p38b
Protein phosphatases inactivate mitogen-activated protein kinase (MAPK) signaling pathways by dephosphorylating components of the
MAPK cascade. Two genes encoding protein-tyrosine phosphatases, PTP2, and a new phosphatase, PTP3, have been isolated in a
genetic selection for negative regulators of an osmotic stress response pathway called HOG, for high osmolarity glycerol, in budding
yeast. PTP2 and PTP3 were isolated as multicopy suppressors of a severe growth defect due to hyperactivation of the HOG pathway.
Phosphatase activity is required for suppression because mutation of the catalytic Cys residue in Ptp2 and Ptp3 destroys suppressor
function and biochemical activity. The substrate of these phosphatases is likely to be the MAPK, Hog1. Catalytically inactive Ptp2 and
Ptp3 coprecipitate with Hog1 from yeast extracts. In addition, strains lacking PTP2 and PTP3 do not dephosphorylate
Hog1-phosphotyrosine as well as wild type. The latter suggests that PTP2 and PTP3 play a role in adaptation. Consistent with this
role, osmotic stress induces expression of PTP2 and PTP3 transcripts in a Hog1-dependent manner. Thus Ptp2 and Ptp3 likely act in a
negative feedback loop to inactivate Hog1 (Jacoby, 1997).
Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the
high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1-green fluorescent protein (GFP) fusion reveal that
even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments.
The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon
osmotic challenge, the Hog1-GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an
iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization
correlate with those found for the dual phosphorylation of Hog1-GFP. The duration of Hog1 nuclear residence is modulated by the
presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein
synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution
pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export (Reiser, 1999).
The MAPKKK Ste11p functions in three Saccharomyces cerevisiae MAPK cascades [the high osmolarity glycerol
(HOG), pheromone response, and pseudohyphal/invasive growth pathways], but its activation in response to high
osmolarity stimulates only the HOG pathway. To determine what restricts cross-activation of MAPK cascades (cross
talk), mutants have been studied in which the pheromone response pathway is activated by high osmolarity (1 M
sorbitol). Mutations in the HOG1 gene, encoding the p38-type MAPK of the HOG pathway, and in
the PBS2 gene, encoding the activating kinase for Hog1p, allow osmolarity-induced activation of the pheromone
response pathway. This cross talk requires the osmosensor Sho1p, as well as Ste20p, Ste50p, the pheromone
response MAPK cascade (Ste11p, Ste7p, and Fus3p or Kss1p), and Ste12p but not Ste4p or the MAPK scaffold
protein, Ste5p. The cross talk in hog1 mutants induces multiple responses of the pheromone response pathway:
induction of a FUS1::lacZ reporter, morphological changes, and mating in ste4 and ste5 mutants. It is suggested that
Hog1p may prevent osmolarity-induced cross talk by inhibiting Sho1p, perhaps as part of a feedback control on the
HOG pathway. Ste20p and Ste50p function in the Sho1p branch of the HOG pathway;
a second osmosensor in addition to Sho1p may activate Ste11p. Pseudohyphal
growth exhibited by wild-type (HOG1) strains depends on SHO1, suggesting that Sho1p may be a receptor that
feeds into the pseudohyphal growth pathway (O'Rourke, 1998).
The yeast alpha-1,3-mannosyltransferase (Mnn1p) is localized to the Golgi by independent transmembrane and lumenal domain
signals. The lumenal domain is localized to the Golgi complex when expressed as a soluble form (Mnn1-s) by exchange of its
transmembrane domain for a cleavable signal sequence.
Mutants that failed to retain the lumenal domain in the Golgi complex, called lumenal domain retention (ldr) mutants, were isolated by
screening mutagenized yeast colonies for those that secreted Mnn1-s. Two genes were identified by this screen, HOG1, a gene
encoding a mitogen-activated protein kinase (MAPK) that functions in the high osmolarity glycerol (HOG) pathway, and LDR1. Basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions.
Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a
concomitant increase of Mnn1p in later Golgi compartments (Reynolds, 1998).
Compared to mammals, insects, and plants, relatively little is known about innate immune responses in the nematode C. elegans. Salmonella enterica serovars cause a persistent infection in the C. elegans intestine that triggers gonadal programmed cell death (PCD) and that C. elegans cell death (ced) mutants are more susceptible to Salmonella-mediated killing. To further dissect the role of PCD in C. elegans innate immunity, both C. elegans and S. enterica factors were identified that affect the elicitation of Salmonella-induced PCD. Salmonella-elicited PCD requires the C. elegans homolog of the mammalian p38 mitogen-activated protein kinase (MAPK) encoded by the pmk-1 gene. Inactivation of pmk-1 by RNAi blocks Salmonella-elicited PCD, and epistasis analysis shows that CED-9 lies downstream of PMK-1. Wild-type Salmonella lipopolysaccharide (LPS) is required for the elicitation of PCD, as well as for persistence of Salmonella in the C. elegans intestine. However, a presumptive C. elegans TOLL signaling pathway does not appear to be required for the PCD response to Salmonella. These results establish a PMK-1-dependant PCD pathway as a C. elegans innate immune response to Salmonella (Aballay, 2003).
The evolutionarily conserved p38 mitogen-activated protein kinase (MAPK) cascade is an integral part of the response to a variety of environmental stresses. The C. elegans PMK-1 p38 MAPK pathway regulates the oxidative stress response via the Cap-N-Collar (CNC) transcription factor SKN-1. In response to oxidative stress, PMK-1 phosphorylates SKN-1, leading to its accumulation in intestine nuclei, where SKN-1 activates transcription of gcs-1, a phase II detoxification enzyme gene. These results delineate the C. elegans p38 MAPK signaling pathway leading to the nucleus that responds to oxidative stress (Inoue, 2005).
Oxidative stress contributes to the etiology of various degenerative diseases such as ischemia and the process of aging. In vertebrates, a major mechanism of oxidative stress defense is orchestrated by the two NF-E2-related factors Nrf1 and Nrf2, which belong to the CNC family of transcription factors. Nrf proteins induce expression of a battery of phase II detoxification enzymes. Nrf proteins accumulate in cell nuclei in response to oxidative stress. In the nematode Caenorhabditis elegans, the SKN-1 protein is required for oxidative stress resistance. SKN-1 is distantly related to the Nrf proteins and induces phase II detoxification gene transcription. Oxidative stress induces SKN-1 to accumulate in intestinal nuclei. This oxidative stress response thus appears to be widely conserved (Inoue, 2005).
Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase (see Drosophila Highwire), causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1lf mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. These data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade (Nakata, 2005).
Regeneration of injured neurons can restore function, but most neurons regenerate poorly or not at all. The failure to regenerate in some cases is due to a lack of activation of cell-intrinsic regeneration pathways. These pathways might be targeted for the development of therapies that can restore neuron function after injury or disease. This study shows that the DLK-1 mitogen-activated protein (MAP) kinase pathway (Drosophila wallenda is MAP kinase kinase kinase homologous to vertebrate DLK and LZK, see Highwire restrains synaptic growth by attenuating a MAP kinase signal) is essential for regeneration in C. elegans motor neurons. Loss of this pathway eliminates regeneration, whereas activating it improves regeneration. Further, these proteins also regulate the later step of growth cone migration. It is concluded that after axon injury, activation of this MAP kinase cascade is required to switch the mature neuron from an aplastic state to a state capable of growth (Hammarlund, 2009).
