Lethal, hereditary mutants of phospholamban elude phosphorylation by protein kinase A

Abstract

The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.

FIGURE 1.

Fitted curves of normalized ATPase activity as a function of calcium concentration for SERCA reconstitutions were adapted from Ceholski et al. (18) and are shown for comparison with R9H PLN data ± S.E. (▾, n = 9). A and B, SERCA was reconstituted in the absence (black curve) and presence of wild-type PLN (gray curve) or R9C (red curve) or R14del (green curve) (A) or R9L (red curve) or R9H (green curve) PLN (B). All kinetic values are given in Table 1. C, PKA-mediated phosphorylation of wild-type and disease-associated mutants of PLN. Phosphorylation is shown as a percentage of wild-type PLN ± S.E. (100% = complete phosphorylation) (n ≥ 4).

FIGURE 2.

Top panel, primary sequence of PLN residues 1–17, with positions targeted for mutagenesis indicated (bold letters). Bottom panel, PKA-mediated phosphorylation of alanine mutants of these residues is shown as a percentage of wild-type PLN ± S.E. (100% = complete phosphorylation, dashed line) (n ≥ 4). Asterisks indicate comparisons against wild type (p < 0.01).

FIGURE 3.

PKA-mediated phosphorylation of PLN alanine mutants in proteoliposomes with SERCA. Phosphorylation is shown as a percentage of wild-type PLN ± S.E. (100% = complete phosphorylation, dashed line) (n ≥ 4). All phosphorylation values have been corrected for PLN content of the proteoliposomes. Asterisks indicate comparisons against wild type (p < 0.01).

FIGURE 4.

PKA-mediated phosphorylation of disease-mimicking PLN mutants. Phosphorylation is shown as a percentage of wild-type PLN ± S.E. (100% = complete phosphorylation, dashed line) (n ≥ 4). Disease-causing mutations are labeled in bold type and shown with black bars. Asterisks indicate comparisons against wild type (p < 0.01).

FIGURE 5.

Top panel, molecular model for the interaction between PKA (surface representation) and PLN (residues Lys³–Thr¹⁷; stick representation) based on structures of PKA bound to peptides (Protein Data Bank entries 1JLU, 1JBP, and 3O7L). Negatively charged residues in PKA are shown in red (Glu²⁰³ and Asp²⁴¹). The phosphorylated Ser¹⁶ of PLN and ATP are labeled. Bottom panel, a detailed view of the PKA active site containing PLN reveals that Arg⁹ of PLN forms electrostatic interactions with Glu²⁰³ and Asp²⁴¹ of PKA. The backbones of PKA residues are shown in green, and the backbones of PLN residues are shown in yellow. Arg⁹, Arg¹³, Arg¹⁴, and Ser¹⁶ of PLN, Glu²⁰³ and Asp²⁴¹ of PKA, and AMP-PCP are labeled.

FIGURE 6.

Time-dependent phosphorylation of wild-type (●, solid line) or R9S (○, dashed line) PLN ± S.E. by recombinant wild-type (A), E203A (B), or D241A (C) PKA. Concentration of phosphorylated PLN (mm) is shown as a function of time (minutes) (n ≥ 3).

FIGURE 7.

Phosphorylation of wild-type PLN (A) or R9S PLN (B) ± S.E. by recombinant wild-type, E203A, or D241A PKA. Phosphorylation of PLN (black bars) and after an additional double aliquot of PKA (gray bars) are compared (n ≥ 3).

FIGURE 8.

A, time-dependent phosphorylation progress curves of wild-type PKA and R9S PLN and E203A PKA and wild-type PLN were replotted as a function of phosphorylated monomeric equivalent units. Phosphorylation stalled when two or three monomers in a pentamer were phosphorylated. B, SDS-PAGE of wild-type, R9C, PLN-SSS, and R9C-SSS PLN (5 μg/lane). Pentameric (PLN₅) and monomeric (PLN₁) PLN are indicated with arrows. C, PKA-mediated phosphorylation of wild-type PLN versus R9C PLN, monomeric PLN-SSS versus R9C-SSS, and cytoplasmic peptide PLN_1–20 versus R9C_1–20 (n ≥ 3; % phosphorylation ± S.E.). Asterisks indicate comparisons against each respective wild-type construct (p < 0.01). N.S., not significant.