Organization and regulation of mitogen-activated protein kinase signaling pathways
Introduction
The core unit of mitogen-activated protein kinase (MAPK) pathways is a three-member protein kinase cascade. Within the three-kinase module, MAPKs are phosphorylated and activated by MAPK kinases (MKKs). MKKs are characteristically dual specificity kinases which catalyze the phosphorylation of MAPKs on both tyrosine and threonine residues. The MKKs are themselves phosphorylated and activated by serine/threonine kinases that function as MKK kinases (MKKKs). During evolution, many of the components of three kinase MAPK modules have been conserved in yeast and man. To date, 12 member proteins of the MAPK family have been identified in mammalian cells, and these can be grouped into five subfamilies, on the basis of sequence homology and function. Seven MKKs and fourteen MKKKs have been functionally identified in mammalian cells. Current knowledge suggests that low molecular weight GTP binding proteins (i.e., Ras, Rac, Cdc42) and specific kinases that could be considered to be MAPK kinase kinase kinases (MKKKKs) regulate the activity of MKKKs, thus controlling the activation of specific three kinase MAPK modules 1••, 2, 3, 4.
Specificity of MAPK responses is achieved by activation of different MKKK–MKK–MAPK modules in response to different stimuli. MAPK modules are differentially activated by growth factors, hormones and cytokines. In addition, MAPK modules may be activated by cellular stresses including irradiation, heat shock, osmotic imbalance, DNA damage, and bacterial products such as lipopolysaccharide. Activation of MAPKs in response to these stimuli controls gene expression, metabolism, cytoskeletal functions and other cellular regulatory events. MAPKs contribute to complex regulatory events including mitogenesis, differentiation, survival and migration (Figure 1).
The combination of twelve MAPKs, seven MKKs and fourteen MKKKs, some with apparently redundant functions, seems extraordinarily complex. But, on closer inspection, certain themes start to emerge. First, the MKKs represent the fewest number of members in the MAPK module. MKKs also have high specificity for their MAPK substrates, allowing minimal variation of the MKK–MAPK part of the MAPK module. In contrast, the fourteen defined MKKKs are more diverse in structure. The MKKKs have different defined regulatory motifs that are not found in MKKs or MAPKs. These motifs include Pleckstrin homology (PH) domains, proline-rich sequences for binding SH (Src homology) 3 domains, binding sites for GTP-binding proteins, leucine-zipper dimerization sequences, and phosphorylation sites for tyrosine and serine/threonine kinases. Thus, MKKKs can be differentially regulated by a variety of upstream inputs for their selective regulation of MKKs.
The diversity of regulatory domains in different MKKKs gives the family of MAPK modules the flexibility to respond to a wide range of cellular stimuli. But what gives an MKKK the ability to act selectively in MAPK pathways, particularly MKKKs that can phosphorylate and activate multiple MKKs? Emerging evidence indicates that specificity is achieved, in part, by the use of scaffolding or anchoring proteins to coordinate MKKK binding to specific proteins for upstream inputs as well as specific downstream MKK–MAPK complexes. Scaffolding of multicomponent regulatory systems is now recognized as a major mechanism for controlling signal transduction pathways 5, 6, 7, 8, 9•. The orchestration of MAPK modules by scaffolding or anchoring proteins can function by both positive and negative regulatory mechanisms. Scaffolding can bring together proteins for their interaction and regulation or conversely may sequester proteins so they do not interact with other proteins. Scaffolding can also control subcellular location, which can, in turn, be regulated by differential expression of specific scaffolding proteins. These protein interactions may also be post-translationally controlled by phosphorylation or proteolysis of scaffolding proteins or their binding partners. In this review we focus on the evidence for protein scaffolding in controlling MAPK modules.
Section snippets
Scaffolding of mitogen-activated protein kinase modules in yeast
The role of scaffolding proteins in bringing together the components of a specific MAPK module has been most clearly demonstrated in the yeast Saccharomyces cerevisiae 10, 11, 12, 13. In S. cerevisiae, a specific MAPK cascade involved in the mating response of haploid cells is activated in response to binding of a mating pheromone to its receptor (Figure 2a). Because deletion or inactivation of component proteins in this pathway leads to sterility, the genes encoding the mating pathway proteins
Scaffolding of mitogen-activated protein kinase modules in mammalian cells
In 1998, a protein referred to as MP1 (MEK partner 1) was identified that appears to be a scaffold protein for the extracellular signal regulated kinase (ERK) pathway 19••, 20. MP1 was found to specifically bind MEK1 (a MKK) and ERK1 (a MAPK) (Figure 2c). When overexpressed in COS cells, MP1 enhances activation of ERK1. The prediction is that MP1 functions to increase the efficiency of the MKKK activation of the MEK1–ERK1 pathway. The predominant MKKK in this pathway is Raf-1 or B-Raf although
Other proteins that regulate mitogen-activated protein kinase signaling
Genetic screens in Drosophila melanogaster and Caenorhabditis elegans have identified several proteins that stimulate signaling of the Raf–MEK–ERK pathway. One of these proteins is the kinase suppressor of Ras (Ksr) 29, 30, 31. In these two organisms, mutations in Ksr were found to attenuate Ras-mediated activation of the ERK pathway. Ksr is expressed in mammalian cells, and yeast two-hybrid analysis using mouse Ksr (mKsr-1) as bait showed interactions of mKsr-1 with both MEK1 and ERK [32•].
Conclusions
The noncatalytic function of specific proteins to organize signaling complexes certainly extends beyond the MAPKs. In fact, cellomics — the definition of three dimensional organization of proteins in the cell — is one of the major problems to be solved in cell biology. One key to cellomics will be to identify protein scaffolds. Several examples of scaffolding proteins in signal transduction complexes other than MAPKs have been defined. Ina D is a protein in D. melanogaster photoreceptor neurons
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Table 1
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