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Mediated Multimerization of Hippo Pathway Complexes in Drosophila

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Mediated Multimerization of Hippo Pathway Complexes in Drosophila ARTICLE cro Biophysical characterization of SARAH domain–mediated multimerization of Hippo pathway complexes in Drosophila Received for publication, January 16, 2020, and in revised form, March 23, 2020 Published, Papers in Press, March 25, 2020, DOI 10.1074/jbc.RA120.012679 X Leah Cairns‡, Angela Patterson§, Kyler A. Weingartner‡, T. J. Koehler‡, Daniel R. DeAngelis‡, Katherine W. Tripp¶, Brian Bothner§, and X Jennifer M. Kavran‡ʈ**1 From the ‡Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 20215, the §Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, 59717, the ¶T. C. Jenkins Department of Biophysics, Krieger School of Arts and Sciences, Johns Hopkins University, Baltimore, Maryland 201218, the ʈDepartment of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland, 20215, and the **Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 20215 Edited by Enrique M. De La Cruz Hippo pathway signaling limits cell growth and proliferation The Hippo pathway controls tissue growth and differentia- and maintains the stem-cell niche. These cellular events result tion by regulating the rate of cell proliferation and apoptosis from the coordinated activity of a core kinase cassette that is (1–6). Gene expression is silenced upon phosphorylation and regulated, in part, by interactions involving Hippo, Salvador, cytoplasmic retention of the transcriptional co-factor Yorkie (1, and dRassF. These interactions are mediated by a conserved 3–8). Aberrant pathway activity leads to overgrowth pheno- coiled-coil domain, termed SARAH, in each of these proteins. types in both mammalian and fly models and is associated with SARAH domain–mediated homodimerization of Hippo kinase tumorigenesis in humans (1, 3–11). Hippo signal transduction leads to autophosphorylation and activation. Paradoxically, relies on the activity of a core kinase cassette that includes two SARAH domain–mediated heterodimerization between Hippo kinases, Hippo and Warts, and two accessory proteins, Sal- and Salvador enhances Hippo kinase activity in cells, whereas vador and Mats. These proteins, together with Yorkie, form complex formation with dRassF inhibits it. To better under- a core kinase cassette that is responsible for gene regulation. stand the mechanism by which each complex distinctly modu- Each component of this core cassette has a mammalian ho- lates Hippo kinase and pathway activity, here we biophysically molog with similar function. The mechanisms regulating the characterized the entire suite of SARAH domain–mediated activity of this kinase cassette are also most likely conserved complexes. We purified the three SARAH domains from Dro- as disruption of the genes encoding either Hippo (hpo)or sophila melanogaster and performed an unbiased pulldown Mats (mts) in flies results in overgrowth phenotypes that can assay to identify all possible interactions, revealing that iso- be rescued by complementation with the mammalian genes lated SARAH domains are sufficient to recapitulate the cel- encoding the homologous proteins, Mst1/2 or Mob1, respec- lular assemblies and that Hippo is a universal binding part- tively (5, 7, 10). ner. Additionally, we found that the Salvador SARAH domain The activity of the core cassette begins with the activation of homodimerizes and demonstrate that this interaction is con- Hippo kinase, or Mst1/2 in mammals, which involves phos- served in Salvador’s mammalian homolog. Using native MS, phorylation of the activation loop (12, 13). Genetic, cellular, and we show that each of these complexes is dimeric in solution. in vitro data suggest that homodimerization mediated by the We also measured the stability of each SARAH domain com- conserved C-terminal coiled-coil domain, termed SARAH, of plex, finding that despite similarities at both the sequence either Hippo or Mst1/2 promotes autophosphorylation in trans and structural levels, SARAH domain complexes differ in sta- and kinase activation (6, 14–19). Activated Hippo then phos- bility. The identity, stoichiometry, and stability of these phorylates Warts. This phosphorylation event leads to Warts interactions characterized here comprehensively reveal the activation, which also involves autophosphorylation and com- nature of SARAH domain–mediated complex formation and plex formation with Mats (5, 20–22). Warts kinase can then provide mechanistic insights into how SARAH domain– phosphorylate the transcriptional co-factor Yorkie, leading mediated interactions influence Hippo