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Western Blotting-Based Quantitative Measurement of Myosin II Regulatory Light Chain Phosphorylation in Small Amounts of Non-muscle Cells

Biochemistry (Moscow) volume 84, pages 11–19 (2019)Cite this article

Abstract

Myosin II is the main molecular motor in the actomyosin-dependent motility in cells. Phosphorylation of the myosin regulatory light chain (RLC) at Ser19 is a prerequisite for smooth muscle/non-muscle myosin II activation and serves as a biochemical equivalent of myosin II activity. Simultaneous phosphorylation at Thr18 further promotes the myosin II ATPase activity. A number of methods have been developed to measure myosin RLC phosphorylation at Ser19 or di-phosphorylation at Thr18/Ser19. While these methods are straightforward and robust in myosin-rich muscle tissues, they demonstrate limited applicability in non-muscle cells that have low myosin II content and are usually available in lesser amounts than muscle tissue. Because of this, dynamic analysis of RLC phosphorylation in multiple samples of non-muscle cells is difficult and requires large number of cells. The use of phospho-specific antibodies increases detection sensitivity but allows estimation of only relative levels of RLC phosphorylation at specific residues, which makes it difficult to estimate the physiologic relevancy of the observed changes in RLC phosphorylation. To measure RLC phosphorylation in small amounts of non-muscle cells, we used external calibration standards of non-phosphorylated and in vitro phosphorylated RLC in standard SDS-PAGE and Western blot procedures with phospho-specific RLC antibodies. Here, we describe the method in detail and demonstrate its application for quantitative measurement of myosin RLC phosphorylation in endothelial cells in response to natural agonists (thrombin or insulin) and intact human platelets. We discuss the advantages and limitations of the proposed method vs other approaches for measuring myosin RLC phosphorylation in non-muscle cells.

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Abbreviations

CaM:

calmodulin

DFP:

diisopropyl fluorophos-phate

DMEM:

Dulbecco’s modified essential medium

DMSO:

dimethyl sulfoxide

FBS:

fetal bovine serum

IPTG:

isopropyl β-D-thiogalactopyranoside

MLCK:

myosin light chain kinase

MOPS:

3-(N-morpholino)propanesulfonic acid

MYPT1:

myosin phosphatase target subunit 1

NMII:

non-muscle myosin II

PBS:

phosphate buffered saline

PVDF:

polyvinylidene difluoride

RLC:

regulatory light chain

ROCK:

Rho-associated protein kinase

SMMII:

smooth muscle myosin II

TBS:

Tris buffered saline

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Author information

Affiliations

  1. National Medical Research Center for Cardiology, 121552, Moscow, Russia

    O. A. Kazakova, A. Y. Khapchaev & V. P. Shirinsky

  2. Lomonosov Moscow State University, Faculty of Fundamental Medicine, 119192, Moscow, Russia

    A. Y. Khapchaev & V. P. Shirinsky

  3. I. M. Sechenov First Moscow State Medical University, 119991, Moscow, Russia

    A. A. Ragimov & E. L. Salimov

Authors
  1. O. A. Kazakova
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  2. A. Y. Khapchaev
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  3. A. A. Ragimov
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  4. E. L. Salimov
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  5. V. P. Shirinsky
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Corresponding author

Correspondence to A. Y. Khapchaev.

Additional information

Published in Russian in Biokhimiya, 2019, Vol. 84, No. 1, pp. 74–84.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM18-193, October 8, 2018.

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Kazakova, O.A., Khapchaev, A.Y., Ragimov, A.A. et al. Western Blotting-Based Quantitative Measurement of Myosin II Regulatory Light Chain Phosphorylation in Small Amounts of Non-muscle Cells. Biochemistry Moscow 84, 11–19 (2019). https://doi.org/10.1134/S0006297919010024

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  • DOI: https://doi.org/10.1134/S0006297919010024

Keywords

  • myosin regulatory light chain
  • non-muscle cells
  • phosphorylation
  • Western blotting
  • quantitative measurement