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

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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.

Introduction

Phospholamban (PLN), a 52 amino acid residue transmembrane protein of the cardiac sarcoplasmic reticulum (SR), is a critical regulator of Ca2+ cycling and a key determinant of β-adrenergic stimulation in the heart.1 At low concentrations of Ca2+, PLN interacts with the cardiac SR Ca2+-ATPase (SERCA2a) and inhibits its affinity for Ca2+ reversibly.2 The phosphorylation state of PLN also affects SERCA2a activity and cardiac contractility. In its dephosphorylated state, PLN interacts with SERCA2a and inhibits the affinity of the enzyme for Ca2+. Upon β-adrenergic stimulation, phosphorylation of PLN by cAMP-dependent protein kinase (PKA) and Ca2+-calmodulin-dependent protein kinase (CaMKII) relieves this inhibitory effect on SERCA2a, leading to enhanced SR-Ca2+ transport and increased rate of cardiac relaxation.3., 4., 5.

PLN has been predicted to contain three domains. Cytosolic domain IA (amino acid residues 1–20) is largely helical and contains serine 16 and threonine 17, the sites of phosphorylation by PKA and CaMKII, respectively. Domain IB (residues 21–30) is unstructured, with a high portion of amidated residues, whereas domain II (residues 31–52) forms a transmembrane helix.6., 7. On the basis of NMR studies, PLN contains two α-helices connected by a turn formed by residues Ile18, Glu19, Met20, and Pro21, resulting in an angle of ∼ 80° between the helices.8., 9., 10. PLN exists in a pentameric and monomeric form, where the monomer is considered to be the functionally active unit and the pentamer may act as a reservoir.11 Detailed cross-linking and site-directed mutagenesis studies have demonstrated that residues in both the cytoplasmic (IA and IB domains) and the transmembrane portions of PLN can interact directly with SERCA2a.12., 13., 14., 15., 16., 17.

The role of PLN in cardiac function has been elucidated by the generation and characterization of genetically altered mouse models. Overexpression of PLN in the heart resulted in depressed calcium kinetics and contractile parameters, but the inhibitory effects were relieved upon PLN phosphorylation by β-adrenergic agonists.18., 19. In contrast, ablation of PLN was associated with enhanced Ca2+ cycling and contractile parameters, resulting in an overall hypercontractile cardiac function, which persisted throughout aging.20., 21. These findings suggested that PLN may constitute a potentially important therapeutic target in heart failure.

Although experimental evidence suggests that PLN is a major regulator of cardiac function, it is unclear whether PLN acts on its own or whether there are other proteins modulating its activity. In the present study, we identified HS-1 associated protein X-1 (HAX-1), a ubiquitously expressed mitochondrial protein with anti-apoptotic function, as a binding partner for PLN. The PLN/HAX-1 interaction is specific and appears to be modulated by the concentration of Ca2+ and the phosphorylation state of PLN. Importantly, the anti-apoptotic function of HAX-1 was found to be enhanced in the presence of PLN, indicating an important role of the PLN/HAX-1 interaction in cardiac survival.

Section snippets

Identification of HAX-1 as a PLN-interacting protein

To identify PLN-interacting proteins, a yeast two-hybrid screen was performed on a pretransformed Human Heart Matchmaker cDNA library, using a PLN bait construct containing the cytoplasmic region of the protein (residues 1–37). From a total of 1.35 × 106 clones screened under high-stringency conditions, 12 positive clones were identified. Four of these clones contained the full-length cDNA sequence of HAX-1 (Figure 1(a)), a ubiquitously expressed anti-apoptotic protein that was identified

Discussion

The present study identified HAX-1 as a novel PLN-binding partner using the yeast two-hybrid system to screen a human heart cDNA library. The minimal binding region of HAX-1 was mapped to a C-terminal fragment, encoding residues 203–245, whereas the PLN binding region contained residues 16–22, a region that includes both the Ser16 and Thr17 phosphorylation sites. To date, myotonic dystrophy protein kinase (DMPK) is the only protein suggested to associate with this PLN region, and this

Yeast two-hybrid screening

A fragment encoding for the N-terminal cytoplasmic region of human PLN (residues 1–37) was amplified by PCR using PLN primer A (5′ATAATGGAAAAAGTGCAA 3′ sense primer) and primer B (5′GAGGCAGAAATTGATAAATAGG 3′ antisense primer) and the PLN/pIBI30 plasmid (a kind gift from Dr Kobra Haghighi, University of Cincinnati, USA) as DNA template.60 The PCR product was subsequently cloned in the EcoRI/SalI sites of the yeast BD pGBKT7 vector (Matchmaker System, BD Biosciences Clontech, Erembodegem,

Acknowledgements

We are grateful to the Biological Imaging Unit at the Foundation for Biomedical Research for assistance with confocal imaging. This study was supported by research funds from the Foundation of Biomedical Research of the Academy of Athens and from the Leduq Foundation; Trans-Antlantic alliance. D.S. and E.V. are supported by the 6th Framework Program, contract number 037277. We thank Ms S.E. Figueira for excellent secretarial assistance.

This work is patent protected by the Greek Industrial

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    E. G. K. and A. K.-K. contributed equally to this work.

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