How does the MAP kinase PMK-3 stimulate regeneration? The DLK-1 pathway is first required for growth cone formation about 7 hours after a break occurs, a process likely to be mediated by the polymerization of microtubules. Activated p38 MAP kinase regulates microtubule dynamics, and microtubule remodeling is required for growth cone initiation during regeneration. Further, defects in microtubule dynamics contribute to the axon outgrowth phenotype of Phr1 mutant mice. Activated p38 may also control other targets that facilitate axon regeneration. p38 regulates local protein synthesis, which is required for regeneration. p38 is also likely to have functions in the nucleus, because it contributes to injury-induced changes in gene transcription. Activated p38 may reach the nucleus by retrograde transport. Retrograde transport in general is critical for regeneration, and transport of activated MAP kinases from axons to the cell body following axotomy has been observed in Aplysia sensory neurons and in rodent sciatic nerves. Thus, regeneration may require activated PMK-3/p38 at the site of the break to regulate microtubule stability and protein expression and also may require PMK-3 to traffic to the nucleus to regulate gene
transcription. The DLK-1 signaling pathway thus provides a critical link between axon injury and the process of regeneration (Hammarlund, 2009).
Spatiotemporal modulation of the evolutionarily conserved, intercellular Notch signaling pathway is important in the development of many animals. Examples include the regulation of neural-epidermal fate decisions in neurogenic ectoderm of Drosophila and somitogenesis in vertebrate presomitic mesoderm. In both these and most other cases, it appears that Notch-class transmembrane receptors are ubiquitously expressed. Modulation of the pathway is achieved primarily by the localized expression of the activating ligand or by alteration of receptor specificity through a glycosyl transferase. In contrast, this report presents an instance where the abundance of the Notch-class mRNA itself is dynamically regulated. Taking advantage of the long cell cycle of the two-cell-stage embryo of the leech Helobdella robusta, it was shown that this regulation is achieved at the levels of both transcript stability and transcription. Moreover, MAPK signaling plays a significant role in regulating accumulation of the transcript by virtue of its effect on Hro-notch mRNA stability. Intracellular injection of heterologous reporter mRNAs shows that the Hro-notch 3' UTR, containing seven AU-rich elements (AREs), is key to regulating transcript stability. Thus, this study shows that regulation of the Notch pathway can occur at a previously underappreciated level, namely that of transcript stability. Given that AU-rich elements occur in the 3' UTR of Notch-class genes in Drosophila, human, and Caenorhabditis elegans, regulation of Notch signaling by modulation of mRNA levels may be operating in other animals as well (Gonsalves, 2007).
In conclusion, this study shows that transcript levels of a Notch-class gene (Hro-notch) oscillate antiphasically in the AB and CD blastomeres of the two-cell embryo in the leech H. robusta, i.e., high transcript levels in AB are associated with low levels in CD and vice versa. Moreover, the Hro-notch levels are controlled by dynamic activation of one or more of the MAPK (p38MAPK and ERK) signaling pathways. Initially, the Hro-notch level in each cell reflects primarily inherited maternal transcripts. Later, the production and turnover of zygotic transcripts becomes important. The 3' UTR of Hro-notch mRNA confers a relatively short half-life to the transcripts, apparently because of the presence of multiple AREs. This instability is counteracted by the p38MAPK pathway. Thus, the Hro-notch transcript levels are controlled by MAPK signaling at the level of transcript stability and possibly also at the level of transcription. This link between the p38MAPK and Notch pathways persists into later development, because coincident p38MAPK activation and Hro-notch mRNA accumulation has been observed in the ectodermal precursor cell DNOPQ (Gonsalves, 2007).
Another instance where p38MAPK plays a role in early development is in the axial patterning of the Drosophila oocyte, where it regulates the availability of the EGF ligand (encoded by gurken) and thus controls the activation of the ERK pathway in the follicle cells. Although the mechanistic intricacies of combinatorial effects between p38MAPK and other signaling pathways remain to be elucidated, the current observations suggest that these effects might involve the regulation of mRNA stability. It has already been demonstrated that oscillations in Notch signaling can be achieved by modulating transcript levels for the presumptive ligand (deltaC, in fish presomitic mesoderm) and for a receptor glycosylating enzyme (lunatic fringe, in chick PSM). The results reveal yet a third mechanism by which oscillatory modulation of Notch signaling may be achieved, namely regulating transcript levels for Notch receptor itself in the two-cell leech embryo. Does this mechanism of Notch regulation operate in other organisms as well? This question can only be answered empirically, but it is noted that Notch-class genes in a variety of organisms bear several pentameric AREs in their 3' UTRs. In particular, the 3' UTR of human Notch-1, like that of Hro-notch, bears nonameric AREs, which makes it a strong candidate for regulation by p38MAPK. The occurrence of AREs within the 3' UTRs of other Notch genes could be a mere coincidence, and the regulation of Notch transcript stability by p38MAPK could be a novelty restricted to leech. This would be a noteworthy result in and of itself, given the frequency with which genes and signaling pathways discovered in one organism prove to have broadly distributed homologs. A more likely alternative is that the current observations provide a relatively prominent, experimentally accessible example of a regulatory interaction that operates throughout the animal kingdom (Gonsalves, 2007).
MAP kinases (MAPKs) form a complex with MAPK kinases (MAPKKs), MAPK-specific phosphatases (MKPs) and various targets
including MAPKAPKs. These docking interactions contribute to regulation of the specificity and efficiency of the enzymatic reactions. A docking site on MAPKs, termed the CD (common docking) domain, is utilized commonly for docking
interactions with MAPKKs, MKPs and MAPKAPKs. However, the CD domain alone does not determine the docking specificity. A novel site, termed the ED site on p38 (Glu160 and Asp161 of human p38) and the corresponding site on ERK2 (Thr156 and Thr157
of rat ERK2), has been identified that regulates the docking specificity towards MAPKAPKs. Remarkably,
exchange of two amino acids in this site of ERK2 for corresponding residues of p38 converts the docking specificity for
MAPKAPK-3/3pk, which is a dominant target of p38, from the ERK2 type to the p38 type, and vice versa. Furthermore, detailed analyses with a number of
MAPKAPKs and MKPs suggest that a groove in the steric structure of MAPKs, which comprises the CD domain and the site identified here, serves as a common
docking region for various MAPK-interacting molecules (Tanoue, 2001).
What is the role of the docking interaction between MAPKs and their substrates?
While it is often the case that the consensus sequence of the phosphorylation site in
the substrate for protein kinases involves charged amino acids, such as the sequence RXRXXS/TP-Hyd (Hyd is a hydrophobic residue) for Akt/PKB, the sequence S/TPXR/K for cyclin-dependent kinases, a group of acidic amino acids for casein kinase II, etc., the consensus sequence for MAPKs, PXS/TP or S/TP, does not contain charged amino acids. Thus, it can be deduced that MAPKs utilize the docking interactions involving the charged amino acids in order to increase their efficiency and specificity for the substrates. However, this idea does not necessarily explain why the same docking groove is utilized for the docking interactions with MAPKKs and phosphatases. Therefore, the docking interactions in the MAPK cascades might have other roles: (1) as the docking interactions of MAPKs with MAPKKs, phosphatases and MAPKAPKs are mutually exclusive, they may underlie the molecular basis for the sequential and specific activation and inactivation of MAPKs; (2) the docking interactions
may function to determine the subcellular localization of MAPKs and MAPK-interacting molecules. For example, the N-terminal portion of MEK1 is the ERK docking domain, and MEK1 can function as a cytoplasmic anchor for ERK2. (3) In addition, the docking interaction with p38 regulates the subcellular localization of 3pk, a member of the MAPKAPKs (Tanoue, 2001).
