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National Institute of Environmental Health Sciences

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Publication Detail

Title: Phosphorylation of the stress-activated protein kinase, MEKK3, at serine 166.

Authors: Adams, Deanna G; Sachs, Nancy A; Vaillancourt, Richard R

Published In Arch Biochem Biophys, (2002 Nov 01)

Abstract: Much effort has focused on the identification of MAPK cascades that are activated by the MEKK family of protein kinases. However, direct phosphorylation and regulation of the MEKK proteins has not been shown. To address this question, we have expressed recombinant (His)6FLAG.MEKK3 in Sf9 insect cells and tethered the purified protein to Ni-Sepharose so that we could precipitate interacting proteins and then identify such proteins by liquid chromatography and mass spectrometry (LC-MS). We identified 14-3-3 proteins as interacting with MEKK3, which suggested that (His)6FLAG.MEKK3 was phosphorylated on serine since 14-3-3 proteins are known to associate with phosphorylated proteins. We identified two phosphorylated amino acids at Ser166 and Ser337 of tryptic peptides derived from (His)6FLAG.MEKK3 by using LC-MS. Antibodies were developed that recognize the specific phosphorylated amino acid and with these antibodies, we demonstrate that various stimuli (tumor necrosis factor, arsenite, forskolin, and serum) promote phosphorylation of Ser166 and Ser337. However, neither of these phosphorylated amino acids is required for association with 14-3-3 protein or regulation of MEKK3-dependent ERK and JNK activity. Nonetheless, these results suggest that MEKK3 is a convergence point of multiple upstream signaling pathways.

PubMed ID: 12392720 Exiting the NIEHS site

MeSH Terms: 14-3-3 Proteins; 3T3 Cells; Animals; COS Cells; Cells, Cultured; Humans; Insecta/cytology; MAP Kinase Kinase Kinase 3; MAP Kinase Kinase Kinases/genetics; MAP Kinase Kinase Kinases/metabolism*; Mice; Mitogen-Activated Protein Kinase 1/genetics; Mitogen-Activated Protein Kinase 1/metabolism; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases/genetics; Mitogen-Activated Protein Kinases/metabolism; Phosphorylation; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Serine/metabolism*; Tyrosine 3-Monooxygenase/metabolism

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