-expressed with GFP–PERP compared with cells expressing p53-RFP only (*T-test, P=0.04). (b) Representative images showing the predominant cytoplasmic localization of p53 (red) in the absence of GFP–PERP expression (left panel), and the prevalent nuclear p53 localization (red) in the presence of GFP–PERP (green; right panel), following induction of the pMT promoter of p53–RFP with 100 μM ZnCl2. Scale bar=20 μm. (c) ZnCl2 does not influence PERP expression or p53 localization. Low basal expression of p53–RFP observed in the absence of ZnCl2 induction had a predominant nuclear localization in the presence of GFP–PERP expression, whilst 100 μM ZnCl2 had no effect on GFP–PERP expression or localization. Scale bar=20 μm. (d) Increased MDM2 expression occurs when p53 is primarily localized in nucleus. MEL202 cells were co-transfected with p53–RFP and MDM2-YFP and images were taken from three independent transfections at 20-h PT. YFP fluorescence was measured in arbitrary units (AU) in cells expressing p53 predominantly in the nucleus (N>C) or more evenly in the nucleus and cytoplasm (N≤C). Mean fluorescence is indicated (♦) with S.D. MDM2-YFP fluorescence was significantly higher in cells expressing p53 primarily in the nucleus (T-test, P<0.0005). Representative images of the differential localization of p53 (red) and MDM2 (yellow) are shown. Scale bar=20 μm. (e) PERP-induced elevated MDM2 expression exhibits oscillations. MEL202 cells co-transfected with GFP–PERP (green in bright field) and MDM2-YFP (yellow) were monitored by time-lapse fluorescence microscopy. Nuclear MDM2–YFP fluorescence was measured (AU) in single cells over time (hours) and data are presented graphically alongside corresponding time point images. An arrow indicates the relevant cell where necessary
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fig3: p53 cellular localization is influenced by PERP expression and is subject to oscillatory regulation. (a) p53 localizes primarily in the nucleus following PERP expression. MEL202 cells transfected with p53–RFP or p53-RFP and GFP–PERP were monitored by confocal fluorescence microscopy and the analysis of the intracellular distribution of proteins of interest was undertaken at 20-h PT. The number of cells exhibiting a predominant nuclear localization (N>C) or a more even p53 distribution in the nucleus and cytoplasm (N≤C) were counted and are presented as the mean percentage of transfected cells from three independent transfections with S.D. p53–RFP was predominantly nuclear in a significantly higher proportion of cells when co-expressed with GFP–PERP compared with cells expressing p53-RFP only (*T-test, P=0.04). (b) Representative images showing the predominant cytoplasmic localization of p53 (red) in the absence of GFP–PERP expression (left panel), and the prevalent nuclear p53 localization (red) in the presence of GFP–PERP (green; right panel), following induction of the pMT promoter of p53–RFP with 100 μM ZnCl2. Scale bar=20 μm. (c) ZnCl2 does not influence PERP expression or p53 localization. Low basal expression of p53–RFP observed in the absence of ZnCl2 induction had a predominant nuclear localization in the presence of GFP–PERP expression, whilst 100 μM ZnCl2 had no effect on GFP–PERP expression or localization. Scale bar=20 μm. (d) Increased MDM2 expression occurs when p53 is primarily localized in nucleus. MEL202 cells were co-transfected with p53–RFP and MDM2-YFP and images were taken from three independent transfections at 20-h PT. YFP fluorescence was measured in arbitrary units (AU) in cells expressing p53 predominantly in the nucleus (N>C) or more evenly in the nucleus and cytoplasm (N≤C). Mean fluorescence is indicated (♦) with S.D. MDM2-YFP fluorescence was significantly higher in cells expressing p53 primarily in the nucleus (T-test, P<0.0005). Representative images of the differential localization of p53 (red) and MDM2 (yellow) are shown. Scale bar=20 μm. (e) PERP-induced elevated MDM2 expression exhibits oscillations. MEL202 cells co-transfected with GFP–PERP (green in bright field) and MDM2-YFP (yellow) were monitored by time-lapse fluorescence microscopy. Nuclear MDM2–YFP fluorescence was measured (AU) in single cells over time (hours) and data are presented graphically alongside corresponding time point images. An arrow indicates the relevant cell where necessary

