biostudies-literatureYeung EHertz FoundationNational Institutes of HealthRoyal SocietyNIGMS NIH HHSNational Science FoundationEngineering and Physical Sciences Research Council877-882.e6https://www.ebi.ac.uk/biostudies/studies/S-EPMC7085240biostudies-literatureUnknown30(5)Multisite protein phosphorylation plays a critical role in cell regulation [1-3]. It is widely appreciated that the functional capabilities of multisite phosphorylation depend on the order and kinetics of phosphorylation steps, but kinetic aspects of multisite phosphorylation remain poorly understood [4-6]. Here, we focus on what appears to be the simplest scenario, when a protein is phosphorylated on only two sites in a strict, well-defined order. This scenario describes the activation of ERK, a highly conserved cell-signaling enzyme. We use Bayesian parameter inference in a structurally identifiable kinetic model to dissect dual phosphorylation of ERK by MEK, a kinase that is mutated in a large number of human diseases [7-12]. Our results reveal how enzyme processivity and efficiencies oCurrent biology : CBInference of Multisite Phosphorylation Rate Constants and Their Modulation by Pathogenic Mutations.PMC7085240EP/R005125/1R01 GM086537R35 GM128813EP/K041096/1T32 GM007388EP/R018472/1Shvartsman SYFilippi SWuhr MMcFann SHarrington HAMarsh LYeung EDufresne EfalseInference of Multisite Phosphorylation Rate Constants and Their Modulation by Pathogenic Mutations.Multisite protein phosphorylation plays a critical role in cell regulation [1-3]. It is widely appreciated that the functional capabilities of multisite phosphorylation depend on the order and kinetics of phosphorylation steps, but kinetic aspects of multisite phosphorylation remain poorly understood [4-6]. Here, we focus on what appears to be the simplest scenario, when a protein is phosphorylated on only two sites in a strict, well-defined order. This scenario describes the activation of ERK, a highly conserved cell-signaling enzyme. We use Bayesian parameter inference in a structurally identifiable kinetic model to dissect dual phosphorylation of ERK by MEK, a kinase that is mutated in a large number of human diseases [7-12]. Our results reveal how enzyme processivity and efficiencies o2020-01-01T00:00:00Z2020 Mar2026-05-02T14:42:16.65Z2021-03-11T08:19:20ZS-EPMC70852403205976610.1016/j.cub.2019.12.052