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In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase

Amrutha Manikandan1, Sreevidya T S2,3, Narayanan Manoj1

  • 1Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India.

Proteins
|January 16, 2023
PubMed

Insights

Protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) activates AMP-activated protein kinase (AMPK) by dephosphorylating its alpha subunit. This study identifies Tyr232 on AMPKα2 as the dephosphorylation site and shows PTP-PEST Tyr64 phosphorylation enhances binding.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis.
  • AMPK is activated by protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions.
  • The specific dephosphorylation sites targeted by PTP-PEST on AMPK remain unidentified.

Purpose of the Study:

  • To elucidate the structural basis of AMPK regulation by PTP-PEST.
  • To identify the phosphotyrosine residue on AMPKα2 dephosphorylated by PTP-PEST.
  • To investigate the role of PTP-PEST phosphorylation at Tyr64 in modulating its interaction with AMPKα2.

Main Methods:

  • Bioinformatic structure prediction of the AMPKα2-PTP-PEST complex.
  • Molecular dynamics simulations to assess complex stability.
  • Analysis of protein-protein interfaces to identify potential dephosphorylation sites.
  • Site-directed mutagenesis (PTP-PEST-Y64D) and co-immunoprecipitation assays.

Main Results:

  • Bioinformatic analysis predicted Tyr232 on AMPKα2 as the primary dephosphorylation site by PTP-PEST.
  • Phosphorylation of PTP-PEST residue Tyr64 was found to enhance complex stability through electrostatic interactions and WPD loop conformational changes.
  • The PTP-PEST-Y64D mutant demonstrated increased binding affinity to AMPKα2, validating in-silico predictions.

Conclusions:

  • This study provides a structural framework for understanding how PTP-PEST regulates AMPK activity.
  • Phosphorylation of PTP-PEST at Tyr64 significantly enhances its interaction with AMPKα2.
  • The findings highlight the intricate interplay between phosphorylation and dephosphorylation in cellular energy regulation.
Keywords:
AMP-activated protein kinasemolecular docking analysismolecular dynamics simulationphosphorylation sitesprotein tyrosine phosphatase

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