Identification of Phosphorylation and Other Post-Translational Modifications in the Central C4C5 Domains of Murine Cardiac Myosin Binding Protein C

Abstract

Cardiac myosin binding protein C (cMyBPC) is a critical multidomain protein that modulates myosin cross bridge behavior and cardiac contractility. cMyBPC is principally regulated by phosphorylation of the residues within the M-domain of its N-terminus. However, not much is known about the phosphorylation or other post-translational modification (PTM) landscape of the central C4C5 domains. In this study, the presence of phosphorylation outside the M-domain was confirmed in vivo using mouse models expressing cMyBPC with nonphosphorylatable serine (S) to alanine substitutions. Purified recombinant mouse C4C5 domain constructs were incubated with 13 different kinases, and samples from the 6 strongest kinases were chosen for mass spectrometry analysis. A total of 26 unique phosphorylated peptides were found, representing 13 different phosphorylation sites including 10 novel sites. Parallel reaction monitoring and subsequent mutagenesis experiments revealed that the S690 site (UniProtKB O70468) was the predominant target of PKA and PKG1. We also report 6 acetylation and 7 ubiquitination sites not previously described in the literature. These PTMs demonstrate the possibility of additional layers of regulation and potential importance of the central domains of cMyBPC in cardiac health and disease. Data are available via ProteomeXchange with identifier PXD031262.

Figure 1

cMyBPC phosphorylation status of WT/3SA mouse models and KO^FL/4SA-injected hearts. (A) Representative phospho-stained (left) and Coomassie stained (right) cardiac myofibrils from WT and 3SA mouse lines. (B) Quantification of the relative protein phosphorylation of WT and 3SA (n = 4). The intensity of the Pro-Q-band was normalized to the Coomassie band intensity. (C) Representative phospho-stained (left) and Coomassie stained (right) cardiac myofibrils from KO hearts that were injected with FL (KO^FL) and 4SA AAV9 vectors. (D) Quantification of the relative protein phosphorylation of KO^FL and 4SA (n = 4). The intensity of the Pro-Q-band was normalized to the Coomassie band intensity. Values are expressed as mean ± SD. Significance was determined by a two-tailed t test. * p < 0.05 versus either WT or KO^FL group.

Figure 2

Representative phospho-stain (A) and Coomassie stained (B) gels of the C4C5 domains of murine cMyBPC incubated in the presence (+) and absence (−) of the corresponding kinase. Phospho-Tag Phosphoprotein Gel Stain from ABP Biosciences was used for these experiments. (C) Averaged relative protein phosphorylation of C4C5 protein over a 3 and 6 h time course. Data were normalized to the initial incubation at 0 h. Because the experiments were done separately for 3 and 6 h, statistical comparisons between 0 vs 3 h and 0 vs 6 h were made using a two-tailed t test. * indicates statistical significance (p < 0.05). Data presented as mean ± SEM (n = 3–4).

Figure 3

(A) Phospho-stain (top) and coomassie (bottom) of control and PKA-treated C4C5, 1A, 2A, and 4A recombinant proteins. (B) Phospho-stain (top) and coomassie (bottom) of control and PKG1-treated C4C5, 1A, 2A, and 4A recombinant proteins. Pro-Q Diamond Phosphoprotein Gel Stain from Invitrogen was used for these experiments. (C) Scatter plot of normalized relative protein phosphorylation of C4C5, 1A, 2A, and 4A constructs after a 6 h incubation with PKA and PKG1 (n = 3–4). Values are expressed as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05 versus C4C5 group.

Figure 4

Summary of the phosphorylation, acetylation, and ubiquitination landscape in the C4C5 domains of cMyBPC. (A) Collision-induced dissociation (CID) spectra of a representative modified peptide containing the S690 phosphorylation site. A triply charged peptide with a mass of 873.704 Da was identified in the tryptic digest of C4C5 and is within −1.63 ppm of the expected mass for the KASAGPHPDAPEDAGADEEWVFDK + PO₃ peptide. The CID spectra for this peptide contains several C-terminal y ions and the masses of these ions are consistent with the modification at S690. (B) A molecular model of the C4C5 domains of mouse cMyBPC is shown with the S690 site and the kinases (PKA and PKG1) targeting that site. Although the S589, S546, and S730 sites were also targeted by PKA, they are not represented in the model because of their low contribution to the overall phosphorylation of C4C5. Linker and loop regions are labeled. (C) A sequence alignment of the C4C5 domains of human and mouse cMyBPC (UniProtKB Q14896 and O70468) with all known phosphorylation (18), acetylation (6), and ubiquitination (13) sites in the C4 and C5 domains, excluding a nearby ubiquitination site at K539 (Wagner et al., 2012). The C4 domain is defined as residues K540 to M628 and the C5 domain is defined as residues E639 to D766 in this study. Note that although residue 691 is an alanine from our reference sequence, Huttlin et al., 2010 reported T691 phosphorylation in the reference sequence NP_032679.2. All PTMs are bolded and underlined. Phosphorylation, acetylation, and ubiquitination sites are color coded as yellow, red, and blue, respectively. The sites targeted by both acetylation and ubiquitination are highlighted gray. All six acetylated site were shared sites for ubiquitination, so red was not used in the figure. From this study, 10 phosphorylation, 6 acetylation, and 7 ubiquitination sites found seem to be novel. The blue and orange shaded areas represent the linker and the loop regions, respectively. * indicates sequence identity.