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Biochemistry (Moscow)

, Volume 84, Issue 1, pp 11–19 | Cite as

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

  • O. A. Kazakova
  • A. Y. KhapchaevEmail author
  • A. A. Ragimov
  • E. L. Salimov
  • V. P. Shirinsky
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.

Keywords

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

Abbreviation

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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Copyright information

© Pleiades Publishing, Inc. 2019

Authors and Affiliations

  • O. A. Kazakova
    • 1
  • A. Y. Khapchaev
    • 1
    • 2
    Email author
  • A. A. Ragimov
    • 3
  • E. L. Salimov
    • 3
  • V. P. Shirinsky
    • 1
    • 2
  1. 1.National Medical Research Center for CardiologyMoscowRussia
  2. 2.Lomonosov Moscow State UniversityFaculty of Fundamental MedicineMoscowRussia
  3. 3.I. M. Sechenov First Moscow State Medical UniversityMoscowRussia

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