PKCepsilon increases phosphorylation of the cardiac myosin binding protein C at serine 302 both in vitro and in vivo

Abstract

Cardiac myosin binding protein C (cMyBPC) phosphorylation is essential for normal cardiac function. Although PKC was reported to phosphorylate cMyBPC in vitro, the relevant PKC isoforms and functions of PKC-mediated cMyBPC phosphorylation are unknown. We recently reported that a transgenic mouse model with cardiac-specific overexpression of PKCepsilon (PKCepsilon TG) displayed enhanced sarcomeric protein phosphorylation and dilated cardiomyopathy. In the present study, we have investigated cMyBPC phosphorylation in PKCepsilon TG mice. Western blotting and two-dimensional gel electrophoresis demonstrated a significant increase in cMyBPC serine (Ser) phosphorylation in 12-month-old TG mice compared to wild type (WT). In vitro PKCepsilon treatment of myofibrils increased the level of cMyBPC Ser phosphorylation in WT mice to that in TG mice, whereas treatment of TG myofibrils with PKCepsilon showed only a minimal increase in cMyBPC Ser phosphorylation. Three peptide motifs of cMyBPC were identified as the potential PKCepsilon consensus sites including a 100% matched motif at Ser302 and two nearly matched motifs at Ser811 and Ser1203. We treated synthetic peptides corresponding to the sequences of these three motifs with PKCepsilon and determined phosphorylation by mass spectrometry and ELISA assay. PKCepsilon induced phosphorylation at the Ser302 site but not at the Ser811 or Ser1203 sites. A S302A point mutation in the Ser302 peptide abolished the PKCepsilon-dependent phosphorylation. Taken together, our data show that the Ser302 on mouse cMyBPC is a likely PKCepsilon phosphorylation site both in vivo and in vitro and may contribute to the dilated cardiomyopathy associated with increased PKCepsilon activity.

Figure 1

Western blotting on the ventricular myofibrillar proteins extracted from 12-month-old WT or TG mouse hearts for Ser phosphorylation. (A) Representative result of SDS–PAGE gel electrophoresis and Western blotting using p-SerPKC antibody. Ten micrograms of sample was loaded in each lane. Membrane was stripped and then reprobed for actin (42 kDa) as control for loading. Cardiac MyBPC was identified at 150 kDa by Western blotting using anti-cMyBPC antibody. (B) Western blotting on serially diluted sample proteins using p-SerPKC antibody. The result confirmed the finding in panel A. (C) Quantitative densitometry analysis on the results from the Western blotting using p-SerPKC antibody; n = 5 (from five pairs of hearts). The density of the p-Ser of cMyBPC was normalized to that of the stripped and reprobed actin in the same sample. The data were then presented as fold increase in Ser phosphorylation of cMyBPC over that of the WT hearts. The paired, one-tailed t-test was used for statistical analysis. p < 0.05, statistically significant. (D) 2D gel analysis on the Ser phosphorylation of cMyBPC. Equal amounts of the samples from WT or TG hearts (50 µg from each) were labeled with Cy3 and underwent 2D gel electrophoresis on separate gels as shown in the first row. The proteins were transferred simultaneously to the same PVDF membrane, and Western blotting was performed first using p-SerPKC antibody (second row). Cardiac MyBPC was detected at 150 kDa. The membrane was then stripped and reblotted for cMyBPC (third row). n = 2.

Figure 2

Western blotting of the ventricular myofibrillar proteins extracted from 12-month-old WT or TG mouse hearts for Tyr or Thr phosphorylation using the p-Tyr antibody or the p-Thr antibody. Five micrograms of sample was loaded in each lane; Western blotting for actin was used as control for loading. (A) Representative results of Western blotting for Tyr and Thr phosphorylation. (B) Quantitative densitometry analysis of the Western blot results using p-Thr antibody; n = 3 (from three pairs of hearts). The density of the p-Thr at ~150 kDa was normalized to that of the stripped and reprobed actin in the same sample. The data were then presented as fold change in Thr phosphorylation over that of the WT hearts. The paired, one-tailed t-test was used for statistical analysis. p = 0.3213; ns, not significant.

Figure 3

In vitro PKCε assay on purified cardiac myofibrillar proteins from 12-month-old WT and TG mice followed by Western blotting using p-SerPKC antibody. The membrane was then stripped and blotted for cMyBPC. An unknown phosphoprotein identified by the p-SerPKC antibody at ~90 kDa was shown as a positive control. n = 3. (A) Representative result from one pair of mice. Five micrograms of sample was loaded in each lane. (B) Quantitative densitometry analysis. The data were presented as fold increase in Ser phosphorylation of cMyBPC over that of the untreated WT hearts (WT group). One-way ANOVA followed by Bonferroni’s multiple comparison test was used for statistical analysis between the different groups. p < 0.05, statistically significant; ns, not significant.

Figure 4

Identification of phosphorylation on the PKCε-treated synthetic cMyBPC peptides. (A) The three motifs on the mouse cMyBPC coding region identified by NetPhos analysis are identical (S302) or similar (S811 and S1203) to the consensus motif for the anti-p-SerPKC antibody. (B) MS on S302 and S302A peptides with or without PKCε kinase treatment. MS identified only one phosphorylated site on S302 peptide, which was abolished after mutating the Ser302 to an Ala in the S302A peptide, indicating that the phosphorylated Ser in the S302 peptide is Ser302; n = 4. (C) MS on S811 and S1203 peptides with or without PKCε kinase treatment. The study failed to identify any phosphorylation residue in either peptide after PKCε treatment; n = 4. p, with PKCε treatment.

Figure 5

ELISA to identify the phosphorylated Ser residue on the synthetic peptides after in vitro PKCε treatment. (A) ELISA on S302 peptide using p-SerPKC antibody; 80D, 80 daltons; p, with PKCε treatment. (B) ELISA on S302A peptide using p-SerPKC antibody. Data were pooled from three experiments (n = 9). The paired, one-tailed t-test was used for statistical analysis; p < 0.01, statistically significant; ns, not significant.

Figure 6

In vitro PKCε competition kinase assays on intact mouse cMyBPC and the synthetic peptides followed by Western blotting using p-SerPKC antibody. Cardiac myofilaments isolated from 12-month-old WT mice were used for in vitro PKCε kinase assay. Equal amounts (5 µg) of myofilament samples were incubated with PKCε at 30 °C for 30 min in the presence of 0.1 mM synthetic peptide of either S302, the mutant S302A, or a structurally unrelated control peptide TKR (TKRKDEECSTTEHPYKKPYM) before being subjected to Western blot for detection of p-serine or actin. Actin-normalized p-serine density was analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. Data were from three independent experiments and are shown as the fold changes of the PKCε-untreated group. *, p < 0.05 compared to the control group; #, p < 0.05 compared to the group of PKCε + peptide S30.

Figure 7

In vitro PKC assay on purified cardiac myofibrillar proteins from 12-month-old WT mice followed by Western blotting. A representative Western blot result of three independent experiments was shown. Five micrograms of sample was incubated with or without an equal amount of PKCα, PKCδ, or PKCε at 30 °C for 20 min. In order to activate PKCα, the PKCα assay buffer contained 200 µM CaCl₂. The p-Ser and actin were first detected by Western blotting. The membrane was then stripped and reprobed for cMyBPC.