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Updated: Aug 13, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation
Anne Fritz1, Kathryn J Brayer, Nathaniel McCormick
1Department of Pharmacology and Toxicology, University of Arizona College of Pharmacy, Tucson, Arizona 85721, USA.
Abstract:
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.
Insights
MAPK/ERK kinase kinase 3 (MEKK3) phosphorylation at Serine 526 is essential for its activity and signal transduction. This site is autophosphorylated, regulated by PP2A and 14-3-3 proteins, and crucial for cellular responses to stress.
Area of Science:
- Cellular signaling pathways
- Protein phosphorylation
- Signal transduction mechanisms
Background:
- MAPK/ERK kinase kinase 3 (MEKK3) is a key upstream regulator in mitogen-activated protein kinase (MAP) kinase pathways.
- Phosphorylation is critical for MEKK3 function, but specific phosphorylation sites and their regulation remain largely uncharacterized.
- Understanding MEKK3 phosphorylation is vital for deciphering its role in cellular responses.
Purpose of the Study:
- To identify and characterize the critical phosphorylation sites of MEKK3.
- To elucidate the regulatory mechanisms governing MEKK3 activity.
- To investigate the role of MEKK3 phosphorylation in signal transduction.
Main Methods:
- Site-directed mutagenesis of MEKK3 activation loop (T-loop) residues.
- Transfection of MEKK3 mutants into HEK293 cells.
- Assays for NF-kappaB reporter gene activation and MAP kinase activity (ERK, JNK, p38).
- Development of phospho-specific antibodies.
- In vitro kinase assays and phosphatase treatments.
Main Results:
- Serine at position 526 (Ser526) is essential for MEKK3-dependent activation of NF-kappaB and MAP kinases.
- MEKK3 mutants with constitutive activity (S526D, S526E) indicate a negative charge at Ser526 is necessary for activity.
- Ser526 is an autophosphorylation site, confirmed by the lack of phosphorylation in the catalytically inactive mutant (K391M).
- Endogenous MEKK3 phosphorylation at Ser526 is induced by osmotic stress.
- Phosphorylation of Ser526 is required for MKK6 phosphorylation and regulated by Protein Phosphatase 2A (PP2A) and 14-3-3 protein interactions.
Conclusions:
- Serine 526 is a critical autophosphorylation site within the T-loop of MEKK3.
- Phosphorylation at Ser526 is essential for MEKK3 catalytic activity and downstream signaling.
- MEKK3 phosphorylation is dynamically regulated by PP2A and 14-3-3 proteins, linking it to cellular stress responses.
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