Abstract
Myosin light chain kinases (MLCK) phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase (S-MLCK) to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase (L-MLCK) has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules (2Ig) in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased “spike”-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.
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Acknowledgements
We thank Dr James Stull and Dr Kristine Kamm for their generous help and instructive suggestions, Dr Qing Shun Zhao for valuable discussions on the manuscripts, Dr Jiong Chen for editing the manuscript, Min Yan Yu for cell culture help and Peng Yu Gu for technical assistance on confocal microscopy. This work was supported by National Natural Science Foundation of China (No. 30470852) and the National Gongguan Project of China (21001BA710B).
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Yang, C., Chen, H., Chen, C. et al. Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase. Cell Res 16, 367–376 (2006). https://doi.org/10.1038/sj.cr.7310047
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DOI: https://doi.org/10.1038/sj.cr.7310047
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