p38 targets serine/threonine kinases

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).

p38 targets the MEF2 family of transcription factors

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 phiA-X-phiB motif (where phiA-X-phiB 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).

Other transcription factors targeted by p38

Upon transforming growth factor-beta (TGF-beta) binding to its cognate receptor, Smad3 and Smad4 form heterodimers and transduce the TGF-beta signal to the nucleus. In addition to the Smad pathway, another pathway involving a member of the mitogen-activated protein kinase kinase kinase family of kinases, TGF-beta-activated kinase-1 (TAK1: Drosophila homolog TGF-ß activated kinase 1), is required for TGF-beta signaling. However, it is unknown how these pathways function together to synergistically amplify TGF-beta signaling. The transcription factor ATF-2 (also called CRE-BP1) is bound by a hetero-oligomer of Smad3 and Smad4 upon TGF-beta stimulation. ATF-2 is one member of the ATF/CREB family that binds to the cAMP response element, and its activity is enhanced after phosphorylation by stress-activated protein kinases, such as c-Jun N-terminal kinase and p38. The binding between ATF-2 and Smad3/4 is mediated via the MH1 region of the Smad proteins and the basic leucine zipper region of ATF-2. TGF-beta signaling also induces the phosphorylation of ATF-2 via TAK1 and p38. Both of these actions are shown to be responsible for the synergistic stimulation of ATF-2 trans-activating capacity. These results indicate that ATF-2 plays a central role in TGF-beta signaling by acting as a common nuclear target for both the Smad and TAK1 pathways (Sano, 1999).

The transcription factor Pax6 is required for normal development of the central nervous system, eyes, nose, and pancreas. The transactivation domain (TAD) of zebrafish Pax6 is phosphorylated in vitro by the mitogen-activated protein kinases (MAPKs) extracellular-signal regulated kinase (ERK) and p38 kinase, but not by Jun N-terminal kinase (JNK). Three of four putative proline-dependent kinase phosphorylation sites are phosphorylated in vitro. Of these sites, the serine 413 (Ser413) is evolutionary conserved from sea urchin to man. Ser413 is also phosphorylated in vivo upon activation of ERK or p38 kinase. Substitution of Ser413 with alanine strongly decreases the transactivation potential of the Pax6 TAD, whereas substitution with glutamate increases the transactivation. Reporter gene assays with wild-type and mutant Pax6 reveal that transactivation by the full-length Pax6 protein from paired domain-binding sites is strongly enhanced (16-fold) following co-transfection with activated p38 kinase. This enhancement is largely dependent on the Ser413 site. ERK activation, however, produces a 3-fold increase in transactivation, which is partly independent of the Ser413 site. These findings provide a starting point for further studies aimed at elucidating a post-translational regulation of Pax6 following activation of MAPK signaling pathways (Mikkola, 1999).

Bicyclic imidazoles, specific inhibitors of the p38 mitogen-activated protein kinase (MAPK) block cytokine synthesis at the translational level. The role of p38 MAPK in the regulation of the IL-1beta cytokine gene in monocytic cell lines was studied using the bicyclic imidazole SB203580. Addition of SB203580 30 min before stimulation of monocytes with LPS inhibits IL-1beta protein and steady state message in a dose-dependent manner in both RAW264.7 and J774 cell lines. The loss of IL-1beta message is due mainly to inhibition of transcription, since nuclear run-off analysis shows an approximately 80% decrease in specific IL-1 RNA synthesis. In contrast, SB203580 has no effect on the synthesis of TNF-alpha message. LPS-stimulated p38 MAPK activity in the RAW264.7 cells is blocked by SB203580, as measured by the inhibition of MAPKAP2 kinase activity, a downstream target of the p38 MAPK. CCAATT/enhancer binding protein (C/EBP)/NFIL-6-driven chloramphenicol acetyltransferase (CAT) reporter activity is sensitive to SB203580, indicating that C/EBP/NFIL-6 transcription factor(s) are also targets of p38 MAPK. In contrast, transfected CAT constructs containing NF-kappaB elements are only partially inhibited (approximately 35%) at the highest concentration of SB203580 after LPS stimulation. Overall, the results demonstrate a role for p38 MAPK in IL-1beta transcription by acting through C/EBP/NFIL-6 transcription factors (Baldassare, 1999).

