Identifications
of Putative PKA Substrates with Quantitative
Phosphoproteomics and Primary-Sequence-Based Scoring
Version 3 2018-02-09, 23:47
Version 2 2017-10-17, 18:25
Version 1 2017-03-15, 13:39
Posted on 2018-02-09 - 23:47
Protein kinase A
(PKA or cAMP-dependent protein kinase) is a serine/threonine
kinase that plays essential roles in the regulation of proliferation,
differentiation, and apoptosis. To better understand the functions
of PKA, it is necessary to elucidate the direct interplay between
PKA and their substrates in living human cells. To identify kinase
target substrates in a high-throughput manner, we first quantified
the change of phosphoproteome in the cells of which PKA activity was
perturbed by drug stimulations. LC–MS/MS analyses identified
2755 and 3191 phosphopeptides from experiments with activator or inhibitor
of PKA. To exclude potential indirect targets of PKA, we built a computational
model to characterize the kinase sequence specificity toward the substrate
target site based on known kinase–substrate relationships.
Finally, by combining the sequence recognition model with the quantitative
changes in phosphorylation measured in the two drug perturbation experiments,
we identified 29 reliable candidates of PKA targeting residues in
living cells including 8 previously known substrates. Moreover, 18
of these sites were confirmed to be site-specifically phosphorylated
in vitro. Altogether this study proposed a confident list of PKA substrate
candidates, expanding our knowledge of PKA signaling network.
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Imamura, Haruna; Wagih, Omar; Niinae, Tomoya; Sugiyama, Naoyuki; Beltrao, Pedro; Ishihama, Yasushi (2017). Identifications
of Putative PKA Substrates with Quantitative
Phosphoproteomics and Primary-Sequence-Based Scoring. ACS Publications. Collection. https://doi.org/10.1021/acs.jproteome.7b00087
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AUTHORS (6)
HI
Haruna Imamura
OW
Omar Wagih
TN
Tomoya Niinae
NS
Naoyuki Sugiyama
PB
Pedro Beltrao
YI
Yasushi Ishihama
KEYWORDS
substrate target sitekinase target substrateshigh-throughput mannersequence recognition modelPKA activityLCPrimary-Sequence-Based Scoring Protein kinasePKA substrate candidatesdrug perturbation experimentssite-specifically phosphorylatedQuantitative PhosphoproteomicsPutative PKA Substrateskinase sequence specificity3191 phosphopeptidesdrug stimulationscAMP-dependent protein kinase