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.