Effects of Pseudo-Phosphorylation of cTnI by P21 Activated Kinase-3 (PAK3) on Structure and Kinetics of Ca2+-Induced Cardiac Thin Filament Regulation

Abstract

Residue Ser151 of rat cardiac troponin I (cTnI) can be phosphorylated by P21-activated kinase 3 (PAK3). It has been found that PAK3 phosphorylation of cTnI induces an increase in Ca2+ sensitivity of myofilament, but detailed mechanism is unknown. We investigated the structural and kinetic effects of phosphorylation of cTnI PAK3 site Ser151 on the Ca2+-induced thin filament regulation. Using steady-state and time-resolved Forster Resonance Energy Transfer (FRET) measurements and stopped-flow kinetic studies, we measured Ca2+-induced changes in FRET distance from the residues 160 and 167 in the regulatory region of cTnI to the residue 89 of cTnC to monitor cTnC and cTnI interactions. The measurements were done with the reconstituted thin filament containing PAK3 pseudo-phosphorylation of cTnI(S151E). We hypothesized that the charge modification at the interface between troponin C (cTnC) and cTnI caused by the phosphorylation at the N-terminus of the regulatory region of cTnI may affect the binding of the regulatory region of cTnI to cTnC. Our results showed that the pseudo-phosphorylation of cTnI(S151E) favors the binding by shortening the distances between the regulatory region of cTnI and cTnC and increasing Ca2+ sensitivity of the structural change. Furthermore, the pseudo-phosphorylation showed similar kinetic effects as the strongly-bound crossbridges on the thin filament regulation by significantly slowing down the kinetics of the Ca2+ dissociation-induced structural transitions of the regulatory region of cTnI. This is consistent with the decreased tension cost observed in the tension measurements of cardiac muscle fiber bundle reconstituted with the pseudo-phosphorylated cTnI, which suggest a decrease in crossbridge detachment rates. Our results provide novel information on the potential molecular mechanism underlying modulation of cardiac thin filament regulation by PAK3 phosphorylation of cTnI.