For the biological function of Map kinase kinases functioning upstream of p38, see licorne Evolutionary homologs.
The p38 mitogen-activated protein kinase (MAPK) group is represented by four isoforms in mammals (p38alpha, p38beta, p38gamma and p38delta). These p38 MAPK isoforms appear to
mediate distinct functions in vivo due, in part, to differences in substrate phosphorylation by individual p38 MAPKs and
also to selective activation by MAPK kinases (MAPKKs). Two factors have been identified that
contribute to the specificity of p38 MAPK activation. One mechanism of specificity is the selective formation of functional complexes between
MAPKK and different p38 MAPKs. The formation of these complexes requires the presence of a MAPK docking site in the N-terminus of the
MAPKK. The second mechanism that confers signaling specificity is the selective recognition of the activation loop (T-loop) of p38 MAPK
isoforms. Together, these processes provide a mechanism that enables the selective activation of p38 MAPK in response to activated MAPKK (Enslen, 2000).
Two genes encode proteins that act as specific activators of p38: Mkk3 and Mkk6. Targeted gene disruption studies in mice have demonstrated non-redundant functions of the Mkk3 and Mkk6 genes, indicating that the MKK3 and MKK6 protein kinases have distinct biological functions. MKK3 activates the isoforms p38alpha, p38gamma and p38delta, but not p38beta2. However, both MKK6 and MKK3b (a variant form of MKK3, which has an additional 29 amino acids fused to the N-terminus of MKK3) activate the four p38 isoforms p38alpha, p38beta2, p38gamma and p38delta. In this study, a p38 kinase docking site has been identified within the N-terminal region of MKK3b and MKK6. These sequences are highly conserved and are required for p38beta2 activation by these two enzymes. MKK3, which lacks the docking site, does not activate p38beta2. Fusion of the p38 docking site of MKK6 to the N-terminus of MKK3 allows activation of p38beta2. Furthermore, synthetic peptides based on the primary sequence of the docking sites of MKK3b and MKK6 inhibit the activation of p38beta2. These data indicate that the binding of p38beta2 to an N-terminal region of the MAPKK is necessary for p38beta2 activation. In contrast, binding to p38alpha is not a requirement for activation by MKK3b and MKK6 in vitro. However, the binding interaction does serve to potentiate p38alpha activation. These data provide an explanation for the selective activation of p38alpha, but not p38beta2, by MKK3 (Enslen, 2000).
A similar role for a docking mechanism has been described in the yeast Saccharomyces cerevisiae where a high affinity interaction between a MAPKK (Ste7p) and MAPK (Kss1p and Fus3p) is required for MAPK activation. This interaction depends on a MAPK docking site present in the N-terminus of Ste7p. The sequence of the MAPK docking site of Ste7p is related to that identified in MKK3b and MKK6. These sequences are similar to the previously reported MAPK docking site consensus sequence found in MAPK substrates: -Arg/Lys-Xaa3-Leu/Ile-Xaa-Leu/Ile-. Interestingly, the N-terminal regions of MEK1 (32 amino acids), MKK4 (43 amino acids) and MKK7 (73 amino acids) have been demonstrated to be important for binding and activation of ERK and JNK. Sequences similar to the MAPK docking sites of MKK3b and MKK6 are present in the N-terminal regions of MEK1, MKK4 and MKK7. Further studies are required to characterize the MAPK docking site with the N-terminus of MEK1, MKK4 and MKK7. In contrast, this study has established the presence of p38 docking sites on MKK3b and MKK6 by mutational analysis and peptide competition analysis (Enslen, 2000 and references therein).
The MAPK docking sites present in MAPKK appear to be targeted during infection by some pathogens. For example, the lethal factor (LF) of anthrax lethal toxin (the major cause of death in animals infected with anthrax) inhibits the ERK signal transduction pathway. LF is a protease that cleaves the N-terminus of MEK1 and MEK2, causing decreased activity towards ERK. The N-terminal cleavage of MEK1 and MEK2 destroys the putative ERK docking site. Loss of the ERK docking site may account for the ability of LF to block activation of ERK by MEK1 and MEK2 in vivo. Interestingly, sequences similar to the LF cleavage site in MEK1 and MEK2 are present in the N-terminus of MKK3b and MKK6. Cleavage of MKK3b and MKK6 by LF at these sites would remove the p38 docking domain and would therefore be predicted to prevent binding and activation of p38beta2 and markedly decrease activation of p38alpha. Indeed, LF recently was reported to inhibit p38 signaling in macrophages (Enslen, 2000 and references therein).
Short peptide sequences that bind p38alpha and p38beta2 have been identified in the transcription factors MEF2A and MEF2C. These domains are necessary for efficient phosphorylation and activation of MEF2A and MEF2C by p38alpha and p38beta2. Similar MAPK-binding motifs have been characterized in several MAPK substrates, including c-Jun, JunB, Elk-1, NFAT4 and ATF-2. In general, these docking sites conform to the general consensus sequence -Arg/Lys-Xaa-Xaa-Xaa-Xaa-Leu/Ile-Xaa-Leu/Ile-. However, three additional classes of docking sites have been identified. These docking sites correspond to the Phe-Xaa-Phe-Pro motif identified in some Ets transcription factors that binds ERK, the -Leu-Ala-Gln-Arg-Arg-Xaa3-Leu/Ile- motif in Mnk2 and p90rsk that binds ERK and the Leu-hydrophobic-Lys/Arg-Lys/Arg-Lys/Arg-Lys/Arg- motif in PRAK and MAPKAP-K2 that binds p38. Binding sites for MAPK have also been identified in phosphatases that regulate MAPK activation. These considerations indicate that MAPK docking sites are located on the kinases that activate MAPK (MAPKK), on phosphatases that inactivate MAPK and on MAPK substrates (Enslen, 2000 and references therein).
The observation that MAPKs can bind related sequences in both their substrates and enzymes that regulate MAPK activity (MAPKK and phosphatases) is intriguing. Is it possible that the regulatory enzymes and substrates compete for binding to MAPK? This hypothesis predicts that substrate phosphorylation by MAPK would only be observed following release of the activated MAPK from MAPKK. Indeed, evidence in favor of this hypothesis has been reported in studies of yeast MAPK signaling pathways. For example, the S.cerevisiae MAPK Kss1p is not able to phosphorylate exogenous substrates when bound to the MAPKK Kss1p. Similarly, the Schizosaccharomyces pombe MAPKK Pek1p binds to and inhibits the MAPK Pmk1p, but releases activated Pmk1p following stimulation. Similar models for binding and release of activated MAPK in mammals have been reported for JNK activation by MKK4 and ERK activation by MEK1 (Enslen, 2000 and references therein).
MAPKs are activated by dual phosphorylation on threonine and tyrosine. Analysis of p38 MAPK phosphorylation by MKK3 and MKK6 provides evidence for selective phosphorylation on these activating sites. The p38alpha MAPK is phosphorylated preferentially on tyrosine by MAPKK that lacks a MAPK docking site (e.g. MKK3). MAPKK with a MAPK docking site (MKK3b and MKK6) causes similar phosphorylation of p38alpha MAPK on both threonine and tyrosine. These data suggest that the docking interaction may increase the processivity of MAPK phosphorylation, leading to increased dual phosphorylation and, consequently, increased activation. The requirement for dual phosphorylation for MAPK activation is likely to account, in part, for the role of docking interactions between MAPK and MAPKK. Studies of the JNK signaling pathway provide another example of selective phosphorylation of MAPK on threonine or tyrosine. MKK4 phosphorylates JNK1 preferentially on tyrosine, while MKK7 preferentially phosphorylates JNK1 on threonine. The mechanism that accounts for this selectivity has not been established. However, the different specificities of MKK4 and MKK7 in vitro suggest that these MAPKKs may collaborate to activate JNK1 in vivo (Enslen, 2000 and references therein).
