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.

LanguageEnglish
Pages24-34
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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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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