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The Legionella Kinase Legk7 Exploits the Hippo Pathway Scaffold Protein MOB1A for Allostery and Substrate Phosphorylation

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The Legionella Kinase Legk7 Exploits the Hippo Pathway Scaffold Protein MOB1A for Allostery and Substrate Phosphorylation The Legionella kinase LegK7 exploits the Hippo pathway scaffold protein MOB1A for allostery and substrate phosphorylation Pei-Chung Leea,b,1, Ksenia Beyrakhovac,1, Caishuang Xuc, Michal T. Bonieckic, Mitchell H. Leea, Chisom J. Onub, Andrey M. Grishinc, Matthias P. Machnera,2, and Miroslaw Cyglerc,2 aDivision of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892; bDepartment of Biological Sciences, College of Liberal Arts and Sciences, Wayne State University, Detroit, MI 48202; and cDepartment of Biochemistry, University of Saskatchewan, Saskatoon, SK S7N5E5, Canada Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved May 1, 2020 (received for review January 12, 2020) During infection, the bacterial pathogen Legionella pneumophila Active LATS1/2 phosphorylate the cotranscriptional regulator manipulates a variety of host cell signaling pathways, including YAP1 (yes-associated protein 1) and its homolog TAZ (tran- the Hippo pathway which controls cell proliferation and differen- scriptional coactivator with PDZ-binding motif). Phosphorylated tiation in eukaryotes. Our previous studies revealed that L. pneu- YAP1 and TAZ are prevented from entering the nucleus by being mophila encodes the effector kinase LegK7 which phosphorylates either sequestered in the cytosol through binding to 14-3-3 pro- MOB1A, a highly conserved scaffold protein of the Hippo path- teins or targeted for proteolytic degradation (6, 8). Consequently, way. Here, we show that MOB1A, in addition to being a substrate the main outcome of signal transduction along the Hippo pathway of LegK7, also functions as an allosteric activator of its kinase is changes in gene expression (6). – activity. A crystallographic analysis of the LegK7 MOB1A complex MOB1A and MOB1B share 96% sequence identity over 216 revealed that the N-terminal half of LegK7 is structurally similar to amino acid (a.a.) residues and are considered to be functionally eukaryotic protein kinases, and that MOB1A directly binds to the redundant. They play a key role in the Hippo pathway by acting LegK7 kinase domain. Substitution of interface residues critical for as scaffold proteins, coordinating a sequential, multistep phos- complex formation abrogated allosteric activation of LegK7 both MICROBIOLOGY in vitro and within cells and diminished MOB1A phosphorylation. phorylation cascade that runs from MST1/2 to the downstream Importantly, the N-terminal extension (NTE) of MOB1A not only components YAP1/TAZ (10, 11). When not phosphorylated, the regulated complex formation with LegK7 but also served as a flexible N-terminal extension (NTE) of MOB1A occupies the docking site for downstream substrates such as the transcriptional binding surface for LATS/NDR and interferes with binding of coregulator YAP1. Deletion of the NTE from MOB1A or addition of these cellular ligands to MOB1A. MST1/2 catalyzes the phos- NTE peptides as binding competitors attenuated YAP1 recruitment phorylation of two threonine residues, T12 and T35, within the to and phosphorylation by LegK7. By providing mechanistic in- sight into the formation and regulation of the LegK7–MOB1A com- Significance plex, our study unravels a sophisticated molecular mimicry strategy that is used by L. pneumophila to take control of the host In order to establish conditions favorable for their intracellular cell Hippo pathway. survival, microbial pathogens modulate a wide range of host cell processes. The eukaryotic Hippo pathway is best known for microbial pathogen | type IV secretion system | translocated effector | its role in controlling cell proliferation and differentiation, yet crystal structure | MST1/2 we recently discovered that it also plays an important role during infection with Legionella pneumophila, the causative icrobial pathogens manipulate host cell signaling pathways agent of Legionnaires’ pneumonia. We show that the Legion- Mby encoding molecular mimics of protein kinases. Kinases ella kinase LegK7 mimics kinases from the host cell Hippo catalyze target phosphorylation by transferring the gamma pathway in order to exploit the scaffold protein MOB1A for phosphate group of adenosine 5′-triphosphate (ATP) onto side downstream target recruitment and phosphorylation. By chains of substrate proteins, preferentially serine, threonine, or employing this strategy of molecular mimicry, LegK7 can fulfill tyrosine residues, thereby altering the activity, localization, or its function in a wide range of host cells, including amoeba in stability of their substrates. Protein phosphorylation is one of the the environment and alveolar macrophages during infection most abundant and most important posttranslational modifica- in humans. tions in living cells, and mammals encode hundreds of kinases that target thousands of substrate proteins (1, 2). To promote Author contributions: P.