FIGURE 5.

Impact of the R145W RCM mutation on PKC- and PKA-mediated phosphorylation. The ability of PKC or PKA to phosphorylate recombinant human troponin was tested in vitro. Human recombinant troponin (250 μg/ml) was treated for 120 min at 37 °C with either PKCϵ (A), PKCα (B), or PKA (C) using 0–10 μg/ml kinase protein as indicated (b represents boiled in A). The reaction was quenched by addition of SDS sample buffer. Western blots were probed for TnT (top gels; loading controls) or site-specific phosphoantibodies (bottom gels) against Thr(P)-143 (PKC; A and B) or cTnI Ser(P)-23/Ser(P)-24 (PKA; C). Presence of the RCM mutation rendered the Thr-143 PKC phosphorylation motif inactive but notably not the Ser-23/Ser-24 PKA motif. These experiments were performed in triplicate.