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キーワード:
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要旨:
Delineation of phosphorylation-based signaling networks
requires reliable data about the underlying cellular kinasesubstrate
interactions. We report a chemical genetics and
quantitative phosphoproteomics approach that encompasses
cellular kinase activation in combination with
comparative replicate mass spectrometry analyses of
cells expressing either inhibitor-sensitive or resistant kinase
variant. We applied this workflow to Plk1 (Polo-like
kinase 1) in mitotic cells and induced cellular Plk1 activity
by wash-out of the bulky kinase inhibitor 3-MB-PP1,
which targets a mutant kinase version with an enlarged
catalytic pocket while not interfering with wild-type Plk1.
We quantified more than 20,000 distinct phosphorylation
sites by SILAC, approximately half of which were measured
in at least two independent experiments in cells
expressing mutant and wild-type Plk1. Based on replicate
phosphorylation site quantifications in both mutant and
wild-type Plk1 cells, our chemical genetic proteomics
concept enabled stringent comparative statistics by significance
analysis of microarrays, which unveiled more
than 350 cellular downstream targets of Plk1 validated by
full concordance of both statistical and experimental
data. Our data point to hitherto poorly characterized aspects
in Plk1-controlled mitotic progression and provide a
largely extended resource for functional studies. We anticipate
the described strategies to be of general utility for
systematic and confident identification of cellular protein
kinase substrates.
