biostudies-literatureInfield DTNHLBI NIH HHSNational Institutes of HealthNIH HHSNIGMS NIH HHSe202213216https://www.ebi.ac.uk/biostudies/studies/S-EPMC9930130biostudies-literatureUnknown155(4)Phosphoregulation is ubiquitous in biology. 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 <10% to >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.The Journal of general physiologyReal-time observation of functional specialization among phosphorylation sites in CFTR.PMC9930130R01GM106569R35 GM148239R01 GM106569R01 GM122420F32HL149184F32 HL149184Galpin JDAhern CAInfield DTSchene MEFazan FSGalles GDfalseReal-time observation of functional specialization among phosphorylation sites in CFTR.Phosphoregulation is ubiquitous in biology. 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 <10% to >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.2023-01-01T00:00:00Z2023 Apr2026-03-31T11:53:00.014Z2025-04-07T01:29:52.994ZS-EPMC99301303669581310.1085/jgp.202213216