Advertisement

Biochemistry (Moscow)

, Volume 81, Issue 9, pp 1013–1022 | Cite as

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

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

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Schoner, W., and Scheiner-Bobis, G. (2008) Role of endogenous cardiotonic steroids in sodium homeostasis, Nephrol. Dial. Transpl., 23, 2723–2729.CrossRefGoogle Scholar
  2. 2.
    Orlov, S. N., Akimova, O. A., and Hamet, P. (2005) Cardiotonic steroids: novel mechanisms of [Na+]i-mediated and -independent signaling involved in the regulation of gene expression, proliferation and cell death, Curr. Hypertens. Rev., 1, 243–257.CrossRefGoogle Scholar
  3. 3.
    Bagrov, A. Y., Shapiro, J. I., and Fedorova, O. V. (2009) Endogenous cardiotonic steroids: physiology, pharmacology, and novel therapeutic targets, Pharmacol. Rev., 61, 9–38.PubMedGoogle Scholar
  4. 4.
    Tian, J., and Xie, Z. (2008) The Na-K-ATPase and calcium-signaling microdomains, Physiology, 23, 205–211.CrossRefPubMedGoogle Scholar
  5. 5.
    Aperia, A. (2007) New roles for an old Na,K-ATPase emerges as an interesting drug target, J. Intern. Med., 261, 44–52.CrossRefPubMedGoogle Scholar
  6. 6.
    Orlov, S. N., and Hamet, P. (2015) Salt and gene expression: evidence for [Na+]i,[K+]i-mediated signaling pathways, Pfluger’s Arch., 467, 489–498.CrossRefGoogle Scholar
  7. 7.
    Pchejetski, D., Taurin, S., Der Sarkissian, S., Lopina, O. D., Pshezhetsky, A. V., Tremblay, J., De Blois, D., Hamet, P., and Orlov, S. N. (2003) Inhibition of Na+,K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio, Biochem. Biophys. Res. Commun., 301, 735–744.CrossRefPubMedGoogle Scholar
  8. 8.
    Akimova, O. A., Bagrov, A. Y., Lopina, O. D., Kamernitsky, A. V., Tremblay, J., Hamet, P., and Orlov, S. N. (2005) Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells, J. Biol. Chem., 280, 832–839.CrossRefPubMedGoogle Scholar
  9. 9.
    Boehning, D., Patterson, R. L., Sedaghat, L., Glebova, N. O., Kurosaki, T., and Snyder, S. H. (2003) Cytochrome c binds to inositol(1,4,5)triphosphate receptors, amplifying calcium-dependent apoptosis, Nature Cell Biol., 5, 10511061.Google Scholar
  10. 10.
    Akimova, O. A., Tverskoi, A. M., Smolyaninova, L. V., Mongin, A. A., Lopina, O. D., La, J., Dulin, N. O., and Orlov, S. N. (2015) Critical role of the a1-Na+,K+-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain, Apoptosis, 20, 1200–1210.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Akimova, O. A., Lopina, O. D., Hamet, P., and Orlov, S. N. (2005) Search for intermediates of Na+,K+-ATPase-mediated [Na+]i/[K+]i-independent death signaling triggered by cardiotonic steroids, Pathophysiology, 12, 125–135.CrossRefPubMedGoogle Scholar
  12. 12.
    Akimova, O. A., Hamet, P., and Orlov, S. N. (2008) [Na+]i/[K+]i-independent death of ouabain-treated renal epithelial cells is not mediated by Na+,K+-ATPase internalization and de novo gene expression, Pfluger’s Arch., 455, 711–719.CrossRefGoogle Scholar
  13. 13.
    Akimova, O. A., Lopina, O. D., Rubtsov, A. M., Gekle, M., Tremblay, J., Hamet, P., and Orlov, S. N. (2009) Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated [Na+]i/[K+]i-independent signaling, Apoptosis, 14, 1266–1273.CrossRefPubMedGoogle Scholar
  14. 14.
    Akimova, O. A., Pchejetski, D., Hamet, P., and Orlov, S. N. (2006) Modest intracellular acidification suppresses death signaling in ouabain-treated cells, Pfluger’s Arch., 451, 569578.Google Scholar
  15. 15.
    Koob, R., Kraemer, D., Trippe, G., Aebi, U., and Drenckhahn, D. (1990) Association of kidney and parotid Na+,K+-ATPase with actin and analogs of spectrin and ankyrin, Eur. J. Cell Biol., 53, 93–100.PubMedGoogle Scholar
  16. 16.
    Morrow, J. S., Cianci, C. D., Ardito, T., Mann, A. S., and Kashgarin, M. (1989) Ankyrin links fodrin to the a-subunit of Na,K-ATPase in Madin–Darby canine kidney cells and in intact renal tubule cells, J. Cell Biol., 108, 455–465.CrossRefPubMedGoogle Scholar