-C.L., K.B., M.P.M., and M.C. designed research; P.-C.L., K.B., C.X., M.T.B., M.H.L., C.J.O., and A.M.G. performed research; P.-C.L., K.B., M.P.M., and M.C. infection, many pathogens, including bacteria and eukaryotic analyzed data; and P.-C.L., K.B., M.P.M., and M.C. wrote the paper. parasites, encode effector kinases that are translocated into the The authors declare no competing interest. host cytosol where they alter signaling cascades, suggesting that kinase mimicry is a commonly used strategy to exploit host cell This article is a PNAS Direct Submission. function (3, 4). Published under the PNAS license. The Hippo pathway is an ancient kinase signaling cascade Data deposition: The coordinates and structure factors of the nonphosphorylated LegK7(11–530)–MOB1A(33–216) apo complex and phosphorylated LegK7(11–530)– conserved in eukaryotes. It is best known for its role in controlling MOB1A(33–216) complex with ATP analog have been deposited in the Protein Data Bank, developmental processes such as cell-cycle progression, cell pro- https://www.pdb.org (PDB ID codes 6MCQ and 6MCP, respectively). liferation, differentiation, and apoptosis (5, 6). Mammalian Ste20- 1P.-C.L. and K.B. contributed equally to this work. like kinases 1/2 (MST1/2), orthologs of the Drosophila Hippo ki- 2To whom correspondence may be addressed. Email: [email protected] or miroslaw. nase, phosphorylate MOB1A/B proteins (Mps one binder kinase [email protected]. activator 1A/B) (7), which in turn form a complex with LATS1/2 This article contains supporting information online at https://www.pnas.org/lookup/suppl/ (large tumor suppressor kinases 1/2) or NDR1/2 (nuclear Dbf2- doi:10.1073/pnas.2000497117/-/DCSupplemental. related kinase 1/2) and assist in their allosteric activation (8, 9). First published June 8, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2000497117 PNAS | June 23, 2020 | vol. 117 | no. 25 | 14433–14443 Downloaded by guest on September 24, 2021 NTE, thus destabilizing interaction with the remainder of (Fig. 1B), suggesting that MOB1A stimulates the kinase activity MOB1A and exposing the LATS/NDR-binding epitope (8, 12). of LegK7 in a dose-dependent manner, most likely by binding Only upon complex formation with MOB1A can allosterically to LegK7. activated LATS/NDR phosphorylate their downstream targets LegK7 is composed of 930 a.a. residues. Since the predicted ki- (5), though the exact molecular mechanism underlying YAP1/ nase domain (amino acids 183 to 462) is located within the TAZ phosphorylation, in particular the role of MOB1A in this N-terminal half (13), we investigated whether the C-terminal half of process, has yet to be determined. LegK7 (∼400 a.a. residues in size) plays a role in allosteric activation We recently showed that the intracellular pathogen Legionella of LegK7 by MOB1A. We purified several C-terminally truncated pneumophila alters the host Hippo signaling pathway during in- LegK7 variants, each encompassing the predicted kinase domain. fection of macrophages (13). This gram-negative bacterium thrives The best-behaved construct was LegK7(11–530), which, in addition within freshwater amoebae in the environment but, upon in- to the kinase domain, contained the flanking amino- and carboxyl- halation by humans, can also infect and replicate within alveolar terminal regions of unknown function. Importantly, LegK7(11–530) macrophages, a scenario that in immunocompromised individuals ’ exhibited kinase activity and robustly phosphorylated MOB1A and often results in a potentially fatal pneumonia called Legionnaires itself, indicating that, similar to the full-length protein, this truncated disease (14, 15). L. pneumophila encodes the effector LegK7, variant was allosterically activated by MOB1A (SI Appendix,Fig. which functions as a molecular mimic of MST1/2 (13). Upon S1A). Hence, in the following, we focused our investigation on the translocation into host cells by the L. pneumophila Dot/Icm type interaction between LegK7(11–530) and MOB1A. IV secretion system (T4SS), LegK7 phosphorylates MOB1A on We examined a possible complex formation between MOB1A T12 and T35, thereby triggering a signaling cascade that results in – – the degradation of the downstream substrates YAP1/TAZ (13). and LegK7(11 530) in vitro. While LegK7(11 530) (61 kDa) and Consequently, the expression of host genes controlled by several MOB1A (25 kDa) eluted according to their expected molecular transcription factors is altered in ways that render macrophages masses during gel-permeation chromatography (Fig. 1C), an – more susceptible to L. pneumophila infection (13). equimolar mixture
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