4 research outputs found
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
Site-Specific Mapping and Time-Resolved Monitoring of Lysine Methylation by High-Resolution NMR Spectroscopy
Methylation and acetylation of protein lysine residues
constitute
abundant post-translational modifications (PTMs) that regulate a plethora
of biological processes. In eukaryotic proteins, lysines are often
mono-, di-, or trimethylated, which may signal different biological
outcomes. Deconvoluting these different PTM types and PTM states is
not easily accomplished with existing analytical tools. Here, we demonstrate
the unique ability of NMR spectroscopy to discriminate between lysine
acetylation and mono-, di-, or trimethylation in a site-specific and
quantitative manner. This enables mapping and monitoring of lysine
acetylation and methylation reactions in a nondisruptive and continuous
fashion. Time-resolved NMR measurements of different methylation events
in complex environments including cell extracts contribute to our
understanding of how these PTMs are established <i>in vitro</i> and <i>in vivo</i>
Site-Specific Mapping and Time-Resolved Monitoring of Lysine Methylation by High-Resolution NMR Spectroscopy
Methylation and acetylation of protein lysine residues
constitute
abundant post-translational modifications (PTMs) that regulate a plethora
of biological processes. In eukaryotic proteins, lysines are often
mono-, di-, or trimethylated, which may signal different biological
outcomes. Deconvoluting these different PTM types and PTM states is
not easily accomplished with existing analytical tools. Here, we demonstrate
the unique ability of NMR spectroscopy to discriminate between lysine
acetylation and mono-, di-, or trimethylation in a site-specific and
quantitative manner. This enables mapping and monitoring of lysine
acetylation and methylation reactions in a nondisruptive and continuous
fashion. Time-resolved NMR measurements of different methylation events
in complex environments including cell extracts contribute to our
understanding of how these PTMs are established <i>in vitro</i> and <i>in vivo</i>
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