NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China; Department of Gastroenterology and Hepatology, Shanghai Institute of Liver Diseases, Zhongshan Hospital, Fudan University, Shanghai, China.
NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
J Biol Chem. 2021 Jan-Jun;296:100730. doi: 10.1016/j.jbc.2021.100730. Epub 2021 Apr 30.
Proper dendrite morphogenesis and synapse formation are essential for neuronal development and function. Dasm1, a member of the immunoglobulin superfamily, is known to promote dendrite outgrowth and excitatory synapse maturation in vitro. However, the in vivo function of Dasm1 in neuronal development and the underlying mechanisms are not well understood. To learn more, Dasm1 knockout mice were constructed and employed to confirm that Dasm1 regulates dendrite arborization and spine formation in vivo. We performed a yeast two-hybrid screen using Dasm1, revealing MRCKβ as a putative partner; additional lines of evidence confirmed this interaction and identified cytoplasmic proline-rich region (823-947 aa) of Dasm1 and MRCKβ self-activated kinase domain (CC1, 410-744 aa) as necessary and sufficient for binding. Using co-immunoprecipitation assay, autophosphorylation assay, and BS3 cross-linking assay, we show that Dasm1 binding triggers a change in MRCKβ's conformation and subsequent dimerization, resulting in autophosphorylation and activation. Activated MRCKβ in turn phosphorylates a class 2 regulatory myosin light chain, which leads to enhanced actin rearrangement, causing the dendrite outgrowth and spine formation observed before. Removal of Dasm1 in mice leads to behavioral abnormalities. Together, these results reveal a crucial molecular pathway mediating cell surface and intracellular signaling communication to regulate actin dynamics and neuronal development in the mammalian brain.
适当的树突形态发生和突触形成对于神经元的发育和功能至关重要。Dasm1 是免疫球蛋白超家族的成员，已知它能促进体外树突生长和兴奋性突触成熟。然而，Dasm1 在神经元发育中的体内功能及其潜在机制尚不清楚。为了了解更多信息，构建了 Dasm1 敲除小鼠并进行了研究，以证实 Dasm1 能调节体内树突分支和棘突的形成。我们利用 Dasm1 进行酵母双杂交筛选，发现 MRCKβ 是一个假定的伴侣；进一步的证据证实了这种相互作用，并鉴定出 Dasm1 的细胞质脯氨酸丰富区（823-947 aa）和 MRCKβ 自身激活激酶结构域（CC1，410-744 aa）是结合所必需和充分的。通过共免疫沉淀实验、自动磷酸化实验和 BS3 交联实验，我们表明，Dasm1 结合触发了 MRCKβ 构象的变化和随后的二聚化，导致自动磷酸化和激活。激活的 MRCKβ 反过来磷酸化一种 2 类调节肌球蛋白轻链，导致肌动蛋白重排增强，从而导致之前观察到的树突生长和棘突形成。在小鼠中去除 Dasm1 会导致行为异常。总之，这些结果揭示了一个关键的分子途径，介导细胞表面和细胞内信号通讯，以调节哺乳动物大脑中的肌动蛋白动力学和神经元发育。
