posted on 2014-04-04, 00:00authored byNaganand Rayapuram, Ludovic Bonhomme, Jean Bigeard, Kahina Haddadou, Cédric Przybylski, Heribert Hirt, Delphine Pflieger
Signaling
cascades rely strongly on protein kinase-mediated substrate
phosphorylation. Currently a major challenge in signal transduction
research is to obtain high confidence substrate phosphorylation sites
and assign them to specific kinases. In response to bacterial flagellin,
a pathogen-associated molecular pattern (PAMP), we searched for rapidly
phosphorylated proteins in Arabidopsis thaliana by
combining multistage activation (MSA) and electron transfer dissociation
(ETD) fragmentation modes, which generate complementary spectra and
identify phosphopeptide sites with increased reliability. Of a total
of 825 phosphopeptides, we identified 58 to be differentially phosphorylated.
These peptides harbor kinase motifs of mitogen-activated protein kinases
(MAPKs) and calcium-dependent protein kinases (CDPKs), as well as
yet unknown protein kinases. Importantly, 12 of the phosphopeptides
show reduced phosphorylation upon flagellin treatment. Since protein
abundance levels did not change, these results indicate that flagellin
induces not only various protein kinases but also protein phosphatases,
even though a scenario of inhibited kinase activity may also be possible.