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Binding of 14-3-3 stabilises recombinant AMPKγ2-containing complexes.

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AMP-activated protein kinase (AMPK) long gamma-2 subunits bind to 14-3-3 proteins, reducing AMPK activity. This interaction reveals a new regulatory mechanism for AMPK energy homeostasis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Metabolism

Background:

  • AMP-activated protein kinase (AMPK) is crucial for maintaining cellular energy homeostasis in mammals.
  • AMPK exists as a heterotrimer composed of alpha, beta, and gamma subunits, with multiple isoforms potentially forming 12 distinct complexes.
  • Regulation of AMPK activity is essential for cellular energy balance, but novel regulatory mechanisms are continually being discovered.

Purpose of the Study:

  • To investigate the interaction between long forms of the AMPK gamma-2 subunit (γ2a and γ2c) and 14-3-3 proteins.
  • To characterize the functional consequences of this interaction on AMPK activity and complex formation.
  • To elucidate the structural basis of the 14-3-3 binding to the AMPK gamma-2 subunit.

Main Methods:

  • Bacterial expression of AMPK complexes containing different gamma-2 isoforms.
  • Co-immunoprecipitation assays to detect protein interactions.
  • In vitro binding assays using phosphorylated peptides and 14-3-3 proteins.
  • Crystal structure determination of the 14-3-3-peptide complex.

Main Results:

  • AMPK complexes containing long gamma-2 isoforms (γ2a, γ2c) bind to 14-3-3 proteins.
  • This binding requires prior phosphorylation of Thr172 on the alpha subunit and is dependent on co-expression with 14-3-3.
  • AMPKγ2-14-3-3 complexes exhibit reduced activity compared to AMPKγ1 or short gamma-2 (γ2b) complexes, but retain allosteric activation by AMP and 991.
  • Two phosphorylated sites (T97 and S122) in the N-terminal region of γ2a were identified as 14-3-3 binding sites.

Conclusions:

  • 14-3-3 proteins bind to the N-terminal region of long gamma-2 isoforms (γ2a/c), representing a novel regulatory mechanism for AMPK.
  • This interaction leads to reduced AMPK activity, impacting cellular energy homeostasis.
  • Further studies are needed to determine the biological significance of this regulatory pathway in vivo.