TI - PKCepsilon increases phosphorylation of the cardiac myosin binding protein C at serine 302 both in vitro and in vivo . AB - 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 phosphorylation in WT mice to that in TG mice , whereas treatment of TG myofibrils with PKCepsilon showed only a minimal increase in 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 .