pathway activity. to the accumulation of phosphorylated Yorkie in the cyto- plasm (7, 8). SARAH domains were first identified as a conserved coiled- This work is supported by National Institutes of Health Grants R01GM134000 coil region located at the C termini of three members of the (to J. M. K.), T32 GM007445 (to L. C.), T32 GM080189 (to T. J. K.), and Hippo pathway, Salvador, dRassF, and Hippo, and were named T32CA009110 (to K. W. and D. D. R.). The authors declare that they have no after each (23). Dimeric, trimeric, and tetrameric assemblies of conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the coiled-coils have been proposed, but the preponderance of evi- official views of the National Institutes of Health. dence supports a dimeric assembly with limited evidence for This article contains Fig. S1. tetrameric assemblies and no evidence for trimeric ones (14, 18, 1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins Uni- 23–34). Structural studies have each revealed a conserved versity, 615 N. Wolfe St., Baltimore, MD 21205. E-mail: [email protected]. arrangement of two antiparallel coiled-coils with each mono- This is an Open Access article under the CC BY license. 6202 J. Biol. Chem. (2020) 295(18) 6202–6213 © 2020 Cairns et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Biophysical characterization of SARAH domains mer adopting a short 3/10-helix followed by a turn and a long ␣-helix (18, 25–32). The dimer interface is mediated by inter- strand interactions along the length of each ␣-helix and pro- vides a rationale for why each SARAH domain–mediated com- plex is mutually exclusive. The SARAH domain of Salvador contains an extended N-terminal region that forms an addi- tional ␣-helix that enlarges the dimer interface with Hippo (31, 32), and Salvador variants lacking this extension bind less tightly to Hippo SARAH domain (32). Together, these data sug- gest that Hippo:Salvador complexes are tighter than other SARAH domain–mediated complexes. Analysis of the interac- tions of SARAH domain complexes have largely focused on the mammalian homologs of Hippo and dRassF with little to no data for Salvador SARAH domain, and reported dissociation constants vary widely, from nanomolar to millimolar (29, 30, Figure 1. Purification of the entire suite of SARAH domain complexes. 35–37). There has been no systematic study of the entire set Coomassie-stained SDS-polyacrylamide gel of purified SARAH domain pro- of possible SARAH domain–mediated interactions, making it teins following gel-filtration chromatography. From left to right are dRassF, difficult to understand the likelihood of any given complex Hippo, and Salvador followed by complexes between Salvador:Hippo and dRassF:Hippo SARAH domains. The migration of each SARAH domain is indi- forming. cated by a cartoon rectangle with green for dRassF, blue for Hippo, and orange Salvador and dRassF bind Hippo via their respective SARAH for Salvador. domains and yet have opposing roles in regulating the activity of Hippo kinase, with Salvador stimulating pathway activity and dRassF inhibiting it (3, 24). The mechanisms by which SARAH To rationalize the function of SARAH domains in modulat- domain–mediated complex formation influences Hippo kinase ing Hippo pathway activity, we undertook a systematic study of activity are still a matter of investigation. The importance of the biophysical parameters of complex formation by the entire these SARAH domain proteins to Hippo pathway activity is suite of isolated SARAH domain complexes. We chose to work underscored by the observation that flies bearing alleles encod- with the Drosophila SARAH domains because they are a sim- ing variants of either Hippo or Salvador that lack SARAH pler system than their mammalian counterparts; there is only domains display overgrowth phenotypes typical of pathway dis- one homolog of each Drosophila SARAH domain compared ruption (4, 6). Likewise, combination of mutant alleles of with multiple isoforms of the human homologs of Hippo and dRassF and Hippo lacking SARAH domains leads to a stronger dRassF. We began by purifying each SARAH domain individu- overgrowth phenotype than either allele separately (24). Each ally to systematically study the entire set of SARAH domain SARAH domain–mediated complex correlates with a distinct complexes. We report here in vitro binding studies that re- phosphorylation state of Hippo. Immunoprecipitation assays vealed that a previously unidentified interaction between Sal- revealed that unphosphorylated Hippo bound dRassF and phos- vador SARAH domains that we determine is dimeric, mediates phorylated Hippo bound Salvador (24). Evidence
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