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 IκB 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-κB 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-κB 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-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.