Phospholamban Interacts with HAX-1, a Mitochondrial Protein with Anti-apoptotic Function

Elizabeth Vafiadaki, Despina Sanoudou, Demetrios A. Arvanitis, Dawn H. Catino, Evangelia G. Kranias, Aikaterini Kontrogianni-Konstantopoulos

Research output: Contribution to journalArticle

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Abstract

Phospholamban (PLN) is a key regulator of Ca2+ homeostasis and contractility in the heart. Its regulatory effects are mediated through its interaction with the sarcoplasmic reticulum Ca2+-ATPase, (SERCA2a), resulting in alterations of its Ca2+-affinity. To identify additional proteins that may interact with PLN, we used the yeast-two-hybrid system to screen an adult human cardiac cDNA library. HS-1 associated protein X-1 (HAX-1) was identified as a PLN-binding partner. The minimal binding regions were mapped to amino acid residues 203-245 for HAX-1 and residues 16-22 for PLN. The interaction between the two proteins was confirmed using GST-HAX-1, bound to the glutathione-matrix, which specifically adsorbed native PLN from human or mouse cardiac homogenates, while in reciprocal binding studies, recombinant His-HAX-1 bound GST-PLN. Kinetic studies using surface plasmon resonance yielded a KD of ∼ 1 μM as the binding affinity for the PLN/HAX-1 complex. Phosphorylation of PLN by cAMP-dependent protein kinase reduced binding to HAX-1, while increasing concentrations of Ca2+ diminished the PLN/HAX-1 interaction in a dose-dependent manner. HAX-1 concentrated to mitochondria, but upon transient co-transfection of HEK 293 cells with PLN, HAX-1 redistributed and co-localized with PLN at the endoplasmic reticulum. Analysis of the anti-apoptotic function of HAX-1 revealed that the presence of PLN enhanced the HAX-1 protective effects from hypoxia/reoxygenation-induced cell death. These findings suggest a possible link between the Ca2+ handling by the sarcoplasmic reticulum and cell survival mediated by the PLN/HAX-1 interaction.

Original languageEnglish
Pages (from-to)65-79
Number of pages15
JournalJournal of Molecular Biology
Volume367
Issue number1
DOIs
Publication statusPublished - 16 Mar 2007
Externally publishedYes

Fingerprint

Mitochondrial Proteins
Proteins
Sarcoplasmic Reticulum
phospholamban
Myocardial Contraction
Two-Hybrid System Techniques
Surface Plasmon Resonance
Calcium-Transporting ATPases
HEK293 Cells
Cyclic AMP-Dependent Protein Kinases
Gene Library
Endoplasmic Reticulum
Transfection
Glutathione
Cell Survival
Mitochondria
Homeostasis
Cell Death

Keywords

  • calcium homeostasis
  • cardiac muscle
  • HAX-1
  • phospholamban
  • sarcoplasmic reticulum

ASJC Scopus subject areas

  • Virology

Cite this

Vafiadaki, E., Sanoudou, D., Arvanitis, D. A., Catino, D. H., Kranias, E. G., & Kontrogianni-Konstantopoulos, A. (2007). Phospholamban Interacts with HAX-1, a Mitochondrial Protein with Anti-apoptotic Function. Journal of Molecular Biology, 367(1), 65-79. https://doi.org/10.1016/j.jmb.2006.10.057

Phospholamban Interacts with HAX-1, a Mitochondrial Protein with Anti-apoptotic Function. / Vafiadaki, Elizabeth; Sanoudou, Despina; Arvanitis, Demetrios A.; Catino, Dawn H.; Kranias, Evangelia G.; Kontrogianni-Konstantopoulos, Aikaterini.

In: Journal of Molecular Biology, Vol. 367, No. 1, 16.03.2007, p. 65-79.

Research output: Contribution to journalArticle

Vafiadaki, E, Sanoudou, D, Arvanitis, DA, Catino, DH, Kranias, EG & Kontrogianni-Konstantopoulos, A 2007, 'Phospholamban Interacts with HAX-1, a Mitochondrial Protein with Anti-apoptotic Function', Journal of Molecular Biology, vol. 367, no. 1, pp. 65-79. https://doi.org/10.1016/j.jmb.2006.10.057
Vafiadaki E, Sanoudou D, Arvanitis DA, Catino DH, Kranias EG, Kontrogianni-Konstantopoulos A. Phospholamban Interacts with HAX-1, a Mitochondrial Protein with Anti-apoptotic Function. Journal of Molecular Biology. 2007 Mar 16;367(1):65-79. https://doi.org/10.1016/j.jmb.2006.10.057
Vafiadaki, Elizabeth ; Sanoudou, Despina ; Arvanitis, Demetrios A. ; Catino, Dawn H. ; Kranias, Evangelia G. ; Kontrogianni-Konstantopoulos, Aikaterini. / Phospholamban Interacts with HAX-1, a Mitochondrial Protein with Anti-apoptotic Function. In: Journal of Molecular Biology. 2007 ; Vol. 367, No. 1. pp. 65-79.
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