  17. 17.
    Haas, M., Wang, H., Tian, J., and Xie, Z. (2002) Src-mediated inter-receptor cross-talk between the Na+,K+-ATPase and epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases, J. Biol. Chem., 277, 18694–18702.CrossRefPubMedGoogle Scholar
  18. 18.
    Liu, J., Kesiry, R., Periyasamy, S. M., Malhotra, D., Xie, Z., and Shapiro, J. I. (2004) Ouabain-induced endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by clathrin-dependent mechanism, Kidney Int., 66, 227–241.CrossRefPubMedGoogle Scholar
  19. 19.
    Wang, H., Haas, M., Liang, M., Cai, T., Tian, J., Li, S., and Xie, Z. (2004) Ouabain assembles signaling cascade through the caveolar Na+,K+-ATPase, J. Biol. Chem., 279, 17250–17259.CrossRefPubMedGoogle Scholar
  20. 20.
    Miakawa-Naito, A., Uhlen, P., Lal, M., Aizman, O., Mikoshiba, K., Brismar, H., Zelenin, S., and Aperia, A. (2003) Cell signaling microdomain with Na,K-ATPase and inositol-1,4,5-triphosphate receptor generates calcium oscillations, J. Biol. Chem., 278, 50355–50361.CrossRefGoogle Scholar
  21. 21.
    Chibalin, A. V., Zierath, J. R., Katz, A. I., Berggren, P. O., and Bertorello, A. M. (1998) Phosphatidylinositol 3-kinasemediated endocytosis of renal Na+,K+-ATPase a-subunit in response to dopamine, Mol. Biol. Cell, 9, 1209–1220.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Klimanova, E. A., Petrushenko, I. Y., Mitkevich, V. A., Anashkina, A. A., Orlov, S. N., Makarov, A. A., and Lopina, O. D. (2015) Binding of ouabain and marinobufagenin leads to different structural changes in Na,K-ATPase and depends on the enzyme conformation, FEBS Lett., 589, 2668–2674.CrossRefPubMedGoogle Scholar
  23. 23.
    Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silverstained polyacrylamide gels, Anal. Chem., 68, 850–858.CrossRefPubMedGoogle Scholar
  24. 24.
    Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry, Nature, 379, 466–469.CrossRefPubMedGoogle Scholar
  25. 25.
    Zhang, Z., Devarajan, P., Dorfman, A. L., and Morrow, J. S. (1998) Structure of the ankyrin-binding domain of aNa-K-ATPase, J. Biol. Chem., 273, 18681–18684.CrossRefPubMedGoogle Scholar
  26. 26.
    Devarajan, P., Scaramuzzino, D. A., and Morrow, J. S. (1994) Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase a-subunit, Proc. Natl. Acad. Sci. USA, 91, 2965–2969.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Liu, X., Spicarova, X., Rydholm, S., Li, J., Brismar, H., and Aperia, A. (2008) Ankyrin B modulates the function of Na,K-ATPase/inositol-1,4,5-triphosphate receptor signaling microdomain, J. Biol. Chem., 283, 11461–11468.CrossRefPubMedGoogle Scholar
  28. 28.
    Muller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L., and Harrison, S. C. (1995) Structure of the NF-?B p50 homodimer bound to DNA, Nature, 373, 311–317.CrossRefPubMedGoogle Scholar
  29. 29.
    Hagman, J., Gutch, M. J., Lin, H., and Grosschedl, R. (1995) FBF contains a novel zinc coordination motif and multiple dimerization and transcriptional activation domains, EMBO J., 14, 2907–2916.PubMedPubMedCentralGoogle Scholar
  30. 30.
    Bork, P., Doerks, T., Springer, T. A., and Snel, B. (1999) Domains in plexins: links to integrins and transcription factors, Trends Biochem. Sci., 24, 261–263.CrossRefPubMedGoogle Scholar
  31. 31.
    Bouche, N., Scharlat, A., Snedded, W., Bouchez, D., and Fromm, H. (2002) A novel family of calmodulin-binding transcription activators in multicellular organisms, J. Biol. Chem., 277, 21851–21861.CrossRefPubMedGoogle Scholar
  32. 32.
    Koltsova, S. V., Trushina, Y., Haloui, M., Akimova, O. A., Tremblay, J., Hamet, P., and Orlov, S. N. (2012) Ubiquitous [Na+]i/[K+]i-sensitive transcriptome in mammalian cells: evidence for [Ca2+]i-independent excitation–transcription coupling, PLoS One, 7, e38032.CrossRefGoogle Scholar
  33. 33.
