Abstract
We have developed a novel expression screening method for identifying protein kinase substrates. In this method, a lambda phage cDNA expression library is screened by in situ, solid-phase phosphorylation using purified protein kinase and [gamma-32P]ATP. Screening a HeLa cDNA library with ERK1 MAP kinase yielded cDNAs of previously characterized ERK substrates, c-Myc and p90RSK, demonstrating the utility of this method for identifying physiological protein kinase substrates. A novel clone isolated in this screen, designated MNK1, encodes a protein-serine/threonine kinase, which is most similar to MAP kinase-activated protein kinase 2 (MAPKAP-K2), 3pK/MAPKAP-K3 and p90RSK. Bacterially expressed MNK1 was phosphorylated and activated in vitro by ERK1 and p38 MAP kinases but not by JNK/SAPK. Further, MNK1 was activated upon stimulation of HeLa cells with 12-O-tetradecanoylphorbol-13-acetate, fetal calf serum, anisomycin, UV irradiation, tumor necrosis factor-alpha, interleukin-1beta, or osmotic shock, and the activation by these stimuli was differentially inhibited by the MEK inhibitor PD098059 or the p38 MAP kinase inhibitor SB202190. Together, these results indicate that MNK1 is a novel class of protein kinase that is activated through both the ERK and p38 MAP kinase signaling pathways.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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Base Sequence
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Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
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Cell Line
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Cloning, Molecular / methods*
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DNA, Complementary / genetics
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Enzyme Activation
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Enzyme Inhibitors / pharmacology
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Flavonoids / pharmacology
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HeLa Cells
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Humans
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Imidazoles / pharmacology
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Intracellular Signaling Peptides and Proteins
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases*
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Molecular Sequence Data
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Phosphorylation
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Pyridines / pharmacology
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Recombinant Fusion Proteins
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Sequence Homology, Amino Acid
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Signal Transduction / physiology
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Spodoptera
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Substrate Specificity
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p38 Mitogen-Activated Protein Kinases
Substances
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DNA, Complementary
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Enzyme Inhibitors
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Flavonoids
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Imidazoles
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Intracellular Signaling Peptides and Proteins
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Pyridines
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Recombinant Fusion Proteins
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MKNK1 protein, human
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Protein Serine-Threonine Kinases
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Calcium-Calmodulin-Dependent Protein Kinases
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases
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4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole
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2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
Associated data
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GENBANK/AB000409
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GENBANK/AB014641