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Identification of Peroxiredoxin 1 as a novel interaction partner for the lifespan regulator protein p66Shc.

Gertz M, Fischer F, Leipelt M, Wolters D, Steegborn C - Aging (Albany NY) (2009)

Bottom Line: Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain.The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture.Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, 44801 Bochum, Germany.

ABSTRACT
Damage caused by reactive oxygen species (ROS) contributes to many aging processes and accompanying diseases. ROS are toxic side products of cellular respiration, but also function as signal, e.g. in the mitochondrial apoptosis pathway. The protein p66Shc, which has been implicated in life-span regulation and aging-related diseases, is a central player in stress-induced apoptosis and the associated ROS burst. Stress signals, such as UV radiation or ROS themselves, activate p66Shc, which was proposed to stimulate its H(2)O(2) forming activity, ultimately triggering mitochondrial disintegration. However, mechanistic details of H(2)O(2) formation and apoptosis induction by p66Shc and regulation of these activities remain to be revealed. Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain. Prx1 was identified in affinity experiments as dominant interaction partner. Complex formation leads to disassembly of Prx1 decamers, which is known to increase its peroxidase activity. The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture. Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.

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Effects of phosphorylation and Pin1 binding on p66Shc. (a-c) The                                            p66CH2CB-dependent ROS-generation is enhanced by phosphorylation of Ser36                                            but inhibited in the presence of Pin1. Changes in fluorescence of 10 μM H2DFFDA                                            were recorded after addition of 20 μM p66CH2CB WT (a) or the                                            p66CH2CB-Ser36Asp mutant simulating Ser36 phosphorylation (a-c),                                            followed by 85 μM Na-dithionite (DN) and 50 μM CuSO4 (Cu) in the                                            presence (b+c) or absence of Pin1 (a-c) and/or the dipeptide                                            Ala-Pro (c). (d) p66CH2CB-induced mitochondrial rupture is                                            inhibited by phosphorylation of Ser36. Mitochondrial rupture was induced                                            after addition of 7 μM CaCl2 by addition of 20 μM p66CH2CB WT or                                            Ser36Asp and monitored photometrically. The initial sensitization with CaCl2                                            was omitted for clarity. (e) H2O2 oxidizes                                            p66CH2CB. 10 μg p66CH2CB were incubated with 0.005 % H2O2                                            and subjected to non-reducing SDS-PAGE
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Figure 1: Effects of phosphorylation and Pin1 binding on p66Shc. (a-c) The p66CH2CB-dependent ROS-generation is enhanced by phosphorylation of Ser36 but inhibited in the presence of Pin1. Changes in fluorescence of 10 μM H2DFFDA were recorded after addition of 20 μM p66CH2CB WT (a) or the p66CH2CB-Ser36Asp mutant simulating Ser36 phosphorylation (a-c), followed by 85 μM Na-dithionite (DN) and 50 μM CuSO4 (Cu) in the presence (b+c) or absence of Pin1 (a-c) and/or the dipeptide Ala-Pro (c). (d) p66CH2CB-induced mitochondrial rupture is inhibited by phosphorylation of Ser36. Mitochondrial rupture was induced after addition of 7 μM CaCl2 by addition of 20 μM p66CH2CB WT or Ser36Asp and monitored photometrically. The initial sensitization with CaCl2 was omitted for clarity. (e) H2O2 oxidizes p66CH2CB. 10 μg p66CH2CB were incubated with 0.005 % H2O2 and subjected to non-reducing SDS-PAGE

Mentions: Cells need to have mechanisms strictly silencing the cytotoxic activities of p66Shc, ROS-generation and apoptosis-induction, during cell cycle but stimulating them during cellular stress. PKCβ and the peptidyl-prolyl isomerase Pin1 have been reported to regulate the relocalization of p66Shc to mitochondria required for apoptosis initiation. PKCβ phosphorylates p66Shc at Ser36 in response to oxidative stress [19], which was reported already to increase cellular ROS levels [24]. We tested whether phosphorylation leads to a direct stimulation of the p66Shc-inherent oxidoreductase activity. We simulated phosphorylation by mutating Ser36 in p66CH2CB to Asp and compared the ROS-generating activity of the Ser36Asp mutant to wildtype (WT) protein in a fluorescence-based H2O2 assay (Figure 1a). The p66CH2CB-Ser36Asp mutant displayed a significantly increased activity compared to WT protein, independent of the dimer/tetramer equilibrium (data not shown) that is known to influence ROS formation [30], showing that phosphorylation at Ser36 indeed directly activates the oxidoreductase activity of p66CH2CB.


