The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry and cell-based assays, including chemically induced dimerization and single-molecule pulldown, we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH domain–mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our work also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

中文翻译：

---

激酶结构域邻近性的增加在河马信号转导过程中促进MST2自磷酸化

Hippo途径在发育生物学中起着重要作用，它通过核心激酶盒的活性控制细胞增殖来介导器官大小。多个上游事件激活了该途径，但是尚不清楚每个事件如何控制该核心激酶盒。核心激酶盒的激活从激酶MST1 / 2（也称为STK3 / 4）的磷酸化开始。在这里，结合使用体外生物化学和基于细胞的测定法，包括化学诱导的二聚化和单分子下拉，我们发现增加相邻激酶结构域的接近度，而不是形成特定的蛋白质装配体，足以触发自身磷酸化。我们在细胞中验证了这种机制，并证明了与活动途径相关的多个事件，包括SARAH域介导的二聚化，膜募集以及与效应蛋白SAV1的复合物形成，均增加了MST2的激酶结构域亲和力和自磷酸化。总之，我们的结果表明，多个不同的上游信号各自利用相同的共同分子机制来刺激MST2自磷酸化。该机制可能在MST2同源物中是保守的。我们的工作还强调了每个激活事件之间河马信号传播的潜在差异，这归因于每个蛋白集合的动力学和调控上的差异，这些蛋白质集合触发MST2自磷酸化以及激活中可能的冗余。我们的结果表明，多个不同的上游信号各自利用相同的共同分子机制来刺激MST2自磷酸化。该机制可能在MST2同源物中是保守的。我们的工作还强调了每个激活事件之间河马信号传播的潜在差异，这归因于每个蛋白集合的动力学和调控上的差异，这些蛋白质集合触发MST2自磷酸化以及激活中可能的冗余。我们的结果表明，多个不同的上游信号各自利用相同的共同分子机制来刺激MST2自磷酸化。该机制可能在MST2同源物中是保守的。我们的工作还强调了每个激活事件之间河马信号传播的潜在差异，这归因于每个蛋白集合的动力学和调控上的差异，这些蛋白质集合触发MST2自磷酸化以及激活中可能的冗余。