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

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.

Activated MST2 kinase is free of kinetic regulation

“…Attempts to purify either full-length SAV1 or the SARAH-domain of SAV1 were unsuccessful, instead we used the SARAH domain from the Drosophila homolog (dSAV-SARAH). Drosophila Salvador shares both sequence, structural, and functional similarity with SAV1, and previous studies of mixed systems (Drosophila Salvador and human MST2) have displayed similar behavior as equivalent mammalian systems 8,18,30,32,35,50 . Then, we confirmed that each purified variant did indeed form a complex with pMST2-FL using an in vitro pull-down assay (Figure 5).…”

“…Genes encoding the full-length human RASSF5 isoform D (hRASSF5, UniProtKB Q8WWW0-2), the full-length human MOB1A (UniProtKB Q9H8S9-1) with two site specific substitutions T12A and T35A (hMOB1A T2A ), or the SARAH domain (residues 531-608) of Salvador from Drosophila melanogaster (dSAV-SARAH)(UniProtKB Q9VCR6)were cloned into a modified pBAT vector downstream from H6 and SUMO tags 44 . Each of the three binding proteins were purified using the same basic protocol, as previously described 30 . Each protein was expressed in T7 Express cells (New England BioLabs, MA) grown in Terrific Broth at 37°C following induction with 0.5mM IPTG.…”

“…The SARAH domain of MST1/2 either forms homodimers, which stimulate autophosphorylation, or heterodimers with the SARAH-domains of either SAV1 or RASSF family members, and each of these interactions tune pathway activity 5,17,30,31 . SAV1 promotes Hippo pathway activity in part by contributing to MST1/2 activation by multiple routes --increasing the effective concentration of MST1/2, translocating it to the membrane, and inhibiting the activity of the STRIPAK phosphatase 8,18,[32][33][34][35] .…”

Expression Patterns of Ras-Association Domain Family Genes and Their Prognostic Values in Gastric Cancer

“…RASSF1-6 each owns their Ras-related domain at the C-terminal 10 and RASSF7-10 at the Nterminal 11 . RASSF1-6 each also possesses the SARAH protein-protein interaction motif, which is a conserved mediator in the Hippo pathway [12][13][14] .…”

The regulatory networks of the Hippo signaling pathway in cancer development

“…In addition to the classical eight cancer-related signaling pathways, with the development of scientific research, more and more signaling pathways have been found to play a significant role in the development of cancer [1]. The Hippo pathway found in Drosophila is a crucial entry point for understanding the molecular mechanisms that control organ growth during development and regeneration [2][3][4]. The intracellular environment's inhibitory growth signal activates the Hippo signaling pathway, causing a series of Kinase cascade phosphorylation.…”