Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Aachen, Germany.
Institute of Biology and Translational Center for Regenerative Medicine, University of Leipzig, Leipzig, Germany.
J Invest Dermatol. 2018 Mar;138(3):627-636. doi: 10.1016/j.jid.2017.10.011. Epub 2017 Dec 6.
A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.
单纯型表皮松解性水疱病的一个特征是角蛋白丝（KF）网络崩溃，这是由基底表皮角蛋白 II（KtyII）K5 和其 I 型伴侣角蛋白 14（K14）的聚集引起的。在这里，我们研究了角蛋白磷酸化在 KF 网络重排和细胞功能中的作用。我们检测到含有 R125C K14 突变体的细胞质单纯型表皮松解性水疱病颗粒中 K5 头域残基 T150 的磷酸化。表达磷酸模拟 T150D K5 突变体导致角质形成细胞中 KF 形成受损。该表型在与其他磷酸模拟 K5 头域突变体组合时增强。值得注意的是，将 T150D K5 突变体引入缺乏 KtyII（KtyII）的角蛋白细胞中会完全阻止角蛋白网络的形成。相比之下，磷酸化缺陷的 T150A K5 导致 KF 分支和周转率降低。T150D K5 的组装在异四聚体阶段停滞，同时与热休克蛋白的结合增加。最后，在 T150 突变细胞中观察到细胞活力降低和对应激源的反应增强。总之，我们的发现确定了 T150 K5 磷酸化是 KF 网络形成和功能的重要决定因素，它可能在单纯型表皮松解性水疱病的发病机制中起作用。
