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Phosphoregulation of tropomyosin-actin interaction revealed using a genetic code expansion strategy

Saravanan Palani1, Darius Koester1, Mohan K Balasubramanian1

  • 1Centre for Mechanochemical Cell Biology, Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK.

Wellcome Open Research
|August 18, 2020
PubMed

Insights

Phosphorylation of serine 125 in tropomyosin (Cdc8) reduces its actin binding affinity. This study uses genetic code expansion to directly demonstrate how phosphoserine impacts Cdc8-actin interactions, revealing mechanisms of actin cytoskeleton regulation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Tropomyosins are crucial for actin cytoskeleton stability and function.
  • Phosphorylation of tropomyosin (Cdc8) at serine 125 was previously shown to decrease actin filament affinity.
  • This mechanism regulates actin dynamics by allowing actin-severing proteins access to filaments.

Purpose of the Study:

  • To directly examine the effect of serine 125 phosphorylation on Cdc8-actin filament binding.
  • To validate the role of phosphoserine in modulating tropomyosin affinity for actin.
  • To showcase the utility of genetic code expansion for studying protein modifications and interactions.

Main Methods:

  • Utilized genetic code expansion in *Escherichia coli* to produce Cdc8-tropomyosin with phosphoserine at position 125 (Cdc8 PS125).
  • Employed an orthogonal tRNA-tRNA synthetase pair for site-specific phosphoserine incorporation.
  • Applied total internal reflection (TIRF) microscopy to visualize and quantify Cdc8-actin filament binding.

Main Results:

  • *E. coli*-produced Cdc8 PS125 exhibited no binding to actin filaments.
  • Incubation of Cdc8 PS125 with lambda phosphatase restored actin filament binding.
  • Demonstrated that the phosphoserine moiety at serine 125 directly reduces Cdc8's affinity for actin filaments.

Conclusions:

  • Directly confirmed that a phosphate group on serine 125 of Cdc8 significantly decreases its binding affinity to actin.
  • Established a direct link between tropomyosin phosphorylation and actin filament instability.
  • Highlighted the power of genetic code expansion for probing post-translational modifications and their functional consequences in cytoskeletal dynamics.
Keywords:
ActinCytokinesesSynthetic biologyTropomyosin

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