Table of contents

Activation of MAP kinase pathway by G proteins

The Gi class of heterotrimeric G proteins has been implicated in transmitting mitogenic signals from a variety of seven-transmembrane domain receptors. In addition, the alpha subunit of Gi2 (alpha i2) is oncogenic when mutated to a constitutively active form (gip2). The mechanism by which Gi2 stimulates cellular proliferation is unknown, but is believed to involve activation of the mitogen-activated protein kinase (MAPK) signaling cascade. To study Gi2 activation of the cascade, a mutant (pertussis toxin [PTX]-resistant alpha i2) was expressed in Chinese hamster ovary cells. After PTX treatment of these cells, Gi-coupled receptors specifically activated PTX-resistant Gi2 without activating other Gi proteins. Receptor-mediated activation of Gi2 leads to activation of MAPK and its upstream activator, MAPK/ERK-activating kinase (MEK). Activation of MAPK and MEK by Gi2 is blocked by expression of a dominant-negative mutant of Ras. Gi2 activation does not, however, detectably increase the proportion of Ras protein in the GTP-bound form. Additional experiments suggest that Gi2 stimulates the MAPK pathway, at least in part, by mechanisms that involve release of its beta gamma subunit, as well as activation of phosphatidylinositol-3 kinase (Pace, 1995).

Activation of 44 and 42 kDa extracellular signal-regulated kinases (ERK)1/2 by angiotensin II (angII) plays an important role in vascular smooth muscle cell (VSMC) function. The dual specificity mitogen-actived protein (MAP) kinase/ERK kinase (MEK) activates ERK1/2 in response to angII, but the MEK activating kinases remain undefined. Raf is a candidate MEK kinase. However, a kinase other than Raf appears responsible for angII-mediated signal transduction because treatment with phorbol 12, 13-dibutyrate (PDBU) for 24 h completely blocks Raf-Ras association in VSMC but does not inhibit activation of MEK and ERK1/2 by angII. It is thought that an atypical protein kinase C (See Drosophila PKC) isoform, which lacks a phorbol ester binding domain, mediates ERK1/2 activation by angII. All isoforms except PKC-zeta are down-regulated by PDBU for 24 h suggesting that PKC-zeta is responsible for angII-mediated ERK1/2 activation. In response to angII, PKC-zeta associates with Ras as shown by co-precipitation of PKC-zeta with anti-H-Ras antibody. To characterize further the role of PKC-zeta, PKC-zeta protein was depleted specifically by transfection with antisense PKC-zeta oligonucleotides. Antisense PKC-zeta oligonucleotide treatment significantly decreases PKC-zeta protein expression (without effect on other PKC isoforms) and angII-mediated ERK1/2 activation in a concentration-dependent manner. These results demonstrate an important difference in signal transduction by angII compared with PDGF and phorbol ester in VSMC, and suggest a critical role for PKC-zeta and Ras in angII stimulation of ERK1/2 (Liao, 1997).

Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS-dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS-independent manner in CHO cells that overexpress a dominant-negative mutant of the guanine nucleotide exchange protein SOS (CHO-DeltaSOS cells). LPA also induces the activation of MAP kinase kinase (MEK), but not of RAF1, in CHO-DeltaSOS cells. The RAS-independent activation of MAP kinase by LPA is blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) or by overexpression of a dominant-negative mutant of the gamma isoform of PI3K. Furthermore, LPA induces the activation of the atypical zeta isoform of protein kinase C (PKC-zeta) in CHO-DeltaSOS cells in a manner that is sensitive to wortmannin or to the dominant-negative mutant of PI3Kgamma, and overexpression of a dominant-negative mutant of PKC-zeta inhibits LPA-induced activation of MAP kinase. PKC-zeta may directly activate MEK. These observations indicate that Gi protein-coupled receptors induce activation of MEK and MAP kinase through a RAS-independent pathway that involves PI3Kgamma-dependent activation of atypical PKC-zeta (Takeda, 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).