MKK3 activates p38alpha MAPK, but not p38beta2 MAPK. Since MKK3 does not bind to p38alpha or p38beta2, the selectivity of MKK3 in activating p38alpha MAPK is not caused by a difference in docking interactions of MKK3 with p38alpha and p38beta2. Instead, these data suggest that molecular determinants within p38alpha (that are absent in p38beta2) contribute to recognition and activation by MKK3. Studies of chimeric p38alpha/beta2 MAPK indicate that the T-loop is a critical element that determines the extent of activation by MKK3. In contrast, these changes in the T-loop do not alter activation by MKK6. Since the T-loop contains the sites of threonine and tyrosine phosphorylation by MKK3, the finding that the T-loop contributes to the specificity of substrate phosphorylation by MKK3 is intriguing because it suggests that the T-loop may play a direct role in substrate recognition by the active site of MKK3. Previous studies have not indicated that the sequence of the T-loop plays an important role in determining the specificity of MAPK activation by MAPKK. Although an important role for the T-loop in substrate recognition by MKK3 has been established, the data do not exclude a role for other regions of the p38 MAPK in determining specificity (Enslen, 2000 and references therein).
Hypertonicity induces a group of genes that are responsible for the intracellular accumulation of protective organic
osmolytes such as sorbitol and betaine. Two representative genes are the aldose reductase enzyme (AR, EC
1.1.1.21), which is responsible for the conversion of glucose to sorbitol, and the betaine transporter (BGT1),
which mediates Na+-coupled betaine uptake in response to osmotic stress. The induction
of BGT1 mRNA in the renal epithelial Madin-Darby canine kidney cell line is inhibited by SB203580, a specific
p38 kinase inhibitor. The hypertonic induction of aldose reductase mRNA in HepG2
cells as well as the osmotic response element (ORE)-driven reporter gene expression in transfected HepG2 cells
are both inhibited by SB203580, suggesting that p38 kinase mediates the activation and/or binding of the
transcription factor(s) to the ORE. Electrophoretic gel mobility shift assays with cell extracts prepared from
SB203580-treated, hypertonically stressed HepG2 cells further show that the binding of trans-acting factors to the
ORE is prevented and is thus also dependent on the activity of p38 kinase. Similarly, treatment of hypertonically
stressed cells with PD098059, a mitogen-activated extracellular regulated kinase kinase (MEK1) inhibitor, results
in inhibition of the hypertonic induction of aldose reductase mRNA, ORE-driven reporter gene expression, and
the binding of trans-acting factors to the ORE. ORE-driven reporter gene expression is not affected by p38
kinase inhibition or MEK1 inhibition in cells incubated in iso-osmotic media. These data indicate that p38 kinase
and MEK1 are involved in the regulation of the hyperosmotic stress response (Nadkarni, 1999).
Through the action of its membrane-bound type I receptors, TGF-ß elicits a wide range of cellular responses that regulate cell proliferation, differentiation and apoptosis. Many of the signaling responses induced by TGF-ß are mediated by Smad proteins, but certain evidence has suggested that TGF-ß can also signal independently of Smads. In mouse mammary epithelial (NMuMG) cells, which respond to TGF-ß treatment in multiple ways, TGF-ß-induced activation of p38 MAP kinase is required for TGF-ß-induced apoptosis, epithelial-to-mesenchymal transition (EMT), but not growth arrest. Using a mutant type I receptor which is incapable of activating Smads but still retains the kinase activity, it was found that activation of p38 is independent of Smads. This mutant receptor is sufficient to activate p38 and cause NMuMG cells to undergo apoptosis. However, it is not sufficient to induce EMT. These results indicate that TGF-ß receptor signals through multiple intracellular pathways and provide first-hand biochemical evidence for the existence of Smad-independent TGF-ß receptor signaling (Yu, 2002).
In an attempt to identify new genes implicated in the control of programmed cell death during limb development, a cDNA library was generated from the regressing interdigital tissue of chicken embryos.
804 sequences were analzyed from this library and 23 genes were identified involved in apoptosis in different models. One of the genes that came up in the screening was the Bone Morphogenetic Protein family member Bmp5 that had not been previously found to be involved in the control of apoptosis during limb development. In agreement with a possible role in the control of cell death, Bmp5 exhibited a regulated pattern of expression in the interdigital tissue. Transcripts of Bmp5 and BMP5 protein were abundant within the cytoplasm of the fragmenting apoptotic interdigital cells in a way suggesting that delivery of BMPs into the tissue is potentiated during apoptosis. Gain-of-function experiments have demonstrated that BMP5 has the same effect as other interdigital BMPs inducing apoptosis in the undifferentiated mesoderm and growth in the prechondrogenic mesenchyme. Both Smad proteins and MAPK p38 have been characterized as intracellular effectors for the action of BMPs in the developing limb autopod. Activation of Smad signaling involves the receptor-regulated genes Smad1 and -8, and the inhibitory Smad6, and results in both the upregulation of gene transcription and protein phosphorylation with subsequent nuclear translocation. MAPK p38 is also quickly phosphorylated after BMP stimulation in the limb mesoderm. Treatment with the inhibitor of p38, SB203580, revealed that there are interdigital genes induced by BMPs in a p38-dependent manner (DKK, Snail and FGFr3), and genes induced in a p38-independent manner (BAMBI, Msx2 and Smads). Together, these results suggest that Smad and MAPK pathways act synergistically in the BMP pathway controlling limb development (Zuzarte-Luísa, 2004).
CD28 costimulation amplifies TCR-dependent signaling in activated T cells, however, the biochemical mechanism(s)
by which this occurs is not precisely understood. The small GTPase Rac-1 controls the catalytic activity of the
mitogen-activated protein kinases (MAPKs) and cell cycle progression through G1. Rac-1 activation requires the
phospho-tyrosine (p-Tyr)-dependent recruitment of the Vav GDP releasing factor (GRF) to the plasma membrane and
assembly of GTPase/GRF complexes, an event critical for Ag receptor-triggered T cell activation. TCR/CD28 costimulation synergistically induces Rac-1 GDP/GTP exchange. These findings, obtained by using
ZAP-70-negative Jurkat T cells, indicate that CD28 costimulation augments TCR-mediated T cell activation by
increasing the ZAP-70-mediated Tyr phosphorylation of Vav. This event regulates the Rac-1-associated GTP/GDP
exchange activity of Vav and downstream pathway(s) leading to PAK-1 and p38 MAPK activation. CD28 amplifies
TCR-induced ZAP-70 activity and association of Vav with ZAP-70 and linker for activation of T cells (LAT). These
results favor a model in which ZAP-70 regulates the intersection of the TCR and CD28 signaling pathways, which
elicit the coupling of TCR and CD28 to the Rac-1, PAK-1, and p38 MAPK effector molecules (Salojin, 1999).
