Biophysical characterization of SARAH domain–mediated multimerization of Hippo pathway complexes in Drosophila
Weingartner, Kyler A.
Koehler, T. J.
DeAngelis, Daniel R.
Tripp, Katherine W.
Kavran, Jennifer M.
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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 SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH 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 SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH 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 SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.
Cairns, Leah, Angela Patterson, Kyler A. Weingartner, T.J. Koehler, Daniel R. DeAngelis, Katherine W. Tripp, Brian Bothner, and Jennifer M. Kavran. “Biophysical Characterization of SARAH Domain–mediated Multimerization of Hippo Pathway Complexes in Drosophila.” Journal of Biological Chemistry 295, no. 18 (May 2020): 6202–6213. doi:10.1074/jbc.ra120.012679.