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Proteomic identification of p38 MAP kinase substrates using in vitro phosphorylation
Author(s) -
Iida Naoyuki,
Fujita Masayuki,
Miyazawa Kohtaro,
Kobayashi Michimoto,
Hattori Seisuke
Publication year - 2014
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201300392
Subject(s) - map2k7 , mapk14 , ask1 , kinase , microbiology and biotechnology , protein kinase a , mitogen activated protein kinase , phosphorylation , mitogen activated protein kinase kinase , cyclin dependent kinase 2 , protein phosphorylation , phosphoprotein , map kinase kinase kinase , p38 mitogen activated protein kinases , c raf , mapkapk2 , biology , proteomics , biochemistry , gene
Protein phosphorylation is a major mechanism that regulates many basic cellular processes. Identification and characterization of substrates for a given protein kinase can lead to a better understanding of signal transduction pathways. However, it is still difficult to efficiently identify substrates for protein kinases. Here, we propose an integrated proteomic approach consisting of in vitro dephosphorylation and phosphorylation, phosphoprotein enrichment, and 2D‐DIGE. Phosphatase treatment significantly reduced the complexity of the phosphoproteome, which enabled us to efficiently identify the substrates. We employed p38 mitogen‐activated protein kinase (p38 MAP kinase) as a model kinase and identified 23 novel candidate substrates for this kinase. Seven selected candidates were phosphorylated by p38 MAP kinase in vitro and in p38 MAP kinase‐activated cells. This proteomic approach can be applied to any protein kinase, allowing global identification of novel substrates.