SB203580 and SB202190, pyridinyl imidazoles that selectively inhibit p38 mitogen-activated protein (MAP) kinase, are widely utilized to assess the physiological roles of p38. Treatment of 3T3-L1 fibroblasts with these p38 MAP kinase inhibitors prevents fibroblast differentiation into adipocytes as judged by an absence of lipid accumulation, a lack of expression of adipocyte-specific genes, and a fibroblastic morphological appearance. In 3T3-L1 fibroblasts and developing adipocytes, p38 is active. p38 activity decreases dramatically during later stages of differentiation. In accordance with the time course of p38 activity, p38 inhibitor treatment during only the early stages of differentiation is sufficient to block adipogenesis. In addition, a 3T3-L1 cell line harboring an inducible dominant negative p38 mutant was constructed. The induction of this dominant negative mutant of p38 prevents adipocyte differentiation. Thus, it is likely that the antiadipogenic activity of SB203580 and SB202190 is indeed due to inhibition of p38 MAP kinase. This study points out that CCAAT/enhancer-binding protein beta (C/EBPbeta), a transcription factor critical for the initial stages of 3T3-L1 adipogenesis, bears a consensus site for p38 phosphorylation and serves as a substrate for p38 MAP kinase in vitro. Although the induction of C/EBPbeta is not significantly altered in the presence of the p38 inhibitor, the amount of in vivo phosphorylated C/EBPbeta is reduced by SB203580. The transcriptional induction of PPARgamma, a gene whose expression is induced by C/EBPbeta, and a factor critically involved in terminal differentiation of adipocytes, is impaired in the presence of p38 inhibitors. Thus, transcription factors such as C/EBPbeta that promote adipocyte differentiation may be p38 targets during adipogenesis. Collectively, the data in this study suggest that p38 MAP kinase activity is important for proper 3T3-L1 differentiation (Engelman, 1998).

The p53 tumor suppressor protein is a transcription factor that plays a key role in the process of apoptosis and the cell's defense against tumor development. Activation of p53 occurs, at least in part, by phosphorylation of its protein. UV induces a functional activation of p53 via phosphorylation at serine 389. The UV-induced phosphorylation of p53 at serine 389 is mediated by p38 kinase. UVC-induced phosphorylation of p53 at serine 389 is markedly impaired by either pretreatment of cells with p38 kinase inhibitor, SB202190, or stable expression of a dominant negative mutant of p38 kinase. In contrast, there was no inhibition observed in cells treated with a specific MEK1 inhibitor (PD98059) or with stable expression of a dominant negative mutant of ERK2 or JNK1. p38 kinase can be co-immunoprecipitated with p53 by using antibodies against p53. Pretreatment of cells with SB202190 blocks the p53 DNA binding activity and p53-dependent transcription. These results strongly suggest that the p38 kinase is at least one of the most important mediators of p53 phosphorylation at serine 389 induced by UVC radiation (Huang, 1999).

The E2F transcription factor plays a major role in cell cycle regulation, differentiation and apoptosis, but it is not clear how it is regulated by non-mitogenic signaling cascades. Two kinases involved in signal transduction have opposite effects on E2F function: the stress-induced kinase JNK1 inhibits E2F1 activity whereas the related p38 kinase reverses Rb-mediated repression of E2F1. JNK1 phosphorylates E2F1 in vitro, and co-transfection of JNK1 reduces the DNA binding activity of E2F1; treatment of cells with TNFalpha has a similar effect. Fas stimulation of Jurkat cells is known to induce p38 kinase and a pronounced increase in Rb phosphorylation is found within 30 min of Fas stimulation. Phosphorylation of Rb correlates with a dissociation of E2F and increased transcriptional activity. The inactivation of Rb by Fas is blocked by SB203580, a p38-specific inhibitor, as well as a dominant-negative p38 construct; cyclin-dependent kinase (cdk) inhibitors as well as dominant-negative cdks have no effect. These results suggest that Fas-mediated inactivation of Rb is mediated via the p38 kinase, independent of cdks. The Rb/E2F-mediated cell cycle regulatory pathway appears to be a normal target for non-mitogenic signaling cascades and could be involved in mediating the cellular effects of such signals (Wang, 1999).

