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Analysis of Site-Specific Phosphorylation of PTEN by Using Enzyme-Catalyzed Expressed Protein Ligation

Samuel H Henager1, Stephanie Henriquez1, Daniel R Dempsey2

  • 1Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD, 21205, USA.

Chembiochem : a European Journal of Chemical Biology
|June 18, 2019
PubMed

Insights

This study introduces subtiligase-mediated expressed protein ligation (EPL) to create wild-type monophosphorylated PTEN proteins. These PTENs exhibit enhanced autoinhibition and improved antibody recognition, crucial for understanding tumor suppressor activity.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cancer Research

Background:

  • PTEN is a crucial tumor suppressor lipid phosphatase.
  • Its activity and localization are regulated by phosphorylation near the C-terminus.
  • Previous studies using classical EPL were limited by a non-native Cys residue.

Purpose of the Study:

  • To generate wild-type monophosphorylated PTEN proteins (wt 1p-PTENs) using subtiligase-mediated EPL.
  • To characterize the autoinhibition and antibody recognition of these wt 1p-PTENs.
  • To compare these findings with previously studied Cys mutant PTENs.

Main Methods:

  • Subtiligase-mediated expressed protein ligation (EPL) for semisynthesis.
  • Enzymatic characterization of monophosphorylated PTEN variants.
  • Alkaline phosphatase sensitivity analysis.
  • Antibody recognition assays using anti-phospho-PTEN antibody.

Main Results:

  • Subtiligase-mediated EPL successfully created wt 1p-PTENs without a non-native Cys residue.
  • These wt 1p-PTENs display a higher degree of autoinhibition than previously observed.
  • The autoinhibited conformations were found to be kinetically labile.
  • Three of the four wt 1p-PTENs were recognized by a common anti-phospho-PTEN antibody, unlike Cys mutants.

Conclusions:

  • PTEN autoinhibition is more significant than previously appreciated, influenced by Tyr379.
  • Subtiligase-mediated EPL is a valuable tool for studying PTEN phosphorylation.
  • The phosphorylation status of PTEN influences its recognition by specific antibodies, important for diagnostic and research applications.
Keywords:
PTENenzyme kineticsphosphatasesphosphorylationsemisynthesis

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