Biophysical characterization of SARAH domain-mediated multimerization of Hippo pathway complexes in Drosophila.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed in each of these proteins. domain-mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, duanyu1800H domain-mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of duanyu1800H domain-mediated complexes. We purified the three duanyu1800H domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated duanyu1800H domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador duanyu1800H domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each duanyu1800H domain complex, finding that despite similarities at both the sequence and structural levels, duanyu1800H domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of duanyu1800H domain-mediated complex formation and provide mechanistic insights into how duanyu1800H domain-mediated interactions influence Hippo pathway activity. © 2020 Cairns et al.

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