T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28
costimulatory receptor. Using purified human CD4+ peripheral blood T cells, it has been shown that CD28 stimulation alone
activates p38 alpha mitogen-activated protein kinase (p38 alpha). Cell proliferation induced by CD28 stimulation
alone, a response attributed to CD4+CD45RO+ memory T cells, is blocked by the highly specific p38 inhibitors. In contrast, proliferation induced by anti-CD3
plus anti-CD28 mAbs is not blocked. Inhibitors of p38 also block CD4+ T cell production of IL-4 , but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-alpha, and IFN-gamma
production are also inhibited, but to a lesser degree than IL-4. IL-4 production is attributed to CD4+CD45RO+ T
cells, and its induction is suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB
203580 strongly inhibits IL-4 production by Th2 cells (IC50 = 10-80 nM), but only partially inhibits IFN-gamma
and IL-2 production by Th1 cells (<50% inhibition at 1 microM). In both Th1 and Th2 cells, CD28 signaling
activates p38 alpha and is required for cytokine production. These results show that p38 alpha plays an important
role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by
CD4+CD45RO+ T cells and Th2 effector cells suggests that p38 alpha may be important in the generation of
Th2-type responses in humans (Schafer, 1999).
Optimal T cell activation requires two signals: one generated by TCR and another by the CD28 costimulatory
receptor. The regulation of costimulation-induced mitogen-activated protein kinase
(MAPK) activation in primary mouse T cells has been investigated. In contrast to what has been reported for human Jurkat T cells, p38
MAPK, but not Jun NH2-terminal kinase (JNK), is found to be weakly activated upon stimulation with either anti-CD3 or
anti-CD28 in murine thymocytes and splenic T cells. However, p38 MAPK is activated strongly and synergistically
by either CD3/CD28 coligation or PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3/CD28-mediated
signaling. Activation of p38 MAPK correlates closely with the stimulation of T cell proliferation. In contrast,
PMA-induced JNK activation is inhibited by Ca2+ ionophore. T cell proliferation and production of IL-2, IL-4, and
IFN-gamma induced by both CD3 and CD3/CD28 ligation and the nuclear expression of the c-Jun and ATF-2
proteins are each blocked by the p38 MAPK inhibitor SB203580. These findings demonstrate that p38 MAPK (1) plays
an important role in signal integration during costimulation of primary mouse T cells; (2) may be involved in the
induction of c-Jun activation and augmentation of AP-1 transcriptional activity, and (3) regulates whether T cells enter
a state of functional unresponsiveness (Zhang,1999).
The expression of the c-jun proto-oncogene is rapidly induced in response to mitogens acting on a large variety of cell
surface receptors. The resulting functional activity of c-Jun proteins appears to be critical for cell proliferation. A large family of G protein-coupled receptors (GPCRs), represented by the m1
muscarinic receptor, can initiate intracellular signaling cascades that result in the activation of mitogen-activated
protein kinases (MAPK) and c-Jun NH2-terminal kinases (JNK). The activation of JNK but not of MAPK
correlates with a remarkable increase in the expression of c-jun mRNA. GPCRs can potently stimulate the activity of the c-jun promoter through MEF2 transcription factors,
which do not act downstream from JNK. In view of this, the nature of
the signaling pathway linking GPCRs to the c-jun promoter was investigated. Utilizing NIH 3T3 cells, it was found that GPCRs can
activate the c-jun promoter in a JNK-independent manner. These GPCRs can
elevate the activity of novel members of the MAPK family, including ERK5, p38alpha, p38gamma, and p38delta. The activation of certain kinases acting downstream from MEK5 (ERK5) and MKK6 (p38alpha and
p38gamma) is necessary to fully activate the c-jun promoter. Moreover, in addition to JNK, three other signaling proteins, ERK5, p38alpha, and
p38gamma, were found to stimulate the c-jun promoter by acting on distinct responsive elements. Taken together,
these results suggest that the pathway linking GPCRs to the c-jun promoter involves the integration of numerous
signals transduced by a highly complex network of MAPK, rather than resulting from the stimulation of a single
linear protein kinase cascade. These findings suggest that each signaling pathway affects one or more
regulatory elements on the c-jun promoter and that the transcriptional response most likely results from the temporal
integration of each of these biochemical routes (Marinissen, 1999).
Hemopoietic cytokines such as interleukin-3 and granulocyte colony-stimulating factor (G-CSF) are potent
activators of hemopoietic cell growth and strongly induce activation of extracellular signal-regulated kinase
(ERK), c-Jun-N-terminal kinase (JNK), and p38 mitogen-activated protein (MAP) kinases. However, the role of
these kinases is unclear. Using specific chemical inhibitors for MEK and p38, it has been demonstrated that both ERK
and p38 pathways are critically involved in the transduction of a proliferative signal and cooperate in
G-CSF-induced cell proliferation. Like ERK and JNK activation, activation of p38 and its
downstream substrate MAP kinase-activated protein kinase 2 by interleukin-3 or G-CSF requires Ras activation.
Two distinct cytoplasmic regions of the G-CSF receptor are involved in activation of the p38
pathway: a region within the 100 membrane-proximal amino acids is sufficient to induce low levels of p38 and
MAP kinase-activated protein kinase 2 activation, whereas the membrane-distal phosphorylation site Tyr763
mediates strong activation of these kinases. The levels of p38 activation correlate closely with those of Ras
activation by G-CSF, suggesting that the degree of Ras activation is a critical determinant for the extent of p38
activation by hemopoietic cytokines (Rausch, 1999).
The bZIP transcription factor ATF2 regulates gene expression in response to environmental changes. ATF2 binds its target promoter/enhancers as a homodimer or as a heterodimer with a restricted group of other bZip proteins, the most well known of which is the c-jun oncogene product. Upon exposure to cellular stresses, the mitogen-activated protein kinase (MAPK) cascades including SAPK/JNK and p38 can enhance ATF2's transactivating function through phosphorylation of Thr69 and Thr71. However, the mechanism of ATF2 activation by growth factors that are poor activators of JNK and p38 is still elusive. In fibroblasts, insulin, epidermal growth factor (EGF) and serum each activate ATF2 via a so far unknown two-step mechanism involving two distinct Ras effector pathways: the Raf-MEK-ERK pathway induces phosphorylation of ATF2 Thr71, whereas subsequent ATF2 Thr69 phosphorylation requires the Ral-RalGDS-Src-p38 pathway. Cooperation between ERK and p38 was found to be essential for ATF2 activation by these mitogens; the activity of p38 and JNK/SAPK in growth factor-stimulated fibroblasts is insufficient to phosphorylate ATF2 Thr71 or Thr69 + 71 significantly by themselves, while ERK cannot dual phosphorylate ATF2 Thr69 + 71 efficiently. These results reveal a so far unknown mechanism by which distinct MAPK pathways and Ras effector pathways cooperate to activate a transcription factor (Ouwens, 2002).
Sphingolipid products such as ceramide (cer), sphingosine (sph),
and sphingosine-1-phosphate (SPP) are implicated in the regulation of
cell growth and apoptosis. Cer, sph, and
SPP differentially modulate ionic events in cultured oligodendrocytes
(OLGs). Cer but not sph or SPP inhibits the inward rectifier
(IKir) in OLGs. To further
investigate the role of sphingolipid products in OLGs, the
effects of cer, sph, and SPP on OLG survival and on the regulation of
mitogen-activated protein kinases (MAPKs) were studied. Cer, sph, and
SPP differentially modulate OLG survival and activation of MAPK
members. Cer causes OLG apoptosis, sph causes OLG lysis, and SPP does
not affect OLG survival. Cer induces a preferential activation of
p38alpha, whereas sph and SPP induce a preferential activation of
extracellular signal-regulated kinase 2 (ERK2) in OLGs. In
addition, the effect of cer on p38alpha activity is mimicked by the
inhibition of IKir with
Ba2+. In contrast, exposure to cer results in
increased activity of ERK2 but not of p38alpha in astrocytes. Cer-induced
OLG apoptosis is attenuated by a p38 inhibitor, SB203580, and by
expression of a p38alpha dominant negative mutant. It is concluded that
p38alpha is the mediator in cer-induced OLG apoptosis and that
cer-induced IKir inhibition may contribute
to the sustained activation of p38alpha in OLGs (Hida, 1999).
