Phospholamban interacts with HAX-1, a mitochondrial protein with anti-apoptotic function.
Phospholamban (PLN) is a key regulator of Ca(2+) homeostasis and contractility in the heart. Its regulatory effects are mediated through its interaction with the sarcoplasmic reticulum Ca(2+)-ATPase, (SERCA2a), resulting in alterations of its Ca(2+)-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 K(D) of approximately 1 muM 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 Ca(2+) 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 Ca(2+) handling by the sarcoplasmic reticulum and cell survival mediated by the PLN/HAX-1 interaction.
Vafiadaki
Elizabeth
E
Molecular Biology Division, Center for Basic Research, Foundation for Biomedical Research of the Academy of Athens, Soranou Efesiou 4, Athens 115 27, Greece.
Sanoudou
Despina
D
Arvanitis
Demetrios A
DA
Catino
Dawn H
DH
Kranias
Evangelia G
EG
Kontrogianni-Konstantopoulos
Aikaterini
A
D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D006801:Humans; D024101:Mitochondrial Proteins / Q000378:metabolism* / Q000502:physiology; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D000099211:Phospholamban
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D000099211:Phospholamban; C104759:HAX1 protein, human