PKCε 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.

Cardiac Myosin-Binding Protein C Mutations and Hypertrophic Cardiomyopathy

“…31 Apart from PKA, cMyBP-C can be phosphorylated by Ca 2ϩ -calmodulin-dependent kinase (CaMK) 32,33 and PKC. 34,35 Transgenic mice hearts in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines displayed reduced contractility and altered sarcomeric structure, indicating that phosphorylation of cMyBP-C is essential for normal cardiac function. 36 Reduced cMyBP-C phosphorylation has been observed in animal models of cardiac hypertrophy and failure 36,37 and in humans with end-stage idiopathic and ischemic cardiomyopathy.…”

The role of protein kinase C-mediated phosphorylation of sarcomeric proteins in the heart—detrimental or beneficial?

“…These authors also showed that PKCβII phosphorylated the PKA sites Ser23/24 in wild-type myofibrils and demonstrated that such cross-phosphorylation resulted in a rightward shift of the pCa-tension curve (Table 1) (Wang et al 2006). In a more recent study by Lu et al (2010), pseudophosphorylation of Thr143 with glutamic acid (T143E) and of Ser23/24 with aspartic acid (S23D/S24D/T143E) caused no alterations in Ca 2+ -sensitivity upon exchange with cTn(T143E) compared (Xiao et al 2007). -TG mice with alanine substitution of phosphorylation sites displayed depressed cardiac contractility and altered sarcomeric structure (Sakthivel et al 2005).…”

“…Mohamed et al (1998) demonstrated that site Ser1169 in chicken is phosphorylated by PKC to 2.0 mol phosphate/mol. Xiao et al (2007) reported that Ser302 on mouse cMyBP-C is a PKCε phosphorylation site both in vivo and in vitro. A specific role for PKC-mediated phosphorylation of cMyBP-C on the contractility and during heart failure has yet to be determined.…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…For example, mAb specifically recognizing phosphorylated and unphosphorylated TnI at Ser23/24 (human sequence) have been commercially developed, and widely used in studies involving measurement of TnI phosphorylation. It is noteworthy that mAb recognizing phosphorylation within specific sequence motifs have been rapidly developed and increasingly used in myofilament protein studies [94,95]. When a phosphorylation-site-specific antibody is not available, MS can be employed.…”