Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, U.S.A.
The T.C. Jenkins Department of Biophysics, Krieger School of Arts and Sciences, Johns Hopkins University, Baltimore, Maryland, U.S.A.
Biochem J. 2023 Aug 16;480(15):1165-1182. doi: 10.1042/BCJ20230067.
The Hippo pathway controls tissue growth and regulates stem cell fate through the activities of core kinase cassette that begins with the Sterile 20-like kinase MST1/2. Activation of MST1/2 relies on trans-autophosphorylation but the details of the mechanisms regulating that reaction are not fully elucidated. Proposals include dimerization as a first step and include multiple models for potential kinase-domain dimers. Efforts to verify and link these dimers to trans-autophosphorylation were unsuccessful. We explored the link between dimerization and trans-autophosphorylation for MST2 and the entire family of MST kinases. We analyzed crystal lattice contacts of structures of MST kinases and identified an ensemble of kinase-domain dimers compatible with trans-autophosphorylation. These dimers share a common dimerization interface comprised of the activation loop and αG-helix while the arrangements of the kinase-domains within the dimer varied depending on their activation state. We then verified the dimerization interface and determined its function using MST2. Variants bearing alanine substitutions of the αG-helix prevented dimerization of the MST2 kinase domain both in solution and in cells. These substitutions also blocked autophosphorylation of full-length MST2 and its Drosophila homolog Hippo in cells. These variants retain the same secondary structure as wild-type and capacity to phosphorylate a protein substrate, indicating the loss of MST2 activation can be directly attributed to a loss of dimerization rather than loss of either fold or catalytic function. Together this data functionally links dimerization and autophosphorylation for MST2 and suggests this activation mechanism is conserved across both species and the entire MST family.
Hippo 通路通过起始于无菌 20 样激酶 MST1/2 的核心激酶盒的活性来控制组织生长并调节干细胞命运。MST1/2 的激活依赖于转自磷酸化，但调节该反应的机制细节尚未完全阐明。提议包括二聚化作为第一步，并包括潜在激酶结构域二聚体的多个模型。验证这些二聚体并将其与转自磷酸化联系起来的努力均未成功。我们探索了 MST2 和整个 MST 激酶家族中二聚化和转自磷酸化之间的联系。我们分析了 MST 激酶的晶体晶格接触结构，并确定了一组与转自磷酸化相容的激酶结构域二聚体。这些二聚体共享由激活环和αG-螺旋组成的共同二聚化界面，而二聚体中激酶结构域的排列则根据其激活状态而变化。然后，我们使用 MST2 验证了二聚化界面及其功能。带有αG-螺旋丙氨酸取代的变体阻止了 MST2 激酶结构域在溶液中和细胞中的二聚化。这些取代还阻止了全长 MST2 和其果蝇同源物 Hippo 在细胞中的自磷酸化。这些变体保留与野生型相同的二级结构和磷酸化蛋白质底物的能力，表明 MST2 激活的丧失可以直接归因于二聚化的丧失，而不是折叠或催化功能的丧失。这些数据共同将 MST2 的二聚化和自磷酸化功能联系起来，并表明这种激活机制在两个物种和整个 MST 家族中都是保守的。
