The ups and downs of MEK kinase interactions

Abstract

MEK kinases (MEKKs) comprise a family of related serine–threonine protein kinases that regulate mitogen-activated protein kinase (MAPK) signalling pathways leading to c-Jun NH₂-terminal kinase (JNK) and p38 activation, induced by cellular stress (e.g., UV and γ irradiation, osmotic stress, heat shock, protein synthesis inhibitors), inflammatory cytokines (e.g., tumour necrosis factor α, TNFα, and interleukin-1, IL1) and G protein-coupled receptor agonists (e.g., thrombin). These stress-activated kinases have been implicated in apoptosis, oncogenic transformation, and inflammatory responses in various cell types. At present, the signalling events involving MEKKs are not well understood. This review summarises our current knowledge concerning the regulation and function of MEKK family members, with particular emphasis on those factors capable of directly interacting with distinct MEKK isoforms.

Introduction

Regulatory mechanisms controlling the proliferation, differentiation, or apoptosis of cells involve intracellular protein kinases that can transduce signals detected on the cell's surface into changes in gene expression. Most prominent amongst the known signal transduction pathways that control these events are the mitogen-activated protein kinase (MAPK) cascades, whose components are evolutionarily highly conserved in structure and organisation, each consisting of a module of three cytoplasmic kinases: a mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK), an MAP kinase kinase (MAPKK), and the MAP kinase (MAPK) itself. The MAPKKK is a serine–threonine kinase that receives activating signals from a membrane-spanning receptor and then phosphorylates and activates its substrate, an MAPKK. This enzyme is a dual-specificity kinase with the potential to phosphorylate critical threonine and tyrosine residues in its substrate protein, the MAPK. MAPKs represent a family of serine–threonine kinases with the potential to phosphorylate other cytoplasmic proteins and to translocate from the cytoplasm to the nucleus, where they can directly regulate the activity of transcription factors controlling gene expression.

The best-investigated MAPK cascades are those of the budding yeast Saccharomyces cerevisiae. This organism uses five different MAPK modules to regulate mating and sporulation and to respond to environmental changes such as high osmolarity, cell integrity, starvation, and filamentous growth (reviewed in Ref. [1]). In the case of the mating response, pheromone stimulation of haploid cells of the opposite sex (a and α) causes cellular responses that prepare the cell for mating, specifically fusion with the mating partner to form a diploid. These responses include polarised growth towards the mating partner, an arrest of the cell cycle in G₁, an increased expression of proteins required for cell adhesion and cell and nuclear fusion. Pheromone ligand activates a seven transmembrane-spanning receptor that in turn stimulates GTP binding to a heterotrimeric G protein (Fig. 1). Subsequent dissociation of the G protein liberates βγ subunits (Ste4p/Ste18p) that bind to a scaffold protein, Ste5p, which assembles the MAPK module by simultaneous interaction with the MAPKKK, Ste11p, the MAPKK, Ste7p, and the MAPK, Fus3p. The scaffold protein, Ste5p, also has the potential to homo-oligomerise and to interact with elements of the cytoskeleton via other associated proteins, thereby forming a large signalling complex (Fig. 1). G protein βγ subunits also activate Ste20p, an MAPKKK kinase that phosphorylates the MAPKKK, Ste11p. For activation of Ste11p, binding of another protein, Ste50p, is also necessary. These events allow the signal to be transduced via the MAPKK, Ste7p, to the MAPK Fus3p. In unstimulated cells, Fus3p appears to form a complex with three other factors, Dig1p, Dig2p, and Ste12p. Ste12p is a transcription factor and Dig1p and Dig2p are associated inhibitory proteins. Pheromone stimulation causes phosphorylation of these three factors by Fus3p, leading to release of Ste12p from Dig1p and Dig2p, thereby allowing Ste12p to interact with other proteins of the transcriptional machinery and to activate transcription [2].

The best understood MAPK signal transduction pathway of mammalian cells is that formed by the Raf–MAPK/ERK kinase (MEK)–extracellular regulated kinase (ERK) module (Fig. 1). Proliferative signals like growth factors cause activation and autophosphorylation of their cognate receptor tyrosine kinases. The phosphorylated tyrosine residues of the receptor serve as docking sites for the adaptor protein Grb2, which is complexed with Sos. Sos is a GTP exchange factor that activates the small G protein Ras, which thereby recruits the MAPKKK Raf to the plasma membrane where it is activated. The details of Raf activation are not yet fully understood, but activated Raf signals via its substrate MEK to the MAPK, ERK (reviewed in [3], [4]).

Whereas Fus3p and Ste7p in S. cerevisiae are direct homologues of mammalian ERK and MEK, respectively, there are no structural homologues of Raf kinases present in yeast; the MAPKKK's that perform the function of Raf in S. cerevisiae and Schizosaccharomyces pombe are the protein kinases Ste11p and Byr2p, respectively. In order to identify homologues of Ste11p and Byr2p in mammalian cells, a degenerate PCR-based strategy was used, and this led to the cloning of a mammalian MAPKKK, termed MEK kinase 1 (MEKK1) [5], [6]. Subsequently, the same strategy was used to identify three additional homologues, designated MEKK2, MEKK3 [7], and MEKK4 [8]. This review will focus on these four mammalian MAPKKKs of the MEKK family, with emphasis on those proteins shown to interact with them and serve as their upstream regulators or downstream substrates.