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Updated: Jun 3, 2026

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
08:07

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Published on: July 26, 2019

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site

Norah E Franklin1, Gregory S Taylor, Panayiotis O Vacratsis

  • 1Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada.

The Journal of Biological Chemistry
|March 5, 2011
PubMed

Insights

Phosphorylation regulates myotubularin-related protein 2 (MTMR2) localization to endosomes. This regulation impacts MTMR2

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Myotubularin-related protein 2 (MTMR2) is an inositol lipid phosphatase involved in endosome function.
  • Mutations in the MTMR2 gene are linked to Charcot-Marie Tooth disease type 4B.
  • The regulatory mechanisms of MTMR2 activity are not well understood.

Purpose of the Study:

  • To investigate the role of reversible phosphorylation in regulating MTMR2 function.
  • To identify specific phosphorylation sites on MTMR2.
  • To elucidate how phosphorylation affects MTMR2 localization and activity.

Main Methods:

  • Mass spectrometry-based proteomics to identify phosphorylation sites.
  • Site-directed mutagenesis to create phosphorylation-deficient (S58A) and phosphomimetic (S58E) MTMR2 mutants.
  • Confocal microscopy to assess MTMR2 localization in endocytic structures.
  • Analysis of PI(3)P-specific sensor protein localization and signal transduction pathways.

Main Results:

  • A high-stoichiometry phosphorylation site was identified at serine 58 (Ser58) of MTMR2.
  • Phosphorylation or S58E mutation decreased MTMR2 localization to endocytic vesicular structures.
  • The S58A mutant showed constitutive localization to early endocytic structures.
  • Altered localization correlated with PI(3)P sensor displacement and increased signal transduction.

Conclusions:

  • Phosphorylation of MTMR2 at Ser58 is a critical regulatory mechanism.
  • This phosphorylation controls MTMR2's spatial and temporal access to its lipid substrates.
  • Regulation of MTMR2 by phosphorylation influences downstream endosome maturation events.

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