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Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly challenging. We have therefore applied a chemical biology approach to light-control the state of single candidate phosphoserines in the canonical anion channel CFTR while simultaneously measuring channel activity. The data show striking non-equivalency among protein kinase A consensus sites, which vary from &amp;lt;10% to &amp;gt;1,000% changes in channel activity upon phosphorylation. Of note, slow phosphorylation of S813 suggests that this site is rate-limiting to the full activation of CFTR. Further, this approach reveals an unexpected coupling between the phosphorylation of S813 and a nearby site, S795. Overall, these data establish an experimental route to understanding roles of specific phosphoserines within complex phosphoregulatory domains. This strategy may be employed in the study of phosphoregulation of other eukaryotic proteins.<\/jats:p>","DOI":"10.1085\/jgp.202213216","type":"journal-article","created":{"date-parts":[[2023,1,10]],"date-time":"2023-01-10T02:20:49Z","timestamp":1673317249000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":10,"title":["Real-time observation of functional specialization among phosphorylation sites in CFTR"],"prefix":"10.1085","volume":"155","author":[{"given":"Daniel T.","family":"Infield","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Physiology and Biophysics and Iowa Neuroscience Institute, University of Iowa, 1 , Iowa, IA, USA"},{"name":"Carver College of Medicine 1 , Iowa, IA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5653-543X","authenticated-orcid":true,"given":"Miranda E.","family":"Schene","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Physiology and Biophysics and Iowa Neuroscience Institute, University of Iowa, 1 , Iowa, IA, USA"},{"name":"Carver College of Medicine 1 , Iowa, IA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frederico S.","family":"Fazan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Physiology and Biophysics and Iowa Neuroscience Institute, University of Iowa, 1 , Iowa, IA, USA"},{"name":"Carver College of Medicine 1 , Iowa, IA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Grace D.","family":"Galles","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Physiology and Biophysics and Iowa Neuroscience Institute, University of Iowa, 1 , Iowa, IA, USA"},{"name":"Carver College of Medicine 1 , Iowa, IA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1923-5312","authenticated-orcid":true,"given":"Jason D.","family":"Galpin","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Physiology and Biophysics and Iowa Neuroscience Institute, University of Iowa, 1 , Iowa, IA, USA"},{"name":"Carver College of Medicine 1 , Iowa, IA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7975-2744","authenticated-orcid":true,"given":"Christopher A.","family":"Ahern","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Physiology and Biophysics and Iowa Neuroscience Institute, University of Iowa, 1 , Iowa, IA, USA"},{"name":"Carver College of Medicine 1 , Iowa, IA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2023,1,25]]},"reference":[{"key":"2023012514543079900_bib1","doi-asserted-by":"publisher","first-page":"E2086","DOI":"10.1073\/pnas.1613546114","article-title":"Synergy of cAMP and calcium signaling pathways in CFTR regulation","volume":"114","author":"Bozoky","year":"2017","journal-title":"Proc. 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