Identification of Peroxiredoxin 1 as a novel interaction partner for the lifespan regulator protein p66Shc.

Gertz M, Fischer F, Leipelt M, Wolters D, Steegborn C - Aging (Albany NY) (2009)

Effects of phosphorylation and Pin1 binding on p66Shc. (a-c) The                                            p66CH2CB-dependent ROS-generation is enhanced by phosphorylation of Ser36                                            but inhibited in the presence of Pin1. Changes in fluorescence of 10 μM H2DFFDA                                            were recorded after addition of 20 μM p66CH2CB WT (a) or the                                            p66CH2CB-Ser36Asp mutant simulating Ser36 phosphorylation (a-c),                                            followed by 85 μM Na-dithionite (DN) and 50 μM CuSO4 (Cu) in the                                            presence (b+c) or absence of Pin1 (a-c) and/or the dipeptide                                            Ala-Pro (c). (d) p66CH2CB-induced mitochondrial rupture is                                            inhibited by phosphorylation of Ser36. Mitochondrial rupture was induced                                            after addition of 7 μM CaCl2 by addition of 20 μM p66CH2CB WT or                                            Ser36Asp and monitored photometrically. The initial sensitization with CaCl2                                            was omitted for clarity. (e) H2O2 oxidizes                                            p66CH2CB. 10 μg p66CH2CB were incubated with 0.005 % H2O2                                            and subjected to non-reducing SDS-PAGE
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Figure 1: Effects of phosphorylation and Pin1 binding on p66Shc. (a-c) The p66CH2CB-dependent ROS-generation is enhanced by phosphorylation of Ser36 but inhibited in the presence of Pin1. Changes in fluorescence of 10 μM H2DFFDA were recorded after addition of 20 μM p66CH2CB WT (a) or the p66CH2CB-Ser36Asp mutant simulating Ser36 phosphorylation (a-c), followed by 85 μM Na-dithionite (DN) and 50 μM CuSO4 (Cu) in the presence (b+c) or absence of Pin1 (a-c) and/or the dipeptide Ala-Pro (c). (d) p66CH2CB-induced mitochondrial rupture is inhibited by phosphorylation of Ser36. Mitochondrial rupture was induced after addition of 7 μM CaCl2 by addition of 20 μM p66CH2CB WT or Ser36Asp and monitored photometrically. The initial sensitization with CaCl2 was omitted for clarity. (e) H2O2 oxidizes p66CH2CB. 10 μg p66CH2CB were incubated with 0.005 % H2O2 and subjected to non-reducing SDS-PAGE
Mentions: Cells need to have mechanisms strictly silencing the cytotoxic activities of p66Shc, ROS-generation and apoptosis-induction, during cell cycle but stimulating them during cellular stress. PKCβ and the peptidyl-prolyl isomerase Pin1 have been reported to regulate the relocalization of p66Shc to mitochondria required for apoptosis initiation. PKCβ phosphorylates p66Shc at Ser36 in response to oxidative stress [19], which was reported already to increase cellular ROS levels [24]. We tested whether phosphorylation leads to a direct stimulation of the p66Shc-inherent oxidoreductase activity. We simulated phosphorylation by mutating Ser36 in p66CH2CB to Asp and compared the ROS-generating activity of the Ser36Asp mutant to wildtype (WT) protein in a fluorescence-based H2O2 assay (Figure 1a). The p66CH2CB-Ser36Asp mutant displayed a significantly increased activity compared to WT protein, independent of the dimer/tetramer equilibrium (data not shown) that is known to influence ROS formation [30], showing that phosphorylation at Ser36 indeed directly activates the oxidoreductase activity of p66CH2CB.

Bottom Line: Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain.The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture.Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, 44801 Bochum, Germany.

ABSTRACT
Damage caused by reactive oxygen species (ROS) contributes to many aging processes and accompanying diseases. ROS are toxic side products of cellular respiration, but also function as signal, e.g. in the mitochondrial apoptosis pathway. The protein p66Shc, which has been implicated in life-span regulation and aging-related diseases, is a central player in stress-induced apoptosis and the associated ROS burst. Stress signals, such as UV radiation or ROS themselves, activate p66Shc, which was proposed to stimulate its H(2)O(2) forming activity, ultimately triggering mitochondrial disintegration. However, mechanistic details of H(2)O(2) formation and apoptosis induction by p66Shc and regulation of these activities remain to be revealed. Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain. Prx1 was identified in affinity experiments as dominant interaction partner. Complex formation leads to disassembly of Prx1 decamers, which is known to increase its peroxidase activity. The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture. Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.

Show MeSH
Related in: MedlinePlus