Mentions: To monitor the effect of PERP expression on the cellular localization of p53, MEL202 cells were co-transfected with GFP–PERP and an expression plasmid in which p53 was fused to red fluorescent protein (RFP) placed under the control of a zinc-inducible promoter. Cells transfected with p53–RFP-only served as controls. In the absence of GFP–PERP expression, p53–RFP was localized primarily in the cytoplasm with low expression in the nucleus (65% of transfected cells; Figures 3a and b), compared with 35% of cells showing nuclear-only p53–RFP expression. However, following GFP–PERP expression, p53–RFP was localized predominantly in the nucleus in a significantly higher proportion of co-transfected cells (58% T-test, P=0.04; Figures 3a and b). Nuclear expression of p53–RFP when co-expressed with GFP–PERP was also detected in the absence of ZnCl2 induction, albeit at much lower expression levels, and ZnCl2 had no effect on the expression or localization of GFP–PERP (Figure 3c). The PERP-induced nuclear localization of p53 suggests that p53 is likely to wield its transcriptional activity – a scenario that is also supported by the p53-dependent upregulation of MDM2 transcription in the presence of elevated PERP (Figure 2). Furthermore, the highest nuclear MDM2–YFP fluorescence in cells co-transfected with MDM2–YFP and p53–RFP occurred when p53–RFP was also localized primarily in the nucleus, while cytoplasmic p53 was associated (fluorescence colocalized) with cytoplasmic MDM2–YFP (Figure 3d), illustrating the nuclear-cytoplasmic shuttling of both p53 and MDM2 that is central to their activities.15


PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells.

Davies L, Spiller D, White MR, Grierson I, Paraoan L - Cell Death Dis (2011)