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin [5-hydroxytryptamine (5-HT)] 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation is attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation. This is because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src, but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase have no effect on 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis is required for 5-HT1A receptor-mediated Erk1/2 activation, a role for CAM in receptor endocytosis is postulated. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuates 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevents agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. It is concluded that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects CAM's role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2 (Della Rocca, 1999).

Muscarinic acetylcholine receptor (mAChR), a member of the G-protein-coupled receptors (GPCRs) gene superfamily, has been shown to mediate the effects of acetylcholine on differentiation and proliferation in the CNS. However, the mechanism or mechanisms whereby mAChRs regulate cell proliferation remain poorly understood. In vitro bFGF-expanded neural progenitor cells dissociated from rat cortical neuroepithelium express muscarinic acetylcholine receptor subtype mRNAs. Stimulation of these mAChRs with carbachol, a muscarinic agonist, activates extracellular-regulated kinases (Erk1/2) and phosphatidylinositol-3 kinase (PI-3K). This, in turn, stimulates DNA synthesis in neural progenitor cells. MEK inhibitor PD98059 and PI-3K inhibitors wortmannin and LY294002 inhibit a carbachol-induced increase in DNA synthesis. These findings indicate that the activation of both PI-3 kinase and MEK signaling pathways via muscarinic receptors is involved in stimulating DNA synthesis in the neural progenitor cells during early neurogenesis (Bing-Sheng, 2001).

In C. elegans, the RAS/MAPK pathway is used in different tissues to regulate various cell fate decisions. Several positive and negative regulators tightly control the activity of the RAS/MAPK pathway at different steps. A link is demonstrated between a G-protein-coupled receptor signaling pathway and the RAS/MAPK cascade. SRA-13, a member of the SRA family of chemosensory receptors, negatively regulates RAS/MAPK signaling during vulval induction and the olfaction of volatile attractants. Epistasis analysis indicates that SRA-13 inhibits the RAS/MAPK pathway at the level or upstream of MAPK. In both tissues, the vulval precursor cells and the chemosensory neurones, SRA-13 acts through the GPA-5 Galpha protein subunit, suggesting a common mechanism of crosstalk. Moreover, vulval induction is repressed by food withdrawal during larval development and SRA-13 activity is required for the suppression of vulval induction in response to food starvation. Thus, SRA-13 may serve to adapt the activity of the RAS/MAPK pathway to environmental conditions (Battu, 2003).

Activation of 7TM receptors typically causes their phosphorylation with consequent arrestin binding and desensitization. Arrestins also act as scaffolds, mediating signaling to Raf and ERK and, for some receptors, inhibiting nuclear translocation of ERK. GnRH receptors act via Gq/11 to stimulate the PLC/Ca2+/PKC cascade and the Raf/MEK/ERK cassette. Uniquely, type I mammalian GnRHRs lack the C-tails that are found in other 7TM receptors (including non-mammalian GnRHRs) and are implicated in arrestin binding. This study compares ERK signaling by human (h) and Xenopus (X) GnRHRs. In HeLa cells XGnRHRs undergo rapid and arrestin-dependent internalization and cause arrestin/GFP translocation to the membrane and endosomes, whereas hGnRHRs do not. Internalized XGnRHRs co-localize with arrestin/GFP, whereas hGnRHRs do not. Both receptors mediate transient ERK phosphorylation and nuclear translocation (revealed by immunohistochemistry or by imaging of co-transfected ERK2/GFP) and for both, ERK phosphorylation is reduced by PKC inhibition but not by inhibiting EGF receptor autophosphorylation. In the presence of PKC inhibitor, Darrestin(319-418) blocks XGnRHR-mediated, but not hGnRHR-mediated, ERK phosphorylation. When receptor number is varied, hGnRHRs activate PLC and ERK more efficiently than XGnRHRs, but are less efficient at causing ERK2/GFP translocation. At high receptor number, XGnRHRs and hGnRHRs both cause ERK2/GFP translocation to the nucleus but at low receptor number XGnRHRs cause ERK2/GFP translocation whereas hGnRHRs do not. Thus, experiments with XGnRHRs have revealed the first direct evidence of arrestin-mediated (likely G protein-independent) GnRHR signalling, whereas those with hGnRHRs imply that scaffolds other than arrestins can determine GnRHR effects on ERK compartmentalization (Caunt, 2005).

