Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and I kappa B 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-kappa B 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-kappa B luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser(526) was necessary for MEKK3 activity and implicating Ser(526) as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser(526) of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser(526), indicating that phosphorylation of Ser(526) occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser(526) in response to osmotic stress. In addition, phosphorylation of Ser(526) was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser(526) 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 Ser(526) and prevented dephosphorylation of Ser(526). In summary, Ser(526) of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.