Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation
Autor: | Kathryn J. Brayer, Richard R. Vaillancourt, Nathaniel McCormick, Deanna G. Adams, Brian E. Wadzinski, Anne Fritz |
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Rok vydání: | 2006 |
Předmět: |
Threonine
Molecular Sequence Data MAP Kinase Kinase 6 MAP Kinase Kinase Kinase 3 Biology Mitogen-activated protein kinase kinase Biochemistry Catalysis MAP2K7 Cell Line Osmotic Pressure Phosphoprotein Phosphatases Serine Humans Protein phosphorylation Amino Acid Sequence Protein Phosphatase 2 Phosphorylation Extracellular Signal-Regulated MAP Kinases Molecular Biology MAPK14 Serine/threonine-specific protein kinase Alanine MAP kinase kinase kinase Autophosphorylation Cyclin-dependent kinase 2 NF-kappa B Cell Biology Molecular biology Enzyme Activation Isoenzymes 14-3-3 Proteins biology.protein Mutagenesis Site-Directed Signal Transduction |
Zdroj: | The Journal of biological chemistry. 281(10) |
ISSN: | 0021-9258 |
Popis: | MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins. |
Databáze: | OpenAIRE |
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