Beta-arrestin mediates desensitization and internalization of ß-adrenergic receptors (ßARs), but also acts as a scaffold protein in extracellular signal-regulated kinase (ERK) cascade. Thus, the role of ß-arrestin2 was examined in the ßAR-mediated ERK signaling pathways. Isoproterenol stimulation equally activates cytoplasmic and nuclear ERK in COS-7 cells expressing ß1AR or ß2AR. However, the activity of nuclear ERK is enhanced by co-expression of ß-arrestin2 in ß2AR- (but not ß1AR-) expressing cells. Pertussis toxin treatment and blockade of Gßgamma action inhibits ß-arrestin2-enhanced nuclear activation of ERK, suggesting that ß-arrestin2 promotes nuclear ERK localization in a Gßgamma dependent mechanism upon receptor stimulation. ß2AR containing the carboxyl terminal region of ß1AR has lost the ß-arrestin2-promoted nuclear translocation. Since the carboxyl terminal region is important for ß-arrestin binding, these results demonstrate that recruitment of ß-arrestin2 to carboxyl terminal region of ß2AR is important for ERK localization to the nucleus (Kobayashi, 2005).

Physiological effects of ß adrenergic receptor (ß2AR) stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. A novel signaling mechanism has been demonstrated wherein ß-arrestins mediate ß2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the ß2AR expressed in HEK-293 cells has been resolved into two components dependent, respectively, on Gs-Gi/protein kinase A (PKA) or ß-arrestins. G protein-dependent activity is rapid, peaking within 2-5 min, is quite transient, is blocked by pertussis toxin (Gi inhibitor) and H-89 (PKA inhibitor), and is insensitive to depletion of endogenous ß-arrestins by siRNA. ß-Arrestin-dependent activation is slower in onset (peak 5-10 min), less robust, but more sustained and shows little decrement over 30 min. It is insensitive to pertussis toxin and H-89 and sensitive to depletion of either ß-arrestin1 or -2 by small interfering RNA. In Gs knock-out mouse embryonic fibroblasts, wild-type ß2AR recruited ß-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel ß2AR mutant (ß2ART68F,Y132G,Y219A or ß2ARTYY), rationally designed based on Evolutionary Trace analysis, is incapable of G protein activation but can recruit ß-arrestins, undergo ß-arrestin-dependent internalization, and activate ß-arrestin-dependent ERK. Interestingly, overexpression of GRK-5 or -6 increases mutant receptor phosphorylation and ß-arrestin recruitment, leads to the formation of stable receptor-ß-arrestin complexes on endosomes, and increases agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gßgamma release upon G protein activation, is ineffective unless it is constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the ß2AR can signal to ERK via a GRK5/6-ß-arrestin-dependent pathway, which is independent of G protein coupling (Shenoy, 2006).

Regulation of MAPK by phosphatases

Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing phosphotyrosine, dsPTPs can hydrolyze phosphoserine/threonine-containing substrates and have been shown to dephosphorylate activated MAPK. A novel dsPTP from rat hippocampus, rVH6, contains the conserved dsPTP active site sequence, VXVHCX2GX2RSX5AY(L/I)M, and exhibits phosphatase activity against activated MAPK. In PC12 cells, rVH6 mRNA is induced during nerve growth factor-mediated differentiation but not during insulin or epidermal growth factor mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly expressed in proliferating cells and declines rapidly during differentiation. rVH6 expression correlates with the inability of fibroblast growth factor to stimulate MAPK activity in proliferating but not in differentiating MM14 cells. rVH6 protein localizes to the cytoplasm and is the first dsPTP to be localized outside the nucleus. This novel subcellular localization may expose rVH6 to potential substrates that differ from nuclear dsPTPs substrates (Mourey, 1996).