Cachexia is a chronic state of negative energy balance and muscle wasting that is a severe complication of cancer and chronic infection. While cytokines such as IL-1alpha, IL-1ß, and TNFalpha can mediate cachectic states, how these molecules affect energy expenditure is unknown. Many cytokines activate the transcriptional PPAR gamma coactivator-1 (PGC-1) through phosphorylation by p38 kinase, resulting in stabilization and activation of PGC-1 protein. Cytokine or lipopolysaccharide (LPS)-induced activation of PGC-1 in cultured muscle cells or muscle in vivo causes increased respiration and expression of genes linked to mitochondrial uncoupling and energy expenditure. These data illustrate a direct thermogenic action of cytokines and p38 MAP kinase through the transcriptional coactivator PGC-1 (Puigserver, 2001).

The nuclear retinoic acid receptor RARgamma2 undergoes proteasome-dependent degradation upon ligand binding. Evidence is provided that the domains that signal proteasome-mediated degradation overlap with those that activate transcription, i.e. the activation domains AF-1 and AF-2. The AF-1 domain signals RARgamma2 degradation through its phosphorylation by p38MAPK in response to RA. The AF-2 domain acts via the recruitment of SUG-1, which belongs to the 19S regulatory subunit of the 26S proteasome. Blocking RARgamma2 degradation through inhibition of either the p38MAPK pathway or the 26S proteasome function impairs its RA-induced transactivation activity. Thus, the turnover of RARgamma2 is linked to transactivation (Gianni, 2002).

The transcriptional coactivator PPAR gamma coactivator 1 alpha (PGC-1alpha) is a key regulator of metabolic processes such as mitochondrial biogenesis and respiration in muscle and gluconeogenesis in liver. Reduced levels of PGC-1alpha in humans have been associated with type II diabetes. PGC-1alpha contains a negative regulatory domain that attenuates its transcriptional activity. This negative regulation is removed by phosphorylation of PGC-1alpha by p38 MAPK, an important kinase downstream of cytokine signaling in muscle, and ß-adrenergic signaling in brown fat. p160 myb binding protein (p160MBP) has been identified as a repressor of PGC-1alpha. The binding and repression of PGC-1alpha by p160MBP is disrupted by p38 MAPK phosphorylation of PGC-1alpha. Adenoviral expression of p160MBP in myoblasts strongly reduces PGC-1alpha's ability to stimulate mitochondrial respiration and the expression of the genes of the electron transport system. This repression does not require removal of PGC-1alpha from chromatin, suggesting that p160MBP is or recruits a direct transcriptional suppressor. Overall, these data indicate that p160MBP is a powerful negative regulator of PGC-1alpha function and provides a molecular mechanism for the activation of PGC-1alpha by p38 MAPK. The discovery of p160MBP as a PGC-1alpha regulator has important implications for the understanding of energy balance and diabetes (Fan, 2004).

Selective recognition of the E-box sequences on muscle gene promoters by heterodimers of myogenic basic helix-loop-helix (bHLH) transcription factors, such as MyoD, with the ubiquitous bHLH proteins E12 and E47 is a key event in skeletal myogenesis. However, homodimers of MyoD or E47 are incapable of binding to and activating muscle chromatin targets, suggesting that formation of functional MyoD/E47 heterodimers is pivotal in controlling muscle transcription. p38 MAPK, whose activity is essential for myogenesis, regulates MyoD/E47 heterodimerization. Phosphorylation of E47 at Ser140 by p38 induces MyoD/E47 association and activation of muscle-specific transcription, while the nonphosphorylatable E47 mutant Ser140Ala fails to heterodimerize with MyoD and displays impaired myogenic potential. Moreover, inhibition of p38 activity in myocytes precludes E47 phosphorylation at Ser140, which results in reduced MyoD/E47 heterodimerization and inefficient muscle differentiation, as a consequence of the impaired binding of the transcription factors to the E regulatory regions of muscle genes. These findings identify a novel pro-myogenic role of p38 in regulating the formation of functional MyoD/E47 heterodimers that are essential for myogenesis (Lluis, 2005).

p38 has been shown to induce the activity of the muscle coactivator MEF2 and to target the SWI/SNF complex to muscle promoters through the functional MyoD transcription factor. It is proposed that p38 may complex two timely and closely linked muscle transcription mechanisms. By binding to and phosphorylating E47, p38 promotes formation of the functional MyoD/E47 heterodimer, which will allow subsequent recruitment of the SWI/SNF complex on myogenic promoters (Lluis, 2005).

p38 targets various transcription factors via intermediate proteins

Continues: p38b Evolutionary homologs part 3/3 | back to part 1/3 |

p38b: Biological Overview | Regulation | Developmental Biology | References

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