Abstract
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.
Highlights
Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche
We previously determined the domain boundaries for the Drosophila melanogaster Salvador SARAH domain [32], which enabled us to purify each of the three SARAH domains from D. melanogaster, Salvador, Hippo, and dRassF (Fig. 1)
Incubated with hexahistidine (H6)2 and thioredoxin-tagged SARAH domains immobilized on nickel-affinity resin; because the SARAH domains have similar mobility on SDS-PAGE, the thioredoxin tag was added to change the migration of one SARAH domain in each pulldown to help with visualization
Summary
We previously determined the domain boundaries for the Drosophila melanogaster Salvador SARAH domain [32], which enabled us to purify each of the three SARAH domains from D. melanogaster, Salvador, Hippo, and dRassF (Fig. 1). To determine which complexes could be formed by isolated SARAH domains, we performed an unbiased in vitro pulldown assay using purified proteins. The resin was collected and washed, and the amount of untagged SARAH domain bound was monitored by Coomassie-stained SDS-PAGE (Fig. 2). Salvador SARAH domain and dRassF SARAH domain bound only themselves or Hippo SARAH domain. No interactions were detected between Salvador and dRassF SARAH domains, regardless of which protein was the bait or prey in the experiment. To ensure complexes were homogeneous in future assays, SARAH domain heterocomplexes were co-expressed and purified using tandem-affinity purification (Fig. 1)
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