Mitogen-activated protein (MAP) kinases can be grouped into three structural families: ERK, JNK, and p38. Each of these kinases are thought to carry out unique functions within cells. ERK, JNK, and p38 are activated by distinct combinations of stimuli in T cells that simulate full or partial activation through the T cell receptor. These kinases are regulated by reversible phosphorylation on Tyr and Thr. The dual specific phosphatases PAC1 and MKP-1 previously have been implicated in the respective in vivo inactivation of ERK or of ERK and JNK. A new MAP kinase phosphatase, MKP-2, is induced in human peripheral blood T cells with phorbol 12-myristate 13-acetate, and expressed in a variety of nonhematopoietic tissues as well. PAC1 recognizes ERK and p38; MKP-2 recognizes ERK and JNK, and MKP-1 recognizes ERK, p38, and JNK. Thus, individual MAP kinase phosphatases can differentially regulate the potential for cross-talk between the various MAP kinase pathways. A hyperactive allele of ERK2, analogous to the Drosophila sevenmaker gain-of-function mutation of rolled, has significantly reduced sensitivity to all three MAP kinase phosphatases in vivo (Chu, 1996).

PAC-1, an early-response gene originally identified in activated T cells, encodes a dual-specificity mitogen-activated protein kinase phosphatase. PAC-1 mRNA levels rapidly increase in mitogen-stimulated lymphocytes, with the induced expression being transient in B cells but sustained in activated T cells. In T cells, sequences necessary for basal and induced transcription reside within a 200-bp region located immediately upstream of the transcription initiation sites. Basal transcription is regulated in part by an E-box element. PAC-1 transcription induced by phorbol stimulation and the expression of the v-ras or v-raf oncogene is mediated via the E-box motif and an AP-2-related site. The ability of an interfering ERK-2 mutant to block phorbol and v-ras-dependent PAC-1 transcription indicates that mitogen-activated protein kinase activation is necessary for these stimuli to induce transcription of the PAC-1 gene in T cells (Grumont, 1996).

SH-PTP2, the vertebrate homolog of Drosophila corkscrew, associates with several activated growth factor receptors, but its biological function is unknown. The effects of wild-type and mutant SH-PTP2 RNA were examined on Xenopus embryogenesis. An internal phosphatase domain deletion (delta P) acts as a dominant negative mutant, causing severe posterior truncations. This phenotype is rescued by SH-PTP2, but not by the closely related SH-PTP1. In ectodermal explants, delta P blocks fibroblast growth factor (FGF)- and activin-mediated induction of mesoderm and FGF-induced mitogen-activated protein (MAP) kinase activation. These results indicate that SH-PTP2 is required for early vertebrate development, acting as a positive component in FGF signaling downstream of the FGF receptor and upstream of MAP kinase (Tang, 1995).

UV irradiation induces apoptosis in U937 human leukemic cells that is accompanied by the activation of both the stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK) signal transduction pathways. The MAPK phosphatase, MKP-1, is capable of inactivating both SAPK and p38 MAPK in vivo. To determine whether MKP-1-mediated inhibition of SAPK and/or p38 MAPK activity provides cytoprotection against UV-induced apoptosis, a U937 cell line conditionally expressing MKP-1 from the human metallothionein IIa promoter was established. Conditional expression of MKP-1 abolishes UV-induced SAPK and p38 MAPK activity, and inhibits UV-induced apoptosis as judged by both morphological criteria and DNA fragmentation. MKP-1 inhibits other biochemical events associated with apoptosis, including activation of caspase-3 and the proteolytic cleavage of the caspase-3 substrate, poly(ADP ribose) polymerase. These findings demonstrate that MKP-1 acts at a site upstream of caspase activation within the apoptotic program. The cytoprotective properties of MKP-1 do not appear to be mediated by its ability to inhibit p38 MAPK because the p38 MAPK specific inhibitor SB203580 has no effect on UV-induced apoptosis in U937 cells. By titrating the level of MKP-1 expression, it was found that MKP-1 inhibits UV-induced SAPK activity, DNA fragmentation, and caspase-3 activation in a similar dose-dependent manner. The dual-specificity phosphatase, PAC1, which does not inhibit the UV-induced activation of SAPK, does not provide a similar cytoprotection against UV-induced apoptosis. These results are consistent with a model whereby MKP-1 provides cytoprotection against UV-induced apoptosis by inhibiting UV-induced SAPK activity (Franklin, 1998).

