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Vol. 20, Issue 1, 306-318, January 1, 2009

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The Anti-apoptotic Protein HAX-1 Interacts with SERCA2 and Regulates Its Protein Levels to Promote Cell Survival

Elizabeth Vafiadaki^*, Demetrios A. Arvanitis^*, Stamatis N. Pagakis^*, Vasiliki Papalouka^*, Despina Sanoudou^*, Aikaterini Kontrogianni-Konstantopoulos^,, and Evangelia G. Kranias^*,,

*Molecular Biology Division, Biomedical Research Foundation, Academy of Athens, Athens, 115 27, Greece; Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, MD 21201; and Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, OH 45267-0575

Submitted June 11, 2008; Revised September 24, 2008; Accepted October 22, 2008
Monitoring Editor: M. Bishr Omary

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.

These authors contributed equally to this work.

Address correspondence to: Aikaterini Kontrogianni-Konstantopoulos (akons001{at}umaryland.edu) or Evangelia G. Kranias (litsa.kranias{at}uc.edu).

Abbreviations used: GFP, green fluorescent protein; GST, glutathione transferase; HAX-1, HS-1 associated protein X-1; HEK, human embryonic kidney; PBS, phosphate-buffered saline; PLN, phospholamban; SERCA2a, sarcoplasmic reticulum Ca²⁺-ATPase; SR, sarcoplasmic reticulum.