This study demonstrates that cer, sph, and SPP differentially modulate OLG survival and the activation of MAPK members,
although these sphingolipid products are interconvertible. Cer is deacylated to form sph, which is then phosphorylated to form
SPP. The forward reactions are catalyzed by ceramidase and sph kinase, whereas the reverse reactions are catalyzed by
phosphatidate phosphohydrolase and cer synthase, respectively. The mechanisms involved in the regulation of cell growth and survival
by sphingolipid products are not completely understood. Cer and SPP cause OLG depolarization, whereas sph
elicits OLG hyperpolarization. Sph consistently induces Cai
increases in OLGs, whereas Cai
responses are observed infrequently with cer or SPP. In addition, inhibition of OLG IKir
underlies cer-induced depolarization but not SPP-induced depolarization. Both cer and SPP induce OLG
depolarization, yet OLG apoptosis is enhanced only by cer. This study asked whether downstream effectors such as MAPK members
play a role in determining whether conditions are permissive for
apoptotic stimuli. JNK and ERK2 are differentially regulated by
sphingolipid products in airway smooth muscle cells and rat mesangial
cells, supporting the concept that the dynamic balance between ERK2 and
JNK/p38 cascades is important in determining cell survival. Similar although not identical results were observed in OLGs. p38alpha
is activated by cer only, whereas ERK2 is activated by sph and SPP.
There is no difference in the JNK1 activity in cer-, sph-, and
SPP-treated OLGs. One interpretation would be that cer-induced
activation of p38alpha, but not of ERK2, is permissive to OLG apoptosis;
conversely, SPP-induced ERK2 activation, but not p38alpha activation, is
not permissive. In addition, the effect of cer on p38alpha
activity is mimicked by Ba2+, a known
IKir blocker, but not by high
K+, suggesting that
IKir inhibition rather than
depolarization per se is a contributory signal to the differential
activation of MAPK members. Failure of SPP to inhibit
IKir, despite its depolarizing action,
correlates with the absence of p38alpha induction and absence of
apoptosis in SPP-treated cells. Based on MAPK cascades activated by
sph, sph should not induce cell death in OLGs. However, sph also causes
sustained Cai increases in OLGs, which can lead
to cell death. Hence, the mechanisms underlying sph-induced OLG lysis and necrosis differ from those of cer-induced OLG
apoptosis (Hida, 1999 and references).
In general, JNK and p38 kinase pathways are considered key mediators of the inflammatory response and are activated by both Fas and TNF
receptor oligomerization or other stressful stimuli; however, their respective roles in apoptosis
remains controversial. The p38 subfamily consists of at least four
isoforms: p38alpha-delta. p38alpha (also known as p38) and p38beta, but not p38gamma and
p38delta, are inhibited by pyridinyl imidazole compounds, such as SB203580. Activation of p38alpha induces apoptosis in Jurkat
T cells and cardiac myocytes, whereas activation of p38beta inhibits
apoptosis or induces a hypertrophic response. Cer is shown to cause sustained activation of p38alpha
in OLGs; cer-induced apoptosis is inhibited by SB203580 and by p38alpha
dominant negative mutant, indicating that activated p38alpha mediates
cer-induced OLG apoptosis. In view of the uniform, modest activation of
JNK1 by cer, sph, and SPP, the role of JNK1 in cer-induced OLG
apoptosis in this study appears to be less significant than p38alpha.
Other stimuli that activate JNK in OLGs include NGF, TNF-alpha, IL, UV
light, and heat shock. Studies from Jurkat T cells and other cell lines suggest that
JNK activation is associated with apoptosis. But other investigators have stressed that
activation of JNK alone is not sufficient to induce apoptosis. Transfection with c-Jun dominant negative mutant or
with SEK1 dominant negative mutant protects neurons against
apoptosis induced by withdrawal of trophic factors and protects U937
cells against cer-induced apoptosis but does not protect Jurkat T cells or human breast carcinoma cells against Fas- or TNF-induced apoptosis. It is plausible that the role of
JNK1 versus p38alpha in apoptosis depends on the cell type and the
apoptotic trigger. In murine fibroblast cell line L9290cyt16, neither
JNK nor p38alpha appears to be required for Fas- or TNF-induced apoptosis (Hida, 1999 and references).
In contrast to p38alpha and JNK1, activation of ERK1/2 is generally
associated with cell proliferation or differentiation, depending on
whether activation is sustained or transient. ERK1 and ERK2 are activated by mitogenic factors (PDGF and basic
FGF) and phorbol esters in OLGs and progenitors. OLGs treated
with PD098059 have a limited number of processes, suggesting a role for
ERKs in process extension. ERK2 activity is transiently
enhanced by cer in astrocytes but not in OLGs, whereas p38alpha is
enhanced by cer in OLGs but not in astrocytes. These results are in
agreement with the concept that cell survival is regulated by opposing
actions of ERK and p38/JNK pathways. However, simultaneous activation of both ERK1/2
and p38 cascades appears to be required for maximal endotoxin-induced astroglial cell activation and for NGF-induced neuronal differentiation of PC12 cells. One
interpretation of the apparent discrepancy would be that the pattern of
activation of MAPK members is a crucial, but not the sole determinant
of cell survival, activation, and differentiation. Other important
factors that influence cell survival include Bcl2, BAD, and
other related mitochondrial proteins, intracellular glutathione content, and ionic fluxes (Hida, 1999 and references).
Cer has been shown to inhibits
IKir via a ras- and raf-1-dependent
pathway in cultured OLGs. Yet, cer activates
p38alpha instead of ERK2. The experiments with Ba2+ indicate that
IKir inhibition may contribute to
cer-induced activation of p38alpha and perhaps prevent an increase in
ERK2 activity as well. A working model whereby proinflammatory
cytokines, hypoxia, or other apoptotic stimuli lead to OLG apoptosis is presented. Although cer-induced
IKir inhibition and increased p38alpha
activity may constitute two simultaneous independent signals required
for OLG apoptosis, it is proposed that
IKir inhibition contributes to the
cer-induced sustained activation of p38alpha, perhaps via activation of
caspases. Cer-induced IKir inhibition,
by reducing K+ influx, leads to diminished
[K+]i, a condition
linked to caspase activation and apoptosis. Interleukin 1beta-converting enzyme
(ICE) family proteases are required for activation of p38 by Fas
but not by sorbitol or etoposide. These studies
support the concept that sphingomyelin cycle is an important regulator
of cell survival and that the ultimate cellular outcome depends on the
integration of multiple signals, including activation of MAPK members
and modulation of ionic events at the plasma membrane (Hida, 1999 and references).
14-3-3 family members are dimeric phosphoserine-binding proteins that participate in signal transduction and checkpoint control
pathways. In this work, dominant-negative mutant forms of 14-3-3 were used to disrupt 14-3-3 function in cultured cells and in
transgenic animals. Transfection of cultured fibroblasts with the R56A and R60A double mutant form of 14-3-3zeta
(DN-14-3-3zeta) inhibits serum-stimulated ERK MAPK activation, but increases the basal activation of JNK1 and p38
MAPK. Fibroblasts transfected with DN-14-3-3zeta exhibit markedly increased apoptosis in response to UVC irradiation that is blocked by
pre-treatment with a p38 MAPK inhibitor, SB202190. Targeted expression of DN-14-3-3eta to murine postnatal cardiac tissue increases the basal
activation of JNK1 and p38 MAPK, and affects the ability of mice to compensate for pressure overload, which results in increased mortality, dilated
cardiomyopathy and massive cardiomyocyte apoptosis. These results demonstrate that a primary function of mammalian 14-3-3 proteins is to inhibit apoptosis (Xing, 2000).
Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and
excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP)
kinase in neurons. In the present study, it was determined if NO-induced cell death in neurons is dependent on p38 MAP kinase activity.
Sodium nitroprusside (SNP), a NO donor, elevates caspase activity and induces death in human SH-SY5Y neuroblastoma cells and
primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminishes caspase induction
and protects SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor
zVAD-fmk does not provide significant protection. A role for p38 MAP kinase is further substantiated by the observation that SB203580 blocks translocation of
the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct
activator of p38 MAP kinase promotes Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons are resistant to SNP, further
demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death
in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway (Ghatan, 2000).
Oxidative stress is implicated in the nerve cell death that occurs in a variety of neurological disorders, and the loss of protein kinase C (PKC) activity has been coupled to the severity of the damage. The functional relationship between stress, PKC, and cell death is, however, unknown. Using an immortalized hippocampal cell line that is particularly sensitive to oxidative stress, it has been shown that activation of PKC by the phorbol ester tetradecanoylphorbol acetate (TPA) inhibits cell death via the stimulation of a
complex protein phosphorylation pathway. TPA treatment leads to the rapid activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), the inactivation of p38 mitogen-activated protein kinase (MAPK), and the downregulation of PKCdelta. Inhibition of either ERK or JNK activation blocks TPA-mediated protection, whereas p38 MAPK and PKCdelta inhibitors block stress-induced nerve cell death. Both p38 MAPK inactivation and JNK activation appear to be downstream of ERK because an agent that blocks ERK activation also blocks the modulation of these other MAP kinase family members by TPA treatment. Thus, the protection from oxidative stress afforded nerve cells by PKC activity requires the combined modulation of multiple enzyme pathways and suggests why the loss of PKC activity contributes to nerve cell death (Maher, 2001).
Axon guidance cues trigger rapid changes in protein dynamics in retinal growth cones: netrin-1 stimulates both protein synthesis and degradation, while Sema3A elicits synthesis, and LPA induces degradation. What signaling pathways are involved? These studies confirm that p42/44 MAPK mediates netrin-1 responses and further show that inhibiting its activity blocks cue-induced protein synthesis. Unexpectedly, p38 MAPK is also activated by netrin-1 in retinal growth cones and is required for chemotropic responses and translation. Sema3A- and LPA-induced responses, by contrast, require a single MAPK, p42/p44 and p38, respectively. In addition, caspase-3, an apoptotic protease, is rapidly activated by netrin-1 and LPA in a proteasome- and p38-dependent manner and is required for chemotropic responses. These findings suggest that the apoptotic pathway may be used locally to control protein levels in growth cones and that the differential activation of MAPK pathways may underlie cue-directed migration (Campbell, 2003).
These data provide evidence for the presence of caspases in growth cones and identify caspase-3 as a potential target of p38 signaling for mediating both netrin-1-induced turning and LPA-induced growth cone collapse. This suggests that, in addition to their roles in apoptosis, caspase-induced protein degradation may play a role in growth cone guidance. Previous studies identified the netrin receptor DCC in regulating cell survival via the activation of caspase-3 by caspase-9 in the absence of netrin-1 in human embryonic kidney 293T cells. By contrast, in Xenopus retinal growth cones, netrin-1 and LPA induce the rapid activation of caspase-3 independent of caspase-9 via the MAPK- and proteasome-mediated proteolysis pathways. The activation of caspase-3 in the confined cellular compartment of the growth cone might not lead to activation of the full apoptotic cascade and cell death but rather to transient, localized changes in specific proteins. The p42/p44 and PI-3 kinase pathways identified in netrin-1 signaling are known to play roles in mediating cell survival and may ensure tight regulation of caspase activity in the growth cone. A role has been identified for caspases in synaptic plasticity independent of their roles in cell death (Campbell, 2003 and references therein).
Since caspases are proteases, a key question asks which proteins do caspases degrade? Candidate proteins include known caspase substrates, such as actin, actin binding proteins, and signal transduction pathway components. For example, gelsolin, an actin severing protein, is present in growth cones and is activated by caspase-3-mediated cleavage. Netrin-1 and LPA stimulate the rapid caspase-3-dependent cleavage of PARP. In addition to its role in maintaining genomic stability, PARP is able to interact with and activate proteasome-mediated proteolysis. Cleavage of PARP may inactivate itself, providing a possible mechanism by which proteasome-mediated proteolysis may be regulated in the case of netrin-1 and LPA. The netrin-1 receptor DCC is itself a substrate of caspase-3, and caspase-mediated cleavage of DCC may potentially be involved in mediating netrin-1-induced chemotropic responses. During apoptosis, caspase-3 is also able to cleave eukaryotic initiation factor 4G (eIF-4G), a crucial protein required for binding cellular mRNA to ribosomes. This may decrease the rate of translation and provide a possible mechanism for negative regulation of netrin-1-stimulated protein synthesis in growth cones. Since the chemotropic responses of growth cones elicited by netrin-1 and LPA are essentially blocked by inhibition of caspase-3, it is likely that of the caspases, caspase-3 plays a major role in these processes (Campbell, 2003 and references therein).
The ubiquitin-proteasome system is critically involved in apoptosis and in mediating chemotropic responses of growth cones. In neuronal cells, proteasome inhibitors protect against apoptosis by acting upstream of caspase activation. These results have revealed a parallel in retinal growth cones where the activation or cleavage of caspase-3 in response to netrin-1 and LPA requires proteasome function, suggesting that caspase-mediated protein degradation lies downstream of proteasome/ubiquitin-mediated proteolysis. Candidate proteins to undergo proteasome/ubiquitin-mediated proteolysis include the inhibitor of apoptosis (IAP) family of proteins, degradation of which can result in caspase activation. IAPs can also target caspase-3 itself for proteasome/ubiquitin-mediated proteolysis, suggesting a possible mechanism for the transient and localized nature of caspase-3 activation in growth cones (Campbell, 2003 and references therein).
The mitogen-activated protein kinase p38 plays a critical role in inflammation, cell cycle progression, differentiation, and apoptosis. The activity of p38 is stimulated by a variety of extracellular stimuli, such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), and subjected to regulation by other intracellular signaling pathways, including the cyclic AMP (cAMP) pathway. Yet the underlying mechanism by which cAMP inhibits p38 activation is unknown. This study shows that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of p38 activation. cAMP inhibits p38 activation via the protein kinase A-CREB pathway. The inhibition is mediated by the CREB target gene Dlc, whose protein product, DLC, interferes with the formation of the MKK3/6-p38 complex, thereby suppressing p38 phosphorylation activation by MKK3/6. The inhibition of p38 activation by cAMP leads to suppression of NF-kappaB activity and promotion of apoptosis in response to TNF-alpha. Thus, these results identify DLC as a novel inhibitor of the p38 pathway and provide a molecular mechanism by which cAMP suppresses p38 activation and promotes apoptosis (Zhang, 2006).
