PKC phosphorylation of titin's PEVK element: a novel and conserved pathway for modulating myocardial stiffness.
PKC phosphorylation of titin's PEVK element: a novel and conserved pathway for modulating myocardial stiffness.
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DOI：
10.1161/circresaha.109.198465
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发表时间：
2009-09-25
影响因子：
20.1
通讯作者：
Granzier H
中科院分区：
文献类型：
--
作者：
Hidalgo C;Hudson B;Bogomolovas J;Zhu Y;Anderson B;Greaser M;Labeit S;Granzier H
蛋白激酶C(PKC)通过磷酸化细丝和粗丝蛋白来调节心肌细胞的收缩能力。心肌肌节还含有第三种肌丝--肌联蛋白，目前尚不清楚肌联蛋白能否被蛋白激酶C磷酸化，以及它是否影响被动张力。研究PKC对肌动蛋白磷酸化及肌动蛋白被动张力的影响。蛋白激酶Cα的磷酸化分析表明，肌动蛋白在皮肤心肌组织中被磷酸化；这种作用被多肽1预处理而加剧。代表Titin弹簧元件的重组蛋白的体外磷酸化结果表明，PKCα以PEVK弹簧元件为靶点。此外，质谱学和定点突变相结合发现了PEVK区的两个高度保守的位点，它们被PKCα(S11878和S12022)磷酸化；当这两个位点突变为丙氨酸时，磷酸化被有效地取消。对带皮左室心肌的力学实验表明，PKCα显著增加了基于肌动蛋白的被动张力，这一作用被PP1逆转。单分子拉伸曲线显示，PKCα在不改变轮廓长度的情况下缩短了PEVK的持续长度(从1.2 nm减少到0.55 nm)，并且利用串联的蠕虫链模型，我们表明这增加了基于Titin的被动力，其肌节长度依赖于PKCα磷酸化后在皮肤心肌中测量的结果。肌动蛋白的PKC磷酸化是连接心肌信号和心肌僵硬的一条新的、保守的途径。
Protein Kinase C (PKC) regulates contractility of cardiac muscle cells by phosphorylating thin- and thick- filament based proteins. Myocardial sarcomeres also contain a third myofilament, titin, and it is unknown whether titin can be phosphorylated by PKC and if it affects passive tension. Study the effect of PKC on titin phosphorylation and titin-based passive tension. Phosphorylation assays with PKCα revealed that titin is phosphorylated in skinned myocardial tissues; this effect is exacerbated by pre-treating with PP1. In vitro phosphorylation of recombinant protein representing titin's spring elements showed that PKCα targets the PEVK spring element. Furthermore, mass spectrometry in combination with site-directed mutagenesis identified two highly conserved sites in the PEVK region that are phosphorylated by PKCα (S11878 and S12022); when these two sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned LV myocardium revealed that PKCα significantly increases titin-based passive tension, an effect that is reversed by PP1. Single molecule force-extension curves show that PKCα decreases the PEVK persistence length (from 1.20 nm to 0.55 nm), without altering the contour length, and using a serially-linked wormlike chain (WLC) model we show that this increases titin-based passive force with a sarcomere length dependence that is similar to that measured in skinned myocardium following PKCα phosphorylation. PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.