p53 cellular localization is influenced by PERP expression and is subject to oscillatory regulation. (a) p53 localizes primarily in the nucleus following PERP expression. MEL202 cells transfected with p53–RFP or p53-RFP and GFP–PERP were monitored by confocal fluorescence microscopy and the analysis of the intracellular distribution of proteins of interest was undertaken at 20-h PT. The number of cells exhibiting a predominant nuclear localization (N>C) or a more even p53 distribution in the nucleus and cytoplasm (N≤C) were counted and are presented as the mean percentage of transfected cells from three independent transfections with S.D. p53–RFP was predominantly nuclear in a significantly higher proportion of cells when co-expressed with GFP–PERP compared with cells expressing p53-RFP only (*T-test, P=0.04). (b) Representative images showing the predominant cytoplasmic localization of p53 (red) in the absence of GFP–PERP expression (left panel), and the prevalent nuclear p53 localization (red) in the presence of GFP–PERP (green; right panel), following induction of the pMT promoter of p53–RFP with 100 μM ZnCl2. Scale bar=20 μm. (c) ZnCl2 does not influence PERP expression or p53 localization. Low basal expression of p53–RFP observed in the absence of ZnCl2 induction had a predominant nuclear localization in the presence of GFP–PERP expression, whilst 100 μM ZnCl2 had no effect on GFP–PERP expression or localization. Scale bar=20 μm. (d) Increased MDM2 expression occurs when p53 is primarily localized in nucleus. MEL202 cells were co-transfected with p53–RFP and MDM2-YFP and images were taken from three independent transfections at 20-h PT. YFP fluorescence was measured in arbitrary units (AU) in cells expressing p53 predominantly in the nucleus (N>C) or more evenly in the nucleus and cytoplasm (N≤C). Mean fluorescence is indicated (♦) with S.D. MDM2-YFP fluorescence was significantly higher in cells expressing p53 primarily in the nucleus (T-test, P<0.0005). Representative images of the differential localization of p53 (red) and MDM2 (yellow) are shown. Scale bar=20 μm. (e) PERP-induced elevated MDM2 expression exhibits oscillations. MEL202 cells co-transfected with GFP–PERP (green in bright field) and MDM2-YFP (yellow) were monitored by time-lapse fluorescence microscopy. Nuclear MDM2–YFP fluorescence was measured (AU) in single cells over time (hours) and data are presented graphically alongside corresponding time point images. An arrow indicates the relevant cell where necessary
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fig3: p53 cellular localization is influenced by PERP expression and is subject to oscillatory regulation. (a) p53 localizes primarily in the nucleus following PERP expression. MEL202 cells transfected with p53–RFP or p53-RFP and GFP–PERP were monitored by confocal fluorescence microscopy and the analysis of the intracellular distribution of proteins of interest was undertaken at 20-h PT. The number of cells exhibiting a predominant nuclear localization (N>C) or a more even p53 distribution in the nucleus and cytoplasm (N≤C) were counted and are presented as the mean percentage of transfected cells from three independent transfections with S.D. p53–RFP was predominantly nuclear in a significantly higher proportion of cells when co-expressed with GFP–PERP compared with cells expressing p53-RFP only (*T-test, P=0.04). (b) Representative images showing the predominant cytoplasmic localization of p53 (red) in the absence of GFP–PERP expression (left panel), and the prevalent nuclear p53 localization (red) in the presence of GFP–PERP (green; right panel), following induction of the pMT promoter of p53–RFP with 100 μM ZnCl2. Scale bar=20 μm. (c) ZnCl2 does not influence PERP expression or p53 localization. Low basal expression of p53–RFP observed in the absence of ZnCl2 induction had a predominant nuclear localization in the presence of GFP–PERP expression, whilst 100 μM ZnCl2 had no effect on GFP–PERP expression or localization. Scale bar=20 μm. (d) Increased MDM2 expression occurs when p53 is primarily localized in nucleus. MEL202 cells were co-transfected with p53–RFP and MDM2-YFP and images were taken from three independent transfections at 20-h PT. YFP fluorescence was measured in arbitrary units (AU) in cells expressing p53 predominantly in the nucleus (N>C) or more evenly in the nucleus and cytoplasm (N≤C). Mean fluorescence is indicated (♦) with S.D. MDM2-YFP fluorescence was significantly higher in cells expressing p53 primarily in the nucleus (T-test, P<0.0005). Representative images of the differential localization of p53 (red) and MDM2 (yellow) are shown. Scale bar=20 μm. (e) PERP-induced elevated MDM2 expression exhibits oscillations. MEL202 cells co-transfected with GFP–PERP (green in bright field) and MDM2-YFP (yellow) were monitored by time-lapse fluorescence microscopy. Nuclear MDM2–YFP fluorescence was measured (AU) in single cells over time (hours) and data are presented graphically alongside corresponding time point images. An arrow indicates the relevant cell where necessary
Mentions: To monitor the effect of PERP expression on the cellular localization of p53, MEL202 cells were co-transfected with GFP–PERP and an expression plasmid in which p53 was fused to red fluorescent protein (RFP) placed under the control of a zinc-inducible promoter. Cells transfected with p53–RFP-only served as controls. In the absence of GFP–PERP expression, p53–RFP was localized primarily in the cytoplasm with low expression in the nucleus (65% of transfected cells; Figures 3a and b), compared with 35% of cells showing nuclear-only p53–RFP expression. However, following GFP–PERP expression, p53–RFP was localized predominantly in the nucleus in a significantly higher proportion of co-transfected cells (58% T-test, P=0.04; Figures 3a and b). Nuclear expression of p53–RFP when co-expressed with GFP–PERP was also detected in the absence of ZnCl2 induction, albeit at much lower expression levels, and ZnCl2 had no effect on the expression or localization of GFP–PERP (Figure 3c). The PERP-induced nuclear localization of p53 suggests that p53 is likely to wield its transcriptional activity – a scenario that is also supported by the p53-dependent upregulation of MDM2 transcription in the presence of elevated PERP (Figure 2). Furthermore, the highest nuclear MDM2–YFP fluorescence in cells co-transfected with MDM2–YFP and p53–RFP occurred when p53–RFP was also localized primarily in the nucleus, while cytoplasmic p53 was associated (fluorescence colocalized) with cytoplasmic MDM2–YFP (Figure 3d), illustrating the nuclear-cytoplasmic shuttling of both p53 and MDM2 that is central to their activities.15

Bottom Line: PERP (p53 apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest.Here, we identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53.These results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Eye and Vision Sciences, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.

ABSTRACT
The activation and regulation of target genes by the tumour-suppressor p53 dictates the fate of a cell, with cell cycle arrest or apoptosis being two distinct outcomes. PERP (p53 apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest. Downregulation of PERP is associated with the aggressive, monosomy 3-type of uveal melanoma (UM), the most common primary intraocular tumour in adults, and increased PERP expression has a pro-apoptotic effect in UM cells. Here, we identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53. Using fluorescent fusion proteins of PERP, p53 and MDM2, we demonstrate in single living UM cells that PERP expression significantly enhances p53 activity and its nuclear localization, increases p53-dependent transcription (including that of MDM2) while allowing oscillatory nucleo-cytoplasmic shuttling of p53/MDM2 complexes. Phosphorylation of p53 serine residues that interfere with the interaction between p53 and its negative regulator MDM2 and enhance pro-apoptotic gene transcription also occurs subsequent to PERP expression. These results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.

Show MeSH
Related in: MedlinePlus