Integration of Cardiac Troponin I Phosphorylations to Modulate Function

Abstract

The troponin complex is a critical molecular switch involved in transducing the calcium activating signal into contraction. Troponin I (TnI), the inhibitory subunit, is phosphorylated as a key mechanism to alter the calcium regulation of contraction. The canonical phosphorylation of TnI at serine-23/24 (Ser-23/24) is a significant modulator of this regulation, and may be fine-tuned by other TnI phosphorylation events. A novel TnI phosphorylation at tyrosine 26 (Tyr-26) was identified in the human heart and shown to decrease in heart failure. We have recently demonstrated that this novel TnI Tyr-26 phosphorylation decreases thin filament calcium sensitivity and accelerates calcium dissociation by itself, identical to the functional effects of Ser-23/24. We further demonstrated the integration of these two modifications causes an additive effect on calcium dissociation while maintaining a decreased calcium sensitivity. These data stress the importance of Tyr-26 function, especially in the presence of Ser-23/24. These data further highlight how the integration of TnI phosphorylation exhibits differential effects when compared to studying these events in isolation. Importantly, the integration of Ser-23/24 and Tyr-26 phosphorylation may not be limited to functional effects alone, but may manifest in signaling as well. Considering the tyrosine kinase recognition sequence (EEXY) is similar to that surrounding TnI Tyr-26 when Ser-23/24 are occupied with phosphate(S(p)S(p)NY), we hypothesized that Ser-23/24 phosphorylation would exhibit crosstalk, altering phosphorylation rate of Tyr-26. To assess this hypothesis, we developed an antibody against pTnI Tyr-26 as a tool to measure changes in Tyr-26 phosphorylation. We validated the specificity and purity of this antibody and are currently elucidating signaling integration by assessing the ability of Ser-23/24 phosphorylation to regulate phosphorylation of Tyr-26 by tyrosine kinases. The functional and signaling integration of TnI phosphorylation may therefore be a significant mechanism to modulate Ser-23/24 phosphorylation function.