Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA.
J Mol Biol. 2010 Jul 30;400(5):1036-45. doi: 10.1016/j.jmb.2010.06.007. Epub 2010 Jun 9.
Residue Ser151 of cardiac troponin I (cTnI) is known to be phosphorylated by p21-activated kinase 3 (PAK3). It has been found that PAK3-mediated phosphorylation of cTnI induces an increase in the sensitivity of myofilament to Ca(2+), but the detailed mechanism is unknown. We investigated how the structural and kinetic effects mediated by pseudo-phosphorylation of cTnI (S151E) modulates Ca(2+)-induced activation of cardiac thin filaments. Using steady-state, time-resolved Förster resonance energy transfer (FRET) and stopped-flow kinetic measurements, we monitored Ca(2+)-induced changes in cTnI-cTnC interactions. Measurements were done using reconstituted thin filaments, which contained the pseudo-phosphorylated cTnI(S151E). We hypothesized that the thin filament regulation is modulated by altered cTnC-cTnI interactions due to charge modification caused by the phosphorylation of Ser151 in cTnI. Our results showed that the pseudo-phosphorylation of cTnI (S151E) sensitizes structural changes to Ca(2+) by shortening the intersite distances between cTnC and cTnI. Furthermore, kinetic rates of Ca(2+) dissociation-induced structural change in the regulatory region of cTnI were reduced significantly by cTnI (S151E). The aforementioned effects of pseudo-phosphorylation of cTnI were similar to those of strong crossbridges on structural changes in cTnI. Our results provide novel information on how cardiac thin filament regulation is modulated by PAK3 phosphorylation of cTnI.
肌钙蛋白 I（cTnI）的残基 151 丝氨酸已知可被 p21 激活激酶 3（PAK3）磷酸化。已经发现，PAK3 介导的 cTnI 磷酸化可增加肌球蛋白丝对 Ca2+的敏感性，但详细的机制尚不清楚。我们研究了 cTnI（S151E）假磷酸化介导的结构和动力学效应如何调节 Ca2+诱导的心肌细肌丝激活。使用稳态、时间分辨的Förster 共振能量转移（FRET）和停流动力学测量，我们监测了 cTnI-cTnC 相互作用的 Ca2+诱导变化。使用含有假磷酸化 cTnI（S151E）的重组细肌丝进行了测量。我们假设，由于 cTnI 丝氨酸 151 的磷酸化引起的电荷修饰，改变了 cTnC-cTnI 相互作用，从而调节细肌丝的调节。我们的结果表明，cTnI（S151E）的假磷酸化通过缩短 cTnC 和 cTnI 之间的位点间距离，使结构变化对 Ca2+敏感。此外，cTnI（S151E）显著降低了 Ca2+解离诱导的 cTnI 调节区结构变化的动力学速率。cTnI 的上述假磷酸化作用类似于强交联桥对 cTnI 结构变化的作用。我们的结果提供了关于 PAK3 磷酸化 cTnI 如何调节心肌细肌丝调节的新信息。
