Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins

Ivan Verrastro, Karina Tveen-Jensen, Rudiger Woscholski, Corinne M. Spickett, Andrew R. Pitt

Research output: Contribution to journalArticle

Abstract

Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

Original languageEnglish
Pages (from-to)24-34
Number of pages11
JournalFree Radical Biology and Medicine
Volume90
Early online date10 Nov 2015
DOIs
Publication statusPublished - 1 Jan 2016

Fingerprint

Phosphoric Monoester Hydrolases
Oxidation
Oxidation-Reduction
Proteins
Dual-Specificity Phosphatases
Tensins
Phosphoprotein Phosphatases
Cell proliferation
Phosphatidylinositol 3-Kinases
Metabolism
Sepharose
Glutathione
Labels
Tumors
Cell Survival
Resins
Western Blotting
Cell Proliferation
Apoptosis

Bibliographical note

© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Funding: EPSRC (EP/I017887/1 Cross-Disciplinary Research Landscape Award).
Data associated with this paper can be obtained by contacting the corresponding author.

Supplementary material avaialble on the journal website

Keywords

  • DNA-binding proteins
  • disulfides
  • glutathione
  • HCT116 cells
  • oxidation-reduction
  • PTEN phosphohydrolase
  • peroxiredoxins
  • proteomics
  • signal transduction
  • thioredoxins

Cite this

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abstract = "Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.",
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note = "{\circledC} 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funding: EPSRC (EP/I017887/1 Cross-Disciplinary Research Landscape Award). Data associated with this paper can be obtained by contacting the corresponding author. Supplementary material avaialble on the journal website",
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AU - Verrastro, Ivan

AU - Tveen-Jensen, Karina

AU - Woscholski, Rudiger

AU - Spickett, Corinne M.

AU - Pitt, Andrew R.

N1 - © 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funding: EPSRC (EP/I017887/1 Cross-Disciplinary Research Landscape Award). Data associated with this paper can be obtained by contacting the corresponding author. Supplementary material avaialble on the journal website

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N2 - Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

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KW - proteomics

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