Biochemistry (Moscow)

, Volume 81, Issue 9, pp 1013–1022

Identification of proteins whose interaction with Na+,K+-ATPase is triggered by ouabain

  • O. A. Akimova
  • L. V. Kapilevich
  • S. N. Orlov
  • O. D. Lopina
Article

DOI: 10.1134/S0006297916090108

Cite this article as:
Akimova, O.A., Kapilevich, L.V., Orlov, S.N. et al. Biochemistry Moscow (2016) 81: 1013. doi:10.1134/S0006297916090108
  • 55 Downloads

Abstract

Prolonged exposure of different epithelial cells (canine renal epithelial cells (MDCK), vascular endothelial cells from porcine aorta (PAEC), human umbilical vein endothelial cells (HUVEC), cervical adenocarcinoma (HeLa), as well as epithelial cells from colon carcinoma (Caco-2)) with ouabain or with other cardiotonic steroids was shown earlier to result in the death of these cells. Intermediates in the cell death signal cascade remain unknown. In the present study, we used proteomics methods for identification of proteins whose interaction with Na+,K+-ATPase is triggered by ouabain. After exposure of Caco-2 human colorectal adenocarcinoma cells with 3 μM of ouabain for 3 h, the protein interacting in complex with Na+,K+-ATPase was coimmunoprecipitated using antibodies against the enzyme α1-subunit. Proteins of coimmunoprecipitates were separated by 2D electrophoresis in polyacrylamide gel. A number of proteins in the coimmunoprecipitates with molecular masses of 71-74, 46, 40-43, 38, and 33-35 kDa was revealed whose binding to Na+,K+-ATPase was activated by ouabain. Analyses conducted by mass spectroscopy allowed us to identify some of them, including seven signal proteins from superfamilies of glucocorticoid receptors, serine/threonine protein kinases, and protein phosphatases 2C, Src-, and Rho-GTPases. The possible participation of these proteins in activation of cell signaling terminated by cell death is discussed.

Keywords

Na+,K+-ATPase ouabain coimmunoprecipitation 2D-PAGE mass spectrometry 

Abbreviations

AKT

protein kinase B

CAMTA1

calmodulinbinding transcription activator 1

CG-1

DNA-binding domain

CTS

cardiotonic steroids

Erk

extracellular signalregulated protein kinase

IP3R

inositol-1,4,5-trisphosphate receptor

IQ

calmodulin-binding motif

Jnk

c-Jun kinase (stress-activated protein kinase)

MAPK

mitogen-activated protein kinase

MEK

protein kinase MAPK/Erk

pHi

intracellular pH

PI3K

inositol-3-phosphate kinase

Raf

serine/threonine protein kinase

Rac and Rho

small GTPbinding proteins

RIPA

buffer for radioimmunoprecipitation

SGK2

serum/glucocorticoid-regulated kinase 2

TIG

transcription factor immunoglobulin/DNA-binding domain

Copyright information

© Pleiades Publishing, Ltd. 2016

Authors and Affiliations

  • O. A. Akimova
    • 1
  • L. V. Kapilevich
    • 2
  • S. N. Orlov
    • 1
    • 2
    • 3
  • O. D. Lopina
    • 1
  1. 1.Lomonosov Moscow State UniversityFaculty of BiologyMoscowRussia
  2. 2.Tomsk State UniversityTomskRussia
  3. 3.Siberian State Medical UniversityTomskRussia

Personalised recommendations