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

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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.

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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

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Authors and Affiliations

  1. Lomonosov Moscow State University, Faculty of Biology, 119991, Moscow, Russia

    O. A. Akimova, S. N. Orlov & O. D. Lopina

  2. Tomsk State University, 634050, Tomsk, Russia

    L. V. Kapilevich & S. N. Orlov

  3. Siberian State Medical University, 634050, Tomsk, Russia

    S. N. Orlov

Authors
  1. O. A. Akimova
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  2. L. V. Kapilevich
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  3. S. N. Orlov
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  4. O. D. Lopina
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Published in Russian in Biokhimiya, 2016, Vol. 81, No. 9, pp. 1269-1279.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM16-094, August 15, 2016.

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Akimova, O.A., Kapilevich, L.V., Orlov, S.N. et al. Identification of proteins whose interaction with Na+,K+-ATPase is triggered by ouabain. Biochemistry Moscow 81, 1013–1022 (2016). https://doi.org/10.1134/S0006297916090108

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  • DOI: https://doi.org/10.1134/S0006297916090108

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