    Tian, J., Cai, T., Yuan, Z., Wang, H., Liu, L., Haas, M., Maksimova, E., Huang, X.-Y., and Xie, Z.-J. (2006) Binding of Src to Na+,K+-ATPase forms a functional signaling complex, Mol. Biol. Cell, 17, 317–326.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Yuan, Z., Cai, T., Tian, J., Ivanov, A. V., Giovannucci, D. R., and Xie, Z. (2005) Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex, Mol. Biol. Cell, 16, 4034–4045.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Ayoub, E., Hall, A., Scott, A. M., Chagnon, M. J., Miquel, G., Halle, M., Noda, M., Bikfalvi, A., and Tremblay, M. L. (2013) Regulation of the Src kinase-associated phosphoprotein 55 homologue by the protein tyrosine phosphatase PTR-PEST in the control cell motility, J. Biol. Chem., 288, 25739–25748.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Akimova, O. A., Lopina, O. D., Tremblay, J., Hamet, P., and Orlov, S. N. (2008) Altered phosphorylation of RRXS*/T* motif in ouabain-treated renal epithelial cells is not mediated by inversion of the [Na+]i/[K+]i ratio, Cell. Physiol. Biochem., 21, 315–324.CrossRefPubMedGoogle Scholar
  37. 37.
    Peterson, R. T., and Schreiber, S. L. (1999) Kinase phosphorylation: keeping it all in the family, Curr. Biol., 9, R521–R524.CrossRefPubMedGoogle Scholar
  38. 38.
    Lang, F., and Cohen, P. (2001) Regulation and physiological roles of serumand glucocorticoid-induced protein kinases isoforms, Sci. STKE, 18, RE17.Google Scholar
  39. 39.
    Vyas, S., Rodrigues, A. J., Silva, J. M., Tronche, F., Almeida, O. F., Sousa, N., and Sotiropoulos, I. (2016) Chronic stress and glucocorticoids: from neuronal plasticity to neurodegeneration, Neural Plast., 2016, 6391686.Google Scholar
  40. 40.
    Matthews, L., Berry, A., Ohanian, V., Ohanian, J., Garside, H., and Ray, D. (2008) Caveolin mediates rapid glucocorticoid effects and couples glucocorticoid action to the antiproliferative program, Mol. Endocrinol., 22, 1320–1330.CrossRefPubMedGoogle Scholar
  41. 41.
    Groeneweg, F. L., Karst, H., De Kloet, E. R., and Joels, M. (2012) Mineralocorticoid and glucocorticoid receptors at the neuronal membrane, regulators of nongenomic corticosteroid signaling, Mol. Cell. Endocrinol., 350, 299–309.CrossRefPubMedGoogle Scholar
  42. 42.
    Samarasinghe, R. A., Witchell, S. F., and DeFranco, D. B. (2012) Cooperativity and complementarity: synergies in non-classic glucocorticoid signaling, Cell Cycle, 11, 28192827.CrossRefGoogle Scholar
  43. 43.
    Strehl, C., Gaber, T., Lowenberg, M., Hommes, D. W., Verhaar, A. P., Schellmann, S., Hahne, M., Fangradt, M., Wagegg, M., Hoff, P., Scheffold, A., Spies, C. M., Burmester, G. R., and Buttgereit, F. (2011) Origin and functional activity of the membrane-bound glucocorticoid receptor, Arthritis Rheum., 63, 3779–3788.CrossRefPubMedGoogle Scholar
  44. 44.
    Lifschitz-Mercer, B., Sheinin, Y., Ben-Meir, D., BramanteSchreiber, L., Leider-Trejo, L., Karby, S., Smorodinsky, N. I., and Lavi, S. (2001) Protein phosphatase 2Ca expression in normal human tissues: an immunohistochemical study, Histochem. Cell Biol., 116, 31–39.PubMedGoogle Scholar
  45. 45.
    Lammers, T., and Lavi, S. (2007) Role of type 2C protein phosphatases in growth regulation and in cellular stress signaling, Crit. Rev. Biochem. Mol. Biol., 42, 437–461.CrossRefPubMedGoogle Scholar
  46. 46.
    Takekawa, M., Maeda, T., and Saito, H. (1998) Protein phosphatase 2Ca inhibits the human stress-responsive p38 and JNK MAPK pathways, EMBO J., 17, 4744–4752.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Alves, D. S., Farr, G. A., Seo-Mayer, P., and Caplan, M. J. (2010) AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression, Mol. Biol. Cell, 21, 4400–4408.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Citi, S., Guerrera, D., Spadaro, D., and Shah, J. (2014) Epithelial junctions and Rho family GTPases: the zonular signalosome, Small GTPases, 5, 1–15.CrossRefPubMedPubMedCentralGoogle Scholar

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