Effect of phosphorylation on the assembly of Hippo signaling regulatory complexes.

Abstract

The Hippo pathway is an evolutionarily conserved signaling network that plays a crucial role in maintaining tissue homeostasis by regulating cell growth/proliferation, differentiation, and apoptosis in response to environmental stimuli. The Hippo pathway integrates a variety of cellular cues including cell contact/density, growth factors, oxidative stress, hormones, and metabolic conditions, to regulate the function of the transcriptional co-activator protein YAP (yes-associated protein) in humans, and its orthologue Yki (yorkie) in Drosophila. Many upstream signals regulate YAP activity by influencing YAP phosphorylation at S127 (S168 in Yki). Although it is understood that phosphorylation of YAP at S127 induces YAP-14-3-3 binding and subsequent cytoplasmic retention, little is known about how phosphorylation at S127 impacts YAP local structure or the assembly of Hippo signaling regulatory complexes. Here we use traditional biophysical techniques such as solution state nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography multi-angle light scattering, to determine the structure, map the binding interface, and characterize the binding thermodynamics of pS127 YAP and Hippo pathway binding partner LATS1 (large tumor suppressor kinase 1).