Dept. of Physics, University of Arizona, Tucson, AZ 85724, USA.
J Struct Biol. 2010 May;170(2):270-7. doi: 10.1016/j.jsb.2010.02.002. Epub 2010 Feb 10.
Post-translational modifications, along with isoform splicing, of titin determine the passive tension development of stretched sarcomeres. It was recently shown that PKCalpha phosphorylates two highly-conserved residues (S26 and S170) of the PEVK region in cardiac titin, resulting in passive tension increase. To determine how each phosphorylated residue affects myocardial stiffness, we generated three recombinant mutant PEVK fragments (S26A, S170A and S170A/S26A), each flanked by Ig domains. Single-molecule force spectroscopy shows that PKCalpha decreases the PEVK persistence length (from 0.99 to 0.68 nm); the majority of this decrease is attributable to phosphorylation of S26. Before PKCalpha, all three mutant PEVK fragments showed at least 40% decrease in persistence length compared to wildtype. Furthermore, Ig domain unfolding force measurements indicate that PEVK's flanking Ig domains are relatively unstable compared to other titin Ig domains. We conclude that phosphorylation of S26 is the primary mechanism through which PKCalpha modulates cardiac stiffness.
肌联蛋白的翻译后修饰以及异构体拼接决定了伸展的肌节的被动张力发展。最近有研究表明，PKCalpha 可使心脏肌联蛋白的 PEVK 区域的两个高度保守残基（S26 和 S170）磷酸化，导致被动张力增加。为了确定每个磷酸化残基如何影响心肌硬度，我们生成了三个重组突变的 PEVK 片段（S26A、S170A 和 S170A/S26A），每个片段都有 Ig 结构域。单分子力谱表明，PKCalpha 降低了 PEVK 的持久长度（从 0.99nm 降至 0.68nm）；这种降低主要归因于 S26 的磷酸化。在 PKCalpha 作用之前，与野生型相比，所有三种突变的 PEVK 片段的持久长度至少降低了 40%。此外，Ig 结构域展开力测量表明，与其他肌联蛋白 Ig 结构域相比，PEVK 的侧翼 Ig 结构域相对不稳定。我们得出结论，S26 的磷酸化是 PKCalpha 调节心脏硬度的主要机制。
