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

Wellcome Open Research

article

Saravanan Palani¹  Darius Koester¹  Mohan K. Balasubramanian¹ 

[1] Centre for Mechanochemical Cell Biology, Division of Biomedical Sciences, Warwick Medical School, University of Warwick

关键词: Cytokineses;    Actin;    Tropomyosin;    Synthetic biology;   

DOI  :  10.12688/wellcomeopenres.16082.1

学科分类：内科医学

来源: Wellcome

【 摘 要 】

Tropomyosins are coiled-coil proteins that regulate the stability and / or function of actin cytoskeleton in muscle and non-muscle cells through direct binding of actin filaments. Recently, using the fission yeast, we discovered a new mechanism by which phosphorylation of serine 125 of tropomyosin (Cdc8), reduced its affinity for actin filaments thereby providing access for the actin severing protein Adf1/Cofilin to actin filaments causing instability of actin filaments. Here we use a genetic code expansion strategy to directly examine this conclusion. We produced inEscherichia coli Cdc8-tropomyosin bearing a phosphate group on Serine-125 (Cdc8PS125), using an orthogonal tRNA-tRNA synthetase pair that directly incorporates phosphoserine into proteins in response to a UAG codon in the corresponding mRNA. We show using total internal reflection (TIRF) microscopy that, whereasE.coli produced Cdc8PS125 does not bind actin filaments, Cdc8PS125 incubated with lambda phosphatase binds actin filaments. This work directly demonstrates that a phosphate moiety present on serine 125 leads to decreased affinity of Cdc8-tropomyosin for actin filaments. We also extend the work to demonstrate the usefulness of the genetic code expansion approach in imaging actin cytoskeletal components.

【 授权许可】

CC BY