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Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2

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† Group of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 100101
‡ University of Chinese Academy of Sciences, Beijing, China 100049
§ Laboratory of Animal Models & Functional Genomics (LAMFG), Research Center of Reverse Vaccinology (RCRV), College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, China 410128
*Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. Telephone: +86-10-6480-6015. E-mail: [email protected]
Cite this: Biochemistry 2017, 56, 32, 4235–4243
Publication Date (Web):July 17, 2017
https://doi.org/10.1021/acs.biochem.7b00311
Copyright © 2017 American Chemical Society
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    Abstract

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    Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 μM for SmM, 3.58 μM for NM2a, 2.30 μM for NM2b, and 1.57 μM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00311.

    • Effects of blebbistatin on the in vitro actin gliding activities of SmM-FL, NM2a-FL, NM2b-FL, and Myo5a-HMM (Figure S1) (PDF)

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