Phosphorylation of Serine 526 Is Required for MEKK3 Activity, and Association with 14-3-3 Blocks Dephosphorylation

Abstract

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IB 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 (MAP2K), and a MAPK kinase kinase (MAP3K). Regulation of the MAP3K, presumably by phosphorylation, provides the impetus for activation of the three-kinase module. Once activated, the MAP3Ks activate MAP2Ks by phosphorylation of two residues within the activation loop. Phosphorylation of MAP2Ks activates these dual specificity kinases to phosphorylate MAPKs on a conserved threonine and tyrosine motif, TXY, also within the activation loop. Once phosphorylated, the MAPKs phosphorylate protein substrates and regulate cellular processes like growth, protein synthesis, gene expression, and nucleotide synthesis (2).Over the last decade, a large body of work has characterized events that occur downstream of the MAPKs. However, little is known regarding the regulatory mechanisms that modulate the MEKK proteins to ultimately regulate the MAPKs. For example, it is known that overexpression of MEKK3 activates the ERK (3, 4), JNK (3-5), p38 (5, 6), ERK5 (7), and NF-B pathways (8 -10). Typically, MEKK3-dependent regulation of these pathways is studied by using transfection studies, and activation of these pathways rarely requires an agonist. Therefore, it appears that some process intrinsic to MEKK3 is critical and sufficient for activation of these pathways.The activation loop of some protein kinase families, such as the arginine-aspartate family, is positioned between subdomains VII and VIII and is phosphorylated by other protein kinases or through autophosphorylation of the kinase itself (11). Phosphorylation within the activation loop of protein kinases results in conformational changes in the protein structure that (i) enhance substrate binding, (ii) correctly position amino acids involved in catalysis, and (iii) relieve steric hindrance within the catalytic domain. Regardless of how the activation loop is phosphorylated, regulation of catalytic activity frequently correlates with phosphorylation of the activation loop.Given that phosphorylation plays a critical role in regulating the MAPKs and the MAP2Ks, we investigated how phosphorylation of MEKK3 might affect its catalytic activity. Since phosphorylation sites within the activation loop of MEKK3 have not been reported, we systematically mutated serine and threonine residues within the activation loop to alanine and monitored MEKK3-dependent activities in HEK293 EBNA cells. Two key amino acids were identified at positions 526 and 530 using a luciferase-based reporter gene assay as well as assays that measure the ERK, JNK, and p38 MAP...

MAP3Ks as central regulators of cell fate during development

“…4) (Deacon and Blank, 1997;Uhlik et al, 2003;Fritz et al, 2006). More recent evidence suggests that it also regulates activities of ERK5 (Chao et al, 1999) and the transcription factor, NF B (Zhao and Lee, 1999;Yang et al, 2001).…”

MEKK2 Kinase Association with 14-3-3 Protein Regulates Activation of c-Jun N-terminal Kinase

“…Upon activating signals, MEKK2/3 undergoes phosphorylation within the activation loop Ser-519/ Ser-526 (23), which is critical for kinase activity. Dimerization of MEKK2/3 is necessary (24,25), suggesting that Ser-519/Ser-526 undergoes a mechanism of trans-autophosphorylation.…”

Homeostatic interactions between MEKK3 and TAK1 involved in NF-κB signaling

“…The activation of MEKK3 or TAK1 catalytic activity requires phosphorylation of critical residues within their activation loops (17,18,21,22). The mechanisms controlling phosphorylation of the activation loop are poorly understood and the upstream kinases remain undefined.…”

“…Serine residues within the activation loop regulate MEKK3 catalytic activity and serve as a sensor of LPS-induced NF-κB signaling (17,18). Although the kinases responsible for in vivo MEKK3 phosphorylation remain poorly defined some evidence suggests phosphorylation of the activation loop may include a mechanism involving dimerization (19) and self-phosphorylation (17,18).…”

“…Phosphorylation of multiple MEKK3 sites was noted following stimulation of cells with TNFα and other exogenous stimuli (16,17). Serine residues within the activation loop regulate MEKK3 catalytic activity and serve as a sensor of LPS-induced NF-κB signaling (17,18).…”