The non-canonical Wnt/cyclic GMP/Ca2+/NF-AT pathway operates via Frizzled-2, a member of the superfamily of G protein-coupled receptors. In scanning for signaling events downstream of the Frizzled-2/Gαt2/PDE6 triad activated in response to Wnt5a, a strong activation of the mitogen-activated protein kinase p38 was observed in mouse F9 teratocarcinoma embryonal cells. The activation of p38 is essential for NF-AT transcriptional activation mediated via Frizzled2. Wnt5a-stimulated p38 activation is rapid, sensitive to pertussis toxin, to siRNA against either Gαt2 or p38α, and to the p38 inhibitor SB203580. Real-time analysis of intracellular cyclic GMP using the Cygnet2 biosensor revealed p38 to act at the level of cyclic GMP, upstream of the mobilization of intracellular Ca2+. Fluorescence resonance energy transfer (FRET) imaging reveals the changes in cyclic GMP in response to Wnt5a predominate about the cell membrane, and likewise sensitive to either siRNA targeting p38 or to treatment with SB203580. Dishevelled is not required for Wnt5a activation of p38; siRNAs targeting Dishevelleds and expression of the Dishevelled antagonist Dapper-1 do not suppress the p38 response to Wnt5a stimulation. These novel results are the first to detail a Dishevelled-independent Wnt response, demonstrating a critical role of the mitogen-activated protein kinase p38 in regulating the Wnt non-canonical pathway (Ma, 2007).
This study reveals a novel role of the p38 MAPK in the Wnt/cyclic GMP/Ca2+/NF-AT transcriptional activation pathway mediated by Frizzled-2. Activation of the non-canonical Wnt/Ca2+ pathway promotes ventral cell fate in the Xenopus embryo. Wnt5a stimulates phosphatidylinositol signaling and Ca2+ transients that are essential to normal development in the zebrafish embryo. Mouse embryonic F9 cells were employed to probe the role of p38 MAPK in the signal linkage map from a proximal step (i.e. activation of Frizzled-2) downstream to the activation of the developmentally regulated, luciferase reporter gene sensitive to NF-AT. The results from these studies provided several key and novel insights about Wnt signaling in the non-canonical pathway (Ma, 2007).
First, although MAPK family members have been implicated in Wnt signaling, the current study is the first report to identify p38 MAPK as downstream in a Wnt-sensitive pathway. Earlier studies of the planar cell polarity pathway in Drosophila and Wnt pathways regulating convergent extension in vertebrate demonstrate the activation of N-terminal c-Jun protein kinase, JNK. Erk1/2 MAPK have not yet been implicated in Wnt signaling, but it is likely that cross-talk must exist between Wnt-sensitive pathways and the MAPK cascade of downstream signaling. For the Wnt5a/cyclic GMP/Ca2+/NF-AT-sensitive transcription pathway, p38 not only regulates the signaling, but is essential for the overall function of the pathway from Wnt5a to the activation of NF-AT (Ma, 2007).
Second, the activation of p38 by Wnt5a feeds into the Wnt5a/cyclic GMP/Ca2+/NF-AT pathway at the level of cyclic GMP, upstream of Ca2+ mobilization. The ability of Wnt5a to activate p38 MAPK itself is not sensitive to the elevation of intracellular cyclic GMP by addition of 8-bromo-cyclic GMP or by inhibition of PDE6 with zaprinast. Furthermore, inhibiting PKG activity does not alter the ability of Wnt5a to activate p38. What is clear is that inhibition of p38 MAPK interrupts the signaling of this pathway at the level of cyclic GMP. This important information was deduced both by read-outs of direct cyclic GMP measurement, as a reflection of PKG activity, and in live cells, making use of the Cygnet2.1 biosensor for cyclic GMP. Current understanding of how p38 MAPK modulates cGMP levels is not complete. Experimental results provide a line of evidence indicating that p38 MAPK is necessary for the PDE6 activation in response to Wnt5a. Although Wnt5a stimulation leads to the activation of Gαt and PDE6, mimicking the pathway in the visual system, the mechanism by which p38 MAPK regulates the PDE6 is not clear (Ma, 2007).
Third, the activation of p38 appears to operate via two interacting signaling paradigms, a GPCR cascade and a traditional MAPK cascade. The Fz2/Gαt2/PDE6 triad operates down to the level of NF-AT-sensitive transcriptional activation, while the MEKK/MKK/MAPK cascade culminates in activation of p38, which is also required for the activation of NF-AT. This configuration has marked similarities to the Fz1-mediated regulation of planar cell polarity, operating in mammals and in flies through Fz1/Gαo/Dvl and downstream to a MEKK/MKK/JNK cascade. Thus GPCRs relay information from Wnt ligands to G proteins and their cognate effectors downstream to MEKKs that control MAPKs and the activity of transcription factors (Ma, 2007).
Finally, this study reveals for the first time the operation of a Wnt-sensitive signaling pathway that to the level of the effecter, p38, operates independent of the phosphoprotein Dvl. Knock-down of Dvl1, Dvl2, Dvl3, or the expression of the Dvl inhibitor Dapper-1 has no effect on the ability of Wnt5a to activate p38, although the signaling to the level of NF-AT does. Taken together, these novel observations reveal an essential role of p38 MAPK in Wnt-sensitive signaling via the non-canonical pathway (Ma, 2007).
The Wnt-β-catenin canonical signaling pathway is crucial for normal embryonic development, and aberrant expression of components of this pathway results in oncogenesis. Upon scanning for the mitogen-activated protein kinase (MAPK) pathways that might intersect with the canonical Wnt-β-catenin signaling pathway in response to Wnt3a, a strong activation of p38 MAPK was observed in mouse F9 teratocarcinoma cells. Wnt3a-induced p38 MAPK activation was sensitive to siRNAs against Gαq or Gαs, but not against either Gαo or Gα11. Activation of p38 MAPK is critical for canonical Wnt-β-catenin signaling. Chemical inhibitors of p38 MAPK (SB203580 or SB239063) and expression of a dominant negative-version of p38 MAPK attenuate Wnt3a-induced accumulation of β-catenin, Lef/Tcf-sensitive gene activation, and primitive endoderm formation. Furthermore, epistasis experiments pinpoint p38 MAPK as operating downstream of Dishevelleds. It was also demonstrated that chemical inhibition of p38 MAPK restores Wnt3a-attenuated GSK3β kinase activity. The involvement of G-proteins and Dishevelleds in Wnt3a-induced p38 MAPK activation was demonstrated, highlighting a critical role for p38 MAPK in canonical Wnt-β-catenin signaling (Bikkavilli, 2008).
Synaptic activity induces changes in the number of dendritic spines. This study reports a pathway of regulated endocytosis triggered by arcadlin, a protocadherin induced by electroconvulsive and other excitatory stimuli in hippocampal neurons. The homophilic binding of extracellular arcadlin domains activates TAO2β, a splice variant of the thousand and one amino acid protein kinase 2, cloned in this study by virtue of its binding to the arcadlin intracellular domain. TAO2β is a MAPKKK that activates the MEK3 MAPKK, which phosphorylates the p38 MAPK. Activation of p38 feeds-back on TAO2β, phosphorylating a key serine required for triggering endocytosis of N-cadherin at the synapse. Arcadlin knockout increases the number of dendritic spines, and the phenotype is rescued by siRNA knockdown of N-cadherin. This pathway of regulated endocytosis of N-cadherin via protocadherin/TAO2β/MEK3/p38 provides a molecular mechanism for transducing neuronal activity into changes in synaptic morphologies (Yasuda, 2007).
Continues: p38b Evolutionary homologs part 2/3 | part 3/3 |
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