The tumor-suppressive Hippo pathway controls tissue homeostasis through balancing cell apoptosis and proliferation. diverse cellular signals. through genetic screens for mutations that caused cells overgrowth and was later on shown to be conserved in mammals (Badouel et al. 2009 Edgar 2006 Halder and Johnson 2011 Harvey and Tapon 2007 Harvey et al. 2013 Pan 2010 Staley and Irvine 2012 Zhao et al. 2010 The ABT-378 core components of the mammalian Hippo pathway include the Ste20 family kinases Mst1/2 the scaffolding protein Salvador (Sav1) the NDR family kinases Lats1/2 and the adaptor protein Mob1. They form a central kinase cascade to transduce signals from cell-surface receptors (Avruch et al. 2012 Hergovich 2012 In the canonical Hippo kinase cascade Mst1/2 in complex with Sav1 phosphorylate and activate the Lats1/2-Mob1 complexes which then phosphorylate the transcriptional co-activator YAP (Yes-associated protein) a major downstream target ABT-378 of the Hippo pathway (Dong et al. 2007 Hao et al. 2008 Hong and Guan 2012 Huang et al. 2005 Zhao et al. 2007 Lats1/2-mediated phosphorylation inhibits YAP in two ways. Phosphorylation of YAP at S127 by Lats1/2 creates a docking site for 14-3-3 proteins. Binding of 14-3-3 causes the cytoplasmic sequestration and inactivation of YAP (Dong et al. 2007 Hao et al. 2008 Zhao et al. 2007 Phosphorylation of YAP at S381 by Lats1/2 promotes its ubiquitination and degradation (Zhao et al. 2010 When the Hippo pathway is definitely turned off YAP is definitely dephosphorylated and translocates into the nucleus. Although YAP does not contain a DNA-binding website it binds to the TEAD family of transcription factors (which consists of a sequence-specific DNA-binding website) to form a functional cross transcription element (Luo 2010 Sudol et al. 2012 Zhao et al. 2008 The YAP-TEAD cross then activates the transcription of Hippo-responsive genes that promote cell growth and proliferation and inhibit apoptosis. Tremendous progress has been made for the dissection of the molecular circuitry of the Hippo pathway and for the understanding of the pathophysiology of this pathway in ABT-378 multiple organisms. By contrast mechanistic and structural studies in this area possess lagged behind. In ABT-378 particular the activation mechanisms of the core Mst1/2-Lats1/2 kinase cascade remain elusive. The upstream kinases Mst1/2 contain an N-terminal kinase domain and a C-terminal SARAH (Salvador/RASSF1A/Hippo) domain (Figure 1A). Mst1 and Mst2 can each form a constitutive homodimer through the SARAH domain and kinase activation requires autophosphorylation of the activation loop (T183 for Mst1 and T180 for Mst2) (Avruch et al. 2012 Creasy et al. 1996 The Mst1/2 regulators Sav1 and RASSF proteins also contain SARAH domains (Figure 1A). The Mst1/2 SARAH domain can form a heterodimer with RASSF SARAH (Hwang et al. 2007 and a heterotetramer with Sav1 SARAH (data not shown). RASSF binding and Sav1 binding to Mst1/2 are mutually exclusive. How RASSFs and Sav1 regulate Mst1/2 activation by forming different SARAH domain-dependent complexes is not understood. Figure 1 Structural Basis for Mst2 Autoactivation RASSFs are important tumor suppressors (Avruch et al. 2009 Richter et al. 2009 Their expression is frequently silenced in human cancers through promoter methylation and reintroduced expression of RASSF1A or RASSF5 inhibits human tumor cell growth (Aoyama et al. 2004 In addition RASSF1A knockout mice have increased spontaneous and chemical-induced tumor susceptibility (Tommasi et al. 2005 The roles of RASSFs in the tumor-suppressive Hippo pathway are far from clear however. In and mammals RASSFs appear to have both negative and positive regulatory functions in the Hippo pathway. Here we report the crystal structures of the human Mst2 kinase domain and Mst2 in complex using PP2Bgamma the SARAH site of RASSF5. SARAH-mediated homodimerization of Mst2 is crucial because of its activation and trans-autophosphorylation. RASSF5 disrupts this dimer prevents and interface Mst2 autoactivation. Oddly enough binding of RASSF5 to Mst2 which has currently undergone autoactivation will not inhibit the kinase activity of Mst2 for the downstream substrate Mob1. This insufficient inhibition of energetic Mst2 might permit RASSF5 to truly have a positive regulatory ABT-378 part in the Hippo signaling. Therefore the purchase of RASSF5 activation-loop and binding phosphorylation determines whether RASSF5 acts mainly because an inhibitor of Mst2. We speculate how the temporal regulation from the binding between RASSFs and Mst1/2 might.