Mosbacher, MaximilianLee, Sung SikYaakov, GiladNadal Ribelles, Mariona, 1984-Nadal Clanchet, Eulàlia devan Drogen, FrankPosas Garriga, FrancescPeter, MatthiasClaassen, Manfred2023-06-142023-06-142023Mosbacher M, Lee SS, Yaakov G, Nadal-Ribelles M, de Nadal E, van Drogen F, Posas F, Peter M, Claassen M. Positive feedback induces switch between distributive and processive phosphorylation of Hog1. Nat Commun. 2023 Apr 29;14(1):2477. DOI: 10.1038/s41467-023-37430-y2041-1723http://hdl.handle.net/10230/57163Cellular decision making often builds on ultrasensitive MAPK pathways. The phosphorylation mechanism of MAP kinase has so far been described as either distributive or processive, with distributive mechanisms generating ultrasensitivity in theoretical analyses. However, the in vivo mechanism of MAP kinase phosphorylation and its activation dynamics remain unclear. Here, we characterize the regulation of the MAP kinase Hog1 in Saccharomyces cerevisiae via topologically different ODE models, parameterized on multimodal activation data. Interestingly, our best fitting model switches between distributive and processive phosphorylation behavior regulated via a positive feedback loop composed of an affinity and a catalytic component targeting the MAP kinase-kinase Pbs2. Indeed, we show that Hog1 directly phosphorylates Pbs2 on serine 248 (S248), that cells expressing a non-phosphorylatable (S248A) or phosphomimetic (S248E) mutant show behavior that is consistent with simulations of disrupted or constitutively active affinity feedback and that Pbs2-S248E shows significantly increased affinity to Hog1 in vitro. Simulations further suggest that this mixed Hog1 activation mechanism is required for full sensitivity to stimuli and to ensure robustness to different perturbations.application/pdfeng© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-023-37430-yComputer modellingDifferential equationsDynamic networksRobustnessinfo:eu-repo/semantics/openAccess