A Hotspot Phosphorylation Site on SHP2 Drives Oncoprotein Activation and Drug Resistance

A Hotspot Phosphorylation Site on SHP2 Drives Oncoprotein Activation and Drug Resistance | 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 Biological Sciences - Article A Hotspot Phosphorylation Site on SHP2 Drives Oncoprotein Activation and Drug Resistance Neil Vasan, Prashath Karunaraj, Remkes Scheele, Malcolm Wells, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7032881/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract SHP2 is a phosphatase and a critical mediator of receptor tyrosine kinase (RTK)-driven RAS/mitogen-activated protein kinase (MAPK) signaling. Despite promising preclinical data, SHP2 inhibitors have shown minimal clinical efficacy, with no defined clinical mechanisms of primary resistance. Here, we elucidate phosphorylation of SHP2 at tyrosine 62 (pY62) as a hotspot phosphorylation site in the proteome and RTK-driven tumor types in patients. We demonstrate that SRC family kinases directly phosphorylate SHP2 at Y62, downstream of but not directly phosphorylated by RTKs. Using biochemical and biophysical analyses, we show that SHP2 Y62D enforces an open, active conformation, resulting in constitutive phosphatase activation that is sufficient to activate MAPK signaling and confer resistance to allosteric SHP2 inhibitors. These findings establish that SHP2 pY62 is a phosphorylation hotspot phenocopying mutational activation, a mechanism of primary resistance to SHP2 inhibitors, and a cancer drug target distinct from wildtype SHP2. Biological sciences/Cell biology/Cell signalling Biological sciences/Biochemistry Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SD3Kinaselibrarypredictions.xlsx Kinase library predictions SupplProtocolSHP2pLX304.docx Suppl Protocol SHP2 - pLX304 SD2NSCLCproteinenrichment.xlsx NSCLC protein enrichment SD1Phosphositefrequency.pdf Phosphosite frequency Cite Share Download PDF Status: Published Journal Publication published 03 Mar, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-7032881","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":489934022,"identity":"1ecb16e4-e928-4894-a293-323d37206a46","order_by":0,"name":"Neil 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Despite promising preclinical data,\r\nSHP2 inhibitors have shown minimal clinical efficacy, with no defined clinical mechanisms of\r\nprimary resistance. Here, we elucidate phosphorylation of SHP2 at tyrosine 62 (pY62) as a\r\nhotspot phosphorylation site in the proteome and RTK-driven tumor types in patients. We\r\ndemonstrate that SRC family kinases directly phosphorylate SHP2 at Y62, downstream of but not\r\ndirectly phosphorylated by RTKs. Using biochemical and biophysical analyses, we show that\r\nSHP2 Y62D enforces an open, active conformation, resulting in constitutive phosphatase\r\nactivation that is sufficient to activate MAPK signaling and confer resistance to allosteric SHP2\r\ninhibitors. These findings establish that SHP2 pY62 is a phosphorylation hotspot phenocopying\r\nmutational activation, a mechanism of primary resistance to SHP2 inhibitors, and a cancer drug\r\ntarget distinct from wildtype SHP2.","manuscriptTitle":"A Hotspot Phosphorylation Site on SHP2 Drives Oncoprotein Activation and Drug\nResistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 02:29:36","doi":"10.21203/rs.3.rs-7032881/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":"bd88dad2-1393-4aa9-8ff1-1feb6603bcf5","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52017073,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":52017074,"name":"Biological sciences/Biochemistry"}],"tags":[],"updatedAt":"2026-04-10T07:12:52+00:00","versionOfRecord":{"articleIdentity":"rs-7032881","link":"https://doi.org/10.1038/s41467-026-70060-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-03-03 05:00:00","publishedOnDateReadable":"March 3rd, 2026"},"versionCreatedAt":"2025-08-05 02:29:36","video":"","vorDoi":"10.1038/s41467-026-70060-8","vorDoiUrl":"https://doi.org/10.1038/s41467-026-70060-8","workflowStages":[]},"version":"v1","identity":"rs-7032881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7032881","identity":"rs-7032881","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}