Efficient Modification of Alpha-Synuclein Serine 129 by Protein Kinase CK1 Requires Phosphorylation of Tyrosine 125 as a Priming Event

S129-phosphorylated alpha-synuclein (α-syn) is abundantly found in Lewy-body inclusions of Parkinson’s disease patients. Residues neighboring S129 include the α-syn tyrosine phosphorylation sites Y125, Y133, and Y136. Here, we use time-resolved NMR spectroscopy to delineate atomic resolution insights into the modification behaviors of different serine and tyrosine kinases targeting these sites and show that Y125 phosphorylation constitutes a necessary priming event for the efficient modification of S129 by CK1, both in reconstituted kinase reactions and mammalian cell lysates. These results suggest that α-syn Y125 phosphorylation augments S129 modification under physiological in vivo conditions

A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time

Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their individual activities in a semicontinuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions