Hsu Jonathan, Weiss Eric L
Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America.
PLoS One. 2013 Oct 21;8(10):e78334. doi: 10.1371/journal.pone.0078334. eCollection 2013.
Hippo pathways are ancient signaling systems that contribute to cell growth and proliferation in a wide diversity of eukaryotes, and have emerged as a conserved regulator of organ size control in metazoans. In budding yeast, a Hippo signaling pathway called the Regulation of Ace2 and Morphogenesis (RAM) network promotes polarized cell growth and the final event in the separation of mother and daughter cells. A crucial regulatory input for RAM network control of cell separation is phosphorylation of a conserved hydrophobic motif (HM) site on the NDR/LATS family kinase Cbk1. Here we provide the first direct evidence that the Hippo-like kinase Kic1 in fact phosphorylates the HM site of Cbk1, and show that Kic1 is allosterically activated by Hym1, a highly conserved protein related to mammalian MO25. Using the structure of mammalian MO25 in complex with the Kic1-related pseudokinase STRAD, we identified conserved residues on Kic1 that are required for interaction with Hym1. We find that Kic1 and Hym1 protein levels remain constant throughout the cell cycle but the proteins' association is regulated, with maximal interaction coinciding with peak Cbk1 HM site phosphorylation. We show that this association is necessary but not sufficient for this phosphorylation, suggesting another level of regulation is required to promote the complex to act upon its substrates. This work presents a previously undiscovered cell cycle regulated interaction between a Hippo kinase and a broadly conserved allosteric activator. Because of the conserved nature of this pathway in higher eukaryotes, this work may also provide insight into the modularity of Hippo signaling pathways.
河马通路是古老的信号系统，在多种真核生物中对细胞生长和增殖起作用，并已成为后生动物器官大小控制的保守调节因子。在芽殖酵母中，一种名为Ace2和形态发生调节（RAM）网络的河马信号通路促进极化细胞生长以及母细胞和子细胞分离的最终事件。RAM网络控制细胞分离的关键调节输入是NDR/LATS家族激酶Cbk1上保守疏水基序（HM）位点的磷酸化。在这里，我们提供了首个直接证据，证明类河马激酶Kic1实际上磷酸化了Cbk1的HM位点，并表明Kic1被Hym1变构激活，Hym1是一种与哺乳动物MO25相关的高度保守蛋白。利用哺乳动物MO25与Kic1相关假激酶STRAD复合物的结构，我们确定了Kic1上与Hym1相互作用所需的保守残基。我们发现Kic1和Hym1蛋白水平在整个细胞周期中保持恒定，但蛋白质的结合受到调控，最大相互作用与Cbk1 HM位点磷酸化峰值一致。我们表明这种结合对于这种磷酸化是必要的但不是充分的，这表明需要另一个调节水平来促进复合物作用于其底物。这项工作展示了河马激酶与广泛保守的变构激活剂之间以前未被发现的细胞周期调节相互作用。由于该通路在高等真核生物中的保守性质，这项工作也可能为河马信号通路的模块化提供见解。
