Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia

Abstract

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias.

Keywords: Fam20C kinase; arrhythmia; heart; histidine-rich calcium-binding protein; phosphorylation.

Fig. 1.

HRC is phosphorylated on Ser96 in human hearts. (A) Schematic depicting HRC domain architecture and phosphorylation sites. Endogenous HRC was immunoprecipitated from human heart extracts and analyzed by phosphoproteomics using liquid chromatography tandem MS. (B) Phosphopeptide derived from MS chromatogram showing human HRC Ser96 phosphorylation (S+80) in vivo. (C) Residues corresponding to the Fam20C S-x-E phosphorylation motif of HRC are highlighted in blue. Numbers represent the first amino acid number in the motif. Accession numbers are as follows: human (Homo sapiens): ACH88003, mouse (Mus musculus): AAD42061, rabbit (Oryctolagus cuniculus): AAA31279, rat (Rattus norvegicus): AAP31904, cow (Bos taurus): NP_001095783, orangutan (Pongo pygmaeus): CAH89847, dog (Canis lupus familiaris): F6UPH0, and pig (Sus scrofa): XP_013854077. (D) Endogenous human HRC immunoprecipitates were treated with λ-phosphatase (λ-phos). Phosphorylated HRC (pHRC) and unphosphorylated HRC are denoted by the arrows and show that λ-phos results in dephosphorylation of HRC.

Fig. S1.

MS chromatogram depicting the phosphopeptide showing human HRC Ser96 phosphorylation (S+80) in vivo.

Fig. 2.

Fam20C is the kinase that phosphorylates HRC. (A) Protein immunoblot of HRC and Fam20C immunoprecipitates (IPs) from cardiac homogenates (1 mg of total protein) from Ser96-HRC mouse hearts showing an association between HRC and Fam20C. The IPs were resolved by SDS/PAGE and immunoblotted with antibodies recognizing Fam20C (Top) and HRC (Bottom). IPs with anti-IgG plus agarose were used as negative controls. HRC-1 and HRC-2 represent IPs from different hearts. (B, Top) FLAG immunoblot of Flag IPs from cell lysates from H9C2 cells coexpressing HRC-FLAG with either WT or the catalytically inactive HA-DA. (B, Bottom) HA immunoblot. The λ-phosphatase (λ-phos) treatment was only applied to HRC-FLAG IPs. Phosphorylated HRC (pHRC) and unphosphorylated HRC are denoted by the arrows and show that λ-phos results in dephosphorylation of HRC. (C, Top) FLAG immunoblot of Flag-IPs from Fam20C KO and WT U20S cells overexpressing HRC-FLAG showing phosphorylation of HRC in WT cells. (C, Bottom) Fam20C immunoblot of conditioned media. (D, Top) Autoradiograph depicting time-dependent incorporation of ³²P from [γ-³²P]ATP into HRC using purified proteins. HRC-FLAG (transiently expressed and purified from Fam20C KO U20S cells) was incubated with WT or D478A (inactive) recombinant Fam20C (DA; purified from baculovirus) in an in vitro kinase assay. (D, Bottom) Parallel anti-FLAG immunoblot.

Fig. 3.

HRC is phosphorylated on Ser96 by Fam20C in cells. (A) Schematic depicting domain architecture and Fam20C-dependent phosphosites. HRC-FLAG was expressed in Fam20C KO and WT U2OS cells and was FLAG-immunoprecipitated, and the phosphorylation sites were mapped by comparative tandem MS (MS/MS). (B) Phosphopeptide derived from MS chromatogram showing Ser96-HRC phosphorylation (S+80) in cells.

Fig. S2.

MS chromatogram depicting the phosphopeptide showing HRC Ser96 phosphorylation (S+80) from cells.

Fig. 4.

HRC phosphomimetic decreases aftercontractions in cardiomyocytes. (A) Aftercontractions in HRC KO mouse cardiomyocytes infected with adenoviruses to express GFP, S96D-HRC, or S96A-HRC under field stimulation of 2 Hz and in the absence or presence of 1 μM isoproterenol. Data are mean ± SEM of the total number of cells per group (n = 22–45 cells from three hearts per group). Comparisons were performed using a t test (*P < 0.05 in comparison to S96D-HRC) and show that S96D-HRC exhibits reduced aftercontractions in either the absence or presence of isoproterenol. (B) Cardiomyocytes were isolated from mutant S96A-HRC mouse hearts and infected as above, and aftercontractions were measured using ±1 μM isoproterenol with field stimulation at 2 Hz (n = 18–37 cells for three S96A hearts). Data are mean ± SEM of the total number of cells per group. Comparisons were performed using a t test (*P < 0.05 in comparison to S96D-HRC), showing that S96D diminishes the aftercontractions in S96A cardiomyocytes.

Fig. 5.

Phosphorylation of HRC regulates its interactions with triadin and SERCA2a. (A) Immunoblot (IB) analysis of HRC-FLAG coexpressed with triadin-V5 and either Fam20C WT or the catalytically inactive DA mutant in H9C2 cells. HRC was Flag-immunoprecipitated from whole-cell lysates, and triadin coimmunoprecipitation was assayed via V5 immunoblotting. The two lower immunoblot panels represent controls for expression levels following immunoprecipitation (IP) of the corresponding epitope. (B) HRC-FLAG coexpressed with SERCA2a-V5 and either Fam20C WT or the catalytically inactive DA mutant. HRC was Flag-immunoprecipitated, and SERCA2a coimmunoprecipitation was assayed via V5 immunoblotting. (C) Results from A and B are expressed as the relative densitometry of V5 IBs of either triadin or SERCA2a in DA samples (n = 3), and show increased binding to triadin but reduced binding to SERCA2a by phosphorylated (WT) HRC. (D) IB analysis of Flag-HRC-S96A or FLAG-HRC-S96D coexpressed with triadin-V5 in H9C2 cells. (Top) HRC was Flag-immunoprecipitated from whole-cell lysates, and triadin co-IP was assayed via V5 immunoblotting. (Middle and Bottom) Immunoblot panels represent controls for expression levels following IP of the corresponding epitope. (E) Either Flag-HRC-S96A or FLAG-HRC-S96D coexpressed with SERCA2a-V5. HRC was Flag-immunoprecipitated, and SERCA2a co-IP was assayed via V5 immunoblotting. (F) Results from A and B are expressed as relative densitometry of V5 IBs of either triadin or SERCA2a (n = 3), and show increased binding to triadin but reduced binding to SERCA2a by the phosphomimetic HRC S96D.

Fig. 6.

Model proposing how Fam20C-mediated phosphorylation of HRC Ser96 affects cardiac Ca homeostasis. The pSer96-HRC binds tighter to triadin than S96A-HRC, which cannot be phosphorylated. Conversely, S96A-HRC or unphosphorylated Ser96-HRC binds tighter to SERCA2a. This suggests that Ser96-HRC phosphorylation (P) regulates HRC’s interactions with the major SR Ca-cycling proteins. Therefore, HRC Ser96 phosphorylation is important for SR Ca homeostasis, preventing arrhythmias.