Randall Centre for Cell and Molecular Biophysics, King's College London, London, United Kingdom.
PLoS One. 2023 Apr 20;18(4):e0284453. doi: 10.1371/journal.pone.0284453. eCollection 2023.
Obscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in cells, in vitro characterization of these interactions using biophysical techniques has been hampered by the intrinsic instability of obscurin GEF domains. To study substrate specificity, mechanism and regulation of obscurin GEF function by individual domains, we successfully optimized recombinant production of obscurin GEF domains and found that MST-family kinases phosphorylate the obscurin DH domain at Thr5798. Despite extensive testing of multiple GEF domain fragments, we did not detect any nucleotide exchange activity in vitro against 9 representative small GTPases. Bioinformatic analyses show that obscurin differs from other Trio-subfamily GEFs in several important aspects. While further research is necessary to evaluate obscurin GEF activity in vivo, our results indicate that obscurin has atypical GEF domains that, if catalytically active at all, are subject to complex regulation.
obscurin 是一种巨大的肌肉蛋白（>800 kDa），具有多个信号结构域，包括来自 Trio 亚家族鸟嘌呤核苷酸交换因子（GEFs）的 SH3-DH-PH 结构域三联体。虽然之前的研究表明这些结构域可以在细胞中激活小 GTPase RhoA 和 RhoQ，但使用生物物理技术对这些相互作用进行体外表征受到 obscurin GEF 结构域固有不稳定性的阻碍。为了研究底物特异性、机制和 obscurin GEF 功能的调节，我们成功地优化了 obscurin GEF 结构域的重组生产，并发现 MST 家族激酶在 Thr5798 处磷酸化 obscurin DH 结构域。尽管对多个 GEF 结构域片段进行了广泛测试，但我们在体外没有检测到任何针对 9 种代表性小 GTPase 的核苷酸交换活性。生物信息学分析表明， obscurin 在几个重要方面与其他 Trio 亚家族 GEFs 不同。虽然有必要进一步研究 obscurin GEF 在体内的活性，但我们的结果表明 obscurin 具有非典型的 GEF 结构域，如果具有催化活性，则受到复杂的调节。
