A phosphorylation switch of Huluwa receptor for embryonic axis induction

A phosphorylation switch of Huluwa receptor for embryonic axis induction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A phosphorylation switch of Huluwa receptor for embryonic axis induction Jing Chen, Yao Li, Yun Yan, Bo Gong, Qianwen Zheng, Haiyan Zhou, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3922628/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Embryonic axis formation is essential for patterning and morphogenesis in vertebrates and is tightly regulated by the dorsal organizer. Previously, we demonstrated that maternally derived Huluwa (Hwa) acts as a dorsal determinant, dictating axis formation by activating β-catenin signaling in zebrafish and Xenopus . However, the mechanism of activation and fine regulation of the Hwa receptor remain unclear. In a systematic mutation screen, a mutation at the 168 th serine (Ser168) in the PPNSP motif of Hwa dramatically abolished its axis-inducing activity. Mechanistically, mutating the Ser168 residue reduced its binding affinity to Tankyrase 1/2 and the degradation of the Axin protein, weakening β-catenin signaling activation. Furthermore, the Ser168 site was confirmed to be phosphorylated via LC-MS/MS and immunoblotting with a phosphorylation-specific antibody. Additionally, several kinases, including Cdk16, Cdk2, and GSK3β, were found to enhance Ser168 phosphorylation in vitro and in vivo , enhancing Hwa's activity in β-catenin signaling and axis induction. Meanwhile, the use of dominant-negative Cdk16 and pHwa (Ser168) antibodies reduced Hwa's function. In summary, Ser168 acts as a phosphorylation switch in Hwa/β-catenin signaling for embryonic axis induction, involving multiple kinases to ensure developmental robustness. Biological sciences/Developmental biology/Embryogenesis/Embryonic induction Biological sciences/Cell biology/Cell signalling Embryonic axis Huluwa(Hwa) Phosphorylation Developmental robustness Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3922628","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":271105658,"identity":"c8ae37d4-bc9e-4d50-81e5-87796c8d901b","order_by":0,"name":"Jing 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induction","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Embryonic axis, Huluwa(Hwa), Phosphorylation, Developmental robustness","lastPublishedDoi":"10.21203/rs.3.rs-3922628/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3922628/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEmbryonic axis formation is essential for patterning and morphogenesis in vertebrates and is tightly regulated by the dorsal organizer. Previously, we demonstrated that maternally derived Huluwa (Hwa) acts as a dorsal determinant, dictating axis formation by activating β-catenin signaling in zebrafish and \u003cem\u003eXenopus\u003c/em\u003e. However, the mechanism of activation and fine regulation of the Hwa receptor remain unclear. In a systematic mutation screen, a mutation at the 168\u003csup\u003eth\u003c/sup\u003e serine (Ser168) in the PPNSP motif of Hwa dramatically abolished its axis-inducing activity. Mechanistically, mutating the Ser168 residue reduced its binding affinity to Tankyrase 1/2 and the degradation of the Axin protein, weakening β-catenin signaling activation. Furthermore, the Ser168 site was confirmed to be phosphorylated via LC-MS/MS and immunoblotting with a phosphorylation-specific antibody. Additionally, several kinases, including Cdk16, Cdk2, and GSK3β, were found to enhance Ser168 phosphorylation \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, enhancing Hwa's activity in β-catenin signaling and axis induction. Meanwhile, the use of dominant-negative Cdk16 and pHwa (Ser168) antibodies reduced Hwa's function. In summary, Ser168 acts as a phosphorylation switch in Hwa/β-catenin signaling for embryonic axis induction, involving multiple kinases to ensure developmental robustness.\u003c/p\u003e","manuscriptTitle":"A phosphorylation switch of Huluwa receptor for embryonic axis induction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-20 11:49:22","doi":"10.21203/rs.3.rs-3922628/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a8d09377-fec7-4b70-86fb-0d9c95cb2207","owner":[],"postedDate":"February 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28571215,"name":"Biological sciences/Developmental biology/Embryogenesis/Embryonic induction"},{"id":28571216,"name":"Biological sciences/Cell biology/Cell signalling"}],"tags":[],"updatedAt":"2024-11-20T08:07:54+00:00","versionOfRecord":{"articleIdentity":"rs-3922628","link":"https://doi.org/10.1038/s41467-024-54450-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-11-19 05:00:00","publishedOnDateReadable":"November 19th, 2024"},"versionCreatedAt":"2024-02-20 11:49:22","video":"","vorDoi":"10.1038/s41467-024-54450-4","vorDoiUrl":"https://doi.org/10.1038/s41467-024-54450-4","workflowStages":[]},"version":"v1","identity":"rs-3922628","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3922628","identity":"rs-3922628","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}