Phosphorylation of trans‐active response DNA‐binding protein‐of 43 kDa promotes its cytoplasmic aggregation and modulates its function in tau mRNA stability and exon 10 alternative splicing
Phosphorylation of trans‐active response DNA‐binding protein‐of 43 kDa promotes its cytoplasmic aggregation and modulates its function in tau mRNA stability and exon 10 alternative splicing
复制标题
DOI：
10.1111/jnc.15450
复制
发表时间：
2021-06
影响因子：
4.7
通讯作者：
Ruozhen Wu;Dingwei Zhou;Xin Shen;F. Chen;Fei Liu;Jianlan Gu
中科院分区：
文献类型：
--
作者：
Ruozhen Wu;Dingwei Zhou;Xin Shen;F. Chen;Fei Liu;Jianlan Gu
43 kDa 的反式活性反应 DNA 结合蛋白 (TDP-43) 促进 tau mRNA 不稳定和 tau 外显子 10 包含。磷酸化 TDP-43 的聚集与肌萎缩侧索硬化症 (ALS) 和额颞叶变性有关。酪蛋白激酶 1ε (CK1ε) 在多个位点磷酸化 TDP-43，增强其细胞质聚集，并调节其在 tau mRNA 加工中的功能。为了确定 TDP-43 位点特异性磷酸化在 tau mRNA 加工中的定位、聚集和功能中的作用，将 TDP-43 在 Ser379、Ser403/404 或 Ser409/410 处突变为丙氨酸或天冬氨酸，以阻断或模拟磷酸化。在体外和培养细胞中研究了 CK1ε 对 TDP-43 及其突变体的位点特异性磷酸化。通过蛋白质印迹分析细胞质和细胞核 TDP-43 和磷酸化 TDP-43。通过免疫染色和放射免疫沉淀测定缓冲液不溶性 TDP-43 的水平来评估 TDP-43 的聚集。使用尾部带有 tau 3'非翻译区和 mini-tau 基因 pCI/SI9-LI10 的绿色荧光蛋白来研究 tau mRNA 稳定性和 tau 外显子 10 的选择性剪接。我们发现 TDP-43 在 Ser379、Ser403/404 或 Ser409/410 处的磷酸阻断突变不能被 CK1ε 有效磷酸化。与 TDP-43 相比，细胞质中磷酸化的 TDP-43 水平更高。这些位点的磷酸化模拟突变增强了 TDP-43 的细胞质聚集。 TDP-43 的磷酸阻断突变体不会抑制绿色荧光蛋白表达，但 Ser379 和 Ser403/404 的磷酸阻断突变进一步增强了 tau 外显子 10 的包含。 TDP-43 在 Ser379、Ser403/404 或 Ser409/410 处的磷酸化引发 CK1ε 的磷酸化，促进 TDP-43 细胞质聚集，并以位点特异性方式调节其在 tau mRNA 加工中的功能。
Trans‐active response DNA‐binding protein of 43 kDa (TDP‐43) promotes tau mRNA instability and tau exon 10 inclusion. Aggregation of phosphorylated TDP‐43 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. Casein kinase 1ε (CK1ε) phosphorylates TDP‐43 at multiple sites, enhances its cytoplasmic aggregation, and modulates its function in tau mRNA processing. To determine roles of TDP‐43 site‐specific phosphorylation in its localization, aggregation, and function in tau mRNA processing, TDP‐43 was mutated to alanine or aspartic acid at Ser379, Ser403/404, or Ser409/410 to block or mimic phosphorylation. Site‐specific phosphorylation of TDP‐43 and its mutants by CK1ε was studied in vitro and in cultured cells. Cytoplasmic and nuclear TDP‐43 and phospho‐TDP‐43 were analyzed by western blots. Aggregation of TDP‐43 was assessed by immunostaining and level of radioimmunoprecipitation assay buffer‐insoluble TDP‐43. Green florescent protein tailed with tau 3′‐untranslated region and mini‐tau gene pCI/SI9‐LI10 were used to study tau mRNA stability and alternative splicing of tau exon 10. We found that phospho‐blocking mutations of TDP‐43 at Ser379, Ser403/404, or Ser409/410 were not effectively phosphorylated by CK1ε. Compared with TDP‐43, higher level of phosphorylated TDP‐43 in the cytoplasm was observed. Phospho‐mimicking mutations at these sites enhanced cytoplasmic aggregation of TDP‐43. Green florescent protein expression was not inhibited by phospho‐blocking mutants of TDP‐43, but tau exon 10 inclusion was further enhanced by phospho‐blocking mutations at Ser379 and Ser403/404. Phosphorylation of TDP‐43 at Ser379, Ser403/404, or Ser409/410 primes its phosphorylation by CK1ε, promotes TDP‐43 cytoplasmic aggregation, and modulates its function in tau mRNA processing in site‐specific manner.