Deciphering the multi-site phos-code of IRBIT underlying its binding to IP3R

Deciphering the multi-site phos-code of IRBIT underlying its binding to IP3R | 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 Deciphering the multi-site phos-code of IRBIT underlying its binding to IP3R Katsuhiko Mikoshiba, Qing Lin, Hao Yang, Qi Feng, Tomohide Sumi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7315114/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract IRBIT has been previously shown to interact with the inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) via a serine-rich segment containing numerous phosphorylation sites. Although the interaction between IRBIT and IP 3 R is phosphorylation dependent, it is unclear which particular phosphoisotypes are directly involved in their interaction. In this study, we investigated the mechanism of how phosphorylation states of IRBIT regulate its interaction with IP 3 R. Site-directed mutagenesis confirmed that S68 is the predominant site of phosphorylation on IRBIT, but it is not required for IP 3 R binding. In vitro kinase assay showed that protein kinase A and casein kinase 2 phosphorylated residues S62/S64/S66 and S80/T82/S84/S85, respectively. Subsequent pulldown assays demonstrated that S71/S74/S77 and S80/S84/S85 provided two binding sites for IP 3 -binding core (IBC) on IP 3 R. Computational estimation showed that the IRBIT-pS80pS84pS85 peptide binds IBC with the lower binding free energy in a mode similar to that of IP 3 . Further studies of Ca 2+ imaging in living cells revealed that IRBIT-S80GS84GS85G mutant was not able to inhibit the activation of IP 3 R, while IRBIT-S68AS80DS84DS85D was sufficient to prohibit IP 3 R-mediated Ca 2+ release. In summary, our results indicate that the phosphorylated S80, S84, and S85 residues on IRBIT may compete with IP 3 for the IP 3 -binding pocket on IP 3 R. Biological sciences/Cell biology/Cell signalling/Calcium signalling Biological sciences/Cell biology/Post-translational modifications/Phosphorylation Full Text Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7315114","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":506344249,"identity":"5c824550-520c-423e-a09a-16dbaa6e5d50","order_by":0,"name":"Katsuhiko 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IP3R","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-7315114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7315114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIRBIT has been previously shown to interact with the inositol 1,4,5-trisphosphate (IP\u003csub\u003e3\u003c/sub\u003e) receptor (IP\u003csub\u003e3\u003c/sub\u003eR) via a serine-rich segment containing numerous phosphorylation sites. Although the interaction between IRBIT and IP\u003csub\u003e3\u003c/sub\u003eR is phosphorylation dependent, it is unclear which particular phosphoisotypes are directly involved in their interaction. In this study, we investigated the mechanism of how phosphorylation states of IRBIT regulate its interaction with IP\u003csub\u003e3\u003c/sub\u003eR. Site-directed mutagenesis confirmed that S68 is the predominant site of phosphorylation on IRBIT, but it is not required for IP\u003csub\u003e3\u003c/sub\u003eR binding. \u003cem\u003eIn vitro\u003c/em\u003e kinase assay showed that protein kinase A and casein kinase 2 phosphorylated residues S62/S64/S66 and S80/T82/S84/S85, respectively. Subsequent pulldown assays demonstrated that S71/S74/S77 and S80/S84/S85 provided two binding sites for IP\u003csub\u003e3\u003c/sub\u003e-binding core (IBC) on IP\u003csub\u003e3\u003c/sub\u003eR. Computational estimation showed that the IRBIT-pS80pS84pS85 peptide binds IBC with the lower binding free energy in a mode similar to that of IP\u003csub\u003e3\u003c/sub\u003e. Further studies of Ca\u003csup\u003e2+\u003c/sup\u003e imaging in living cells revealed that IRBIT-S80GS84GS85G mutant was not able to inhibit the activation of IP\u003csub\u003e3\u003c/sub\u003eR, while IRBIT-S68AS80DS84DS85D was sufficient to prohibit IP\u003csub\u003e3\u003c/sub\u003eR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e release. In summary, our results indicate that the phosphorylated S80, S84, and S85 residues on IRBIT may compete with IP\u003csub\u003e3\u003c/sub\u003e for the IP\u003csub\u003e3\u003c/sub\u003e-binding pocket on IP\u003csub\u003e3\u003c/sub\u003eR.\u003c/p\u003e","manuscriptTitle":"Deciphering the multi-site phos-code of IRBIT underlying its binding to IP3R","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 17:17:02","doi":"10.21203/rs.3.rs-7315114/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e4c83e16-1b5f-4243-a11d-2ec0723e7f07","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53782353,"name":"Biological sciences/Cell biology/Cell signalling/Calcium signalling"},{"id":53782354,"name":"Biological sciences/Cell biology/Post-translational modifications/Phosphorylation"}],"tags":[],"updatedAt":"2026-03-21T04:15:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 17:17:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7315114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7315114","identity":"rs-7315114","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}