Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. A new subfamily of murine serine/threonine kinases has been identified, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases: Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates Mnk in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells (Waskiewicz, 1997).

A novel expression screening method has been developed for identifying protein kinase substrates. In this method, a lambda phage cDNA expression library is screened by in situ, solid-phase phosphorylation using purified protein kinase and [gamma-32P]ATP. Screening a HeLa cDNA library with ERK1 MAP kinase yields cDNAs of previously characterized ERK substrates, c-Myc and p90RSK, demonstrating the utility of this method for identifying physiological protein kinase substrates. A novel clone isolated in this screen, designated MNK1, encodes a protein-serine/threonine kinase, which is most similar to MAP kinase-activated protein kinase 2 (MAPKAP-K2), 3pK/MAPKAP-K3 and p90RSK. Bacterially expressed MNK1 is phosphorylated and activated in vitro by ERK1 and p38 MAP kinases but not by JNK/SAPK. Further, MNK1 is activated upon stimulation of HeLa cells with 12-O-tetradecanoylphorbol-13-acetate, fetal calf serum, anisomycin, UV irradiation, tumor necrosis factor-alpha, interleukin-1beta, or osmotic shock, and the activation by these stimuli is differentially inhibited by the MEK inhibitor PD098059 or the p38 MAP kinase inhibitor SB202190. Together, these results indicate that MNK1 is a novel class of protein kinase that is activated through both the ERK and p38 MAP kinase signaling pathways (Fukunaga, 1997).

NF-κB is activated in response to proinflammatory stimuli, infections, and physical stress. While activation of NF-κB by many stimuli depends on the IκB kinase (IKK) complex, which phosphorylates IκBs at N-terminal sites, the mechanism of NF-κB activation by ultraviolet (UV) radiation remained enigmatic, since it is IKK independent. UV-induced NF-κB activation has been shown to depend on phosphorylation of IκBα at a cluster of C-terminal sites that are recognized by CK2 (formerly casein kinase II). Furthermore, CK2 activity toward IκB is UV inducible through a mechanism that depends on activation of p38 MAP kinase. Inhibition of this pathway prevents UV-induced IκBα degradation and increases UV-induced cell death. Thus, the p38-CK2-NF-κB axis is an important component of the mammalian UV response (Kato, 2003).

Members of the MEF2 family of transcription factors bind as homo- and hetero-dimers to the MEF2 sites found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. The transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. The potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members was examined. MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four mammalian p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein (Zhao, 1999).

Mitogen-activated protein (MAP) kinase-mediated signaling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases that phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. Among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38alpha and p38beta2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to understanding of signaling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38) (Yang, 1999).

Differentiation of muscle cells is regulated by extracellular growth factors that transmit largely unknown signals into the cells. Some of these growth factors induce mitogen-activated protein kinase (MAPK) cascades within muscle cells. The kinase activity of p38 MAPK is induced early during terminal differentiation of L8 cells. Addition of a specific p38 inhibitor (SB 203580) to myoblasts blocks their fusion to multinucleated myotubes and prevents the expression of MyoD and MEF2 family members and myosin light chain 2. The expression of MKK6, a direct activator of p38, or of p38 itself enhances the activity of MyoD in converting 10T1/2 fibroblasts to muscle, whereas treatment with SB 203580 inhibits MyoD. Several lines of evidence suggesting that the involvement of p38 in MyoD activity is mediated via its co-activator MEF2C, a known substrate of p38, are presented. In these experiments MEF2C protein and MEF2-binding sites are shown to be necessary for the p38 MAPK pathway to regulate the transcription of muscle creatine kinase reporter gene. These results indicate that the p38 MAPK pathway promotes skeletal muscle differentiation at least in part via activation of MEF2C (Zetser, 1999).

Myocyte enhancer factor 2 (MEF2) is in the MADS family of transcription factors. Although MEF2 is known as a myogenic factor, the expression pattern of the MEF2 family of genes (MEF2A-D) in developing brain also suggests a role in neurogenesis. Transfection with MEF2C, the predominant form in mammalian cerebral cortex, induces a mixed neuronal/myogenic phenotype in undifferentiated P19 precursor cells. During retinoic acid-induced neurogenesis of these cells, a dominant negative form of MEF2 enhances apoptosis but does not affect cell division. The mitogen-activated protein kinase p38alpha activates MEF2C. Dominant negative p38alpha also enhances apoptotic death of differentiating neurons, but these cells can be rescued from apoptosis by coexpression of constitutively active MEF2C. These findings suggest that the p38alpha/MEF2 pathway prevents cell death during neuronal differentiation (Okamoto, 2000).

A solution has been found to the structural question of MAP kinase p38 in complex with docking site peptides containing a phi_A-X-phi_B motif (where phi_A-X-phi_B are hydrophobic regions), derived from substrate MEF2A and activating enzyme MKK3b. The peptides bind to the same site in the C-terminal domain of the kinase, which is both outside the active site and distinct from the 'CD' domain previously implicated in docking site interactions. Mutational analysis on the interaction of p38 with the docking sites supports the crystallographic models and has uncovered two novel residues on the docking groove that are critical for binding. The two peptides induce similar large conformational changes local to the peptide binding groove. The peptides also induce unexpected and different conformational changes in the active site, as well as structural disorder in the phosphorylation lip. Thus docking site interactions, in addition to providing a specificity determinant, may have a role in the activation of the kinase. (Chang, 2002).

The development and differentiation of distinct cell types is achieved through the sequential expression of subsets of genes; yet, the molecular mechanisms that temporally pattern gene expression remain largely unknown. In skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. p38 activity facilitates MyoD and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits Pol II. Most importantly, expression of late-activated genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that a MyoD-mediated feed-forward circuit temporally patterns gene expression (Penn, 2004).

Temporally patterned gene expression in a complex program of cell differentiation is achieved through a feed-forward mechanism. MyoD initiates the expression of specific Mef2 isoforms and activates the p38 MAPK pathway. p38 activity facilitates MyoD and Mef2 binding at genes expressed late in the myogenic program, and the binding of Mef2D recruits Pol II and correlates with the transcription of these genes. Most importantly, expression of some late-stage genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that the timing of expression is programmed by an intrinsic delay while Mef2 isoforms and p38 activity accumulate, and substantiating the role of a transcriptional feed-forward circuit in temporally patterning gene expression. Because p38 and Mef2D cooperate with MyoD to regulate only a subset of late-stage genes, it is likely that additional sets of genes might require other MyoD-regulated intermediate factors (Penn, 2004).

This study suggests two distinct roles of p38 kinase: (1) as a rate limiting factor in the binding of Mef2 and MyoD, and (2) in facilitating phosphorylation and progression of Pol II. The role of p38 in facilitating the binding of MyoD and Mef2 is likely to be through an effect on chromatin, since it does not alter the binding of these factors in gel-shift assays, and the recent demonstration that the p38 pathway targets the SWI/SNF complex to muscle loci through an interaction with MyoD might account for its effect on factor binding, although other mechanisms, such as histone phosphorylation, might also effect factor binding. The role of p38 in facilitating Pol II phosphorylation and progression is likely to be through the phosphorylation of Mef2D, because prior studies have shown that p38 phosphorylation of the Mef2 activation domain greatly potentiates the transcriptional activity of Mef2. This study shows that the Mef2D isoform is rate limiting for transcription at a subset of late promoters. This suggests that the Mef2D isoform has promoter-specific activities and that the relative abundance of Mef2 isoforms determines which subsets of promoters are actively transcribed (Penn, 2004).