MEKK3 Directly Regulates MEK5 Activity as Part of the Big Mitogen-activated Protein Kinase 1 (BMK1) Signaling Pathway

Abstract

Big mitogen-activated protein (MAP) kinase (BMK1), also known as ERK5, is a member of the MAP kinase family whose cellular activity is elevated in response to growth factors, oxidative stress, and hyperosmolar conditions. Previous studies have identified MEK5 as a cellular kinase directly regulating BMK1 activity; however, signaling molecules that directly regulate MEK5 activity have not yet been defined. Through utilization of a yeast two-hybrid screen, we have identified MEKK3 as a molecule that physically interacts with MEK5. This interaction appears to take place in mammalian cells as evidenced by the fact that cellular MEK5 and MEKK3 co-immunoprecipitate. In addition, we show that a dominant active form of MEKK3 stimulates BMK1 activity through MEK5. Moreover, we demonstrate that MEKK3 activity is required for growth factor mediated cellular activation of endogenous BMK1. Taken together, these results identify MEKK3 as a kinase that regulates the activity of MEK5 and BMK1 during growth factor-induced cellular stimulation.The mitogen-activated protein (MAP) 1 kinase cascades represent a primary mechanism by which cells transduce intracellular signals (1-3). These kinase cascades display a high degree of evolutionary conservation, as evidenced in a variety of eukaryotes ranging from yeast to mammals (2, 4). Three sequentially activated kinases make up the core of the MAP kinase module: a MAP kinase kinase kinase, or MEKK; a MAP kinase kinase, or MEK; and a MAP kinase (5, 6). In addition to delivering signals from extracellular stimuli to intended effectors, these kinase modules harmonize incoming signals from parallel signaling pathways and provide signal amplification as well as biological specificity. To date, four separate MAP kinases have been identified in mammalian cells and are known as ERK, JNK/SAPK, p38, and BMK1/ERK5 (7-13).BMK1/ERK5 represents the newest member of the mammalian MAP kinase family and was independently cloned by our laboratory and another group using different experimental approaches (7,11). We have previously demonstrated that BMK1 is activated by growth factors, oxidative stress, and hyperosmolar conditions (8,9,14). Upon activation, BMK1 stimulates the activity of myocyte enhancer factor 2C (MEF2C), a transcription factor that induces the expression of the proto-oncogene c-jun (9). Through the use of a dominant negative form of BMK1, we have demonstrated that BMK1 is required for growth factor-induced cell proliferation and cell cycle progression (8). Using the yeast two-hybrid system, MEK5 was identified by Zhou et al. (11) as the molecule responsible for regulating BMK1 activity. Subsequently, we have determined that MEK5 specifically activates BMK1 but not other mammalian MAP kinases in vivo (9). In addition, we have shown that MEK5 activity is required for the activation of BMK1 induced by extracellular stimuli (8, 9).The upstream kinase responsible for regulating MEK5 activity within the BMK1 signaling module has not yet been reported. In this regard, studies by English et a...

New insights into the biogenesis of nuclear RNA polymerases?This paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

“…MAP(3)Ks participate in the early steps of a signal transduction pathway that will promote various cellular processes in responses to a number of external stimuli through a sequential MAP(3)K > MAP(2)K > MAPK phosphorylation cascade (Chang and Karin 2001;Pearson et al 2001). In accordance to this general scheme, MEKK3 is known to phosphorylate a number of MAP(2)K (MEK/MKK1-7), which leads to the activation of their specific downstream MAPK (ERK1/2, JNK/SAPK, ERK5, and p38) (Blank et al 1996;Deacon and Blank 1997;Ellinger-Ziegelbauer et al 1997;Chao et al 1999;Deacon and Blank 1999;Uhlik et al 2003). MEKK3 is also involved in the NF-κB pathway where it activates the IKK complex in response to TNFα and LPS (Yang et al 2001;Schmidt et al 2003;Samanta et al 2004).…”

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). MEKK3 knockout mice (MEKK3 Ϫ/Ϫ ) die in utero at approximately E10.5 due to abnormalities in extraembryonic vasculature of the yolk sac, the embryonic vasculature and the heart.…”

ERK5 regulation in naïve T‐cell activation and survival

“…One possibility is MEKK3, as mouse knockouts of MEKK3 [16] have a similar phenotype to that of ERK5 knockouts. In addition, MEKK3 has been shown to activate ERK5 in transfection experiments in Hek293 cells [17] and kinase inactive MEKK3 blocks ERK5 activation in Cos7 cells [18].…”

“…One possibility is MEKK3, as mouse knockouts of MEKK3 [16] have a similar phenotype to that of ERK5 knockouts. In addition, MEKK3 has been shown to activate ERK5 in transfection experiments in Hek293 cells [17] and kinase inactive MEKK3 blocks ERK5 activation in Cos7 cells [18].ERK5 is expressed in most cell types; however, its precise physiological roles are not completely understood. ERK5 has been shown to promote both the proliferation and survival of several cell lines [10,19].…”