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Actin is a highly conserved cytoskeletal protein found abundantly in eukaryotic cells. It constitutes 10% weight of the total cellular protein in muscle cells, while in non-muscle cells, it is lower and makes up around 1–5 percent of the total cell protein. Actin found in the unicellular amoebae and complex multicellular animals is around 80% similar, demonstrating their conservation over a billion years of evolution.  Actin coding genes are conserved within species and across...
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Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function.

Timothy Travers1, Hanshuang Shao2, Brian A Joughin3

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Tandem-site phosphorylation in intrinsically disordered protein regions can act as a switch. Phosphorylation at Tyr(4) of alpha-actinin-4 (ACTN4) exposes Tyr(31) for regulation, impacting actin interaction.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Phosphorylation predominantly occurs in intrinsically disordered regions of eukaryotic proteins.
  • Human alpha-actinin-4 (ACTN4) has intrinsically disordered regions, with specific tyrosines (Tyr(4), Tyr(31)) being phosphorylated upon epidermal growth factor (EGF) stimulation.
  • m-calpain cleavage removes the Tyr(4) site from ACTN4.

Purpose of the Study:

  • To investigate the mechanism of tandem-site phosphorylation in intrinsically disordered regions.
  • To determine the role of Tyr(4) and Tyr(31) phosphorylation in ACTN4's interaction with actin.
  • To elucidate the regulation of ACTN4 phosphorylation by receptor tyrosine kinases.

Main Methods:

  • In vitro biochemical assays to assess protein-protein interactions.
  • Site-directed mutagenesis to create phosphorylation-mimicking mutants.
  • Molecular dynamics simulations to predict protein structure and accessibility.
  • Cell-based assays using fibroblast cells and EGF stimulation.
  • Mathematical modeling to analyze phosphorylation kinetics.
  • Inhibition of TAM family receptor tyrosine kinases (e.g., AXL).

Main Results:

  • Phosphorylation-mimicking mutations at Tyr(31) alone reduced ACTN4-actin interaction in vitro.
  • Molecular dynamics simulations indicated Tyr(31) is buried, and Tyr(4) phosphorylation increases Tyr(31) solvent exposure.
  • EGF stimulation increased ACTN4 phosphorylation at Tyr(4) in mutant forms.
  • m-calpain cleavage product showed background phosphorylation levels, similar to a double mutant.
  • Inhibition of AXL blocked EGF-stimulated ACTN4 tyrosine phosphorylation.
  • Mathematical modeling suggested Tyr(4) kinase affinity dictates Tyr(31) phosphorylation kinetics.

Conclusions:

  • Tandem-site phosphorylation in intrinsically disordered regions acts as a regulatory switch.
  • Phosphorylation of Tyr(4) in ACTN4 enhances the accessibility and subsequent phosphorylation of Tyr(31).
  • This mechanism regulates ACTN4's interaction with actin, influenced by receptor tyrosine kinases like AXL.