The control of cell division in response to mitogens is mediated at least in part by MAP kinase (MAPK) signaling pathways. The p42MAPK and p44MAPK enzymes [extracellular signal-regulated kinase (ERK)-2 and ERK-1] are activated in cells stimulated with mitogens, by phosphorylation on threonine and tyrosine residues within protein kinase subdomain VIII, mediated by a class of MAP kinase (or ERK) kinases typified by MEK1. Inhibition of p42MAPK and p44MAPK blocks cell-cycle reentry and is principally mediated in vivo by members of a family of dual specificity phosphatases, of which MAP kinase phosphatase (MKP-1, also called 3CH134, CL100, or erp) is archetypal. At least nine distinct MKP family members have been cloned; most (if not all of them) are the products of immediate early genes and therefore under tight transcriptional control. MKP-1, MKP-2, and MKP-3 are transiently synthesized after activation of p42MAPK and p44MAPK, suggesting the presence of a negative feedback loop to regulate p42MAPK and p44MAPK. To determine whether expression of the MKP-1 protein is also subject to transcriptional control, the half-life of MKP-1 in CCL39 fibroblast cells was determined. MKP-1 is barely detectable in quiescent CCL39 fibroblasts, but its expression level is increased in cells stimulated with mitogens. Its half-life is on the order of 45 min (Brondello, 1999).

The mitogen-activated protein (MAP) kinase cascade is inactivated at the level of MAP kinase by members of the MAP kinase phosphatase (MKP) family, including MKP-1. MKP-1 is a labile protein in CCL39 hamster fibroblasts; its degradation is attenuated by inhibitors of the ubiquitin-directed proteasome complex. MKP-1 is a target in vivo and in vitro for p42MAPK or p44MAPK, which phosphorylates MKP-1 on two carboxyl-terminal serine residues, Serine 359 and Serine 364. This phosphorylation does not modify MKP-1's intrinsic ability to dephosphorylate p44MAPK but leads to stabilization of the protein. Thus p42MAPK and p44MAPK enzymes have a central role in the capacity of cells to divide in response to growth factors. Activation of p42MAPK and p44MAPK is a prerequisite for cell-cycle reentry. However, inappropriate or constitutive activation of the p42MAPK or p44MAPK cascade may provoke cellular senescence. Taken together, these findings illustrate a complex control mechanism designed to limit undesirable long-term activation of p42MAPK and p44MAPK and further demonstrate the importance of regulated protein degradation to the control of cell division processes. These results illustrate the importance of regulated protein degradation in the control of mitogenic signaling (Brondello, 1999).

The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. High-level expression of Chicken Pyst1/Mkp3 in neural plate correlates with active MAPK. FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). by applying a specific MAP kinase kinase inhibitor (PD184352) it has been shown that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. It is concluded that Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. These results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates (Eblaghie, 2003).

Mitogen-activated protein kinase (MAPK) pathways are major mediators of extracellular signals that are transduced to the nucleus. MAPK signaling is attenuated at several levels, and one class of dual-specificity phosphatases, the MAPK phosphatases (MKPs), inhibit MAPK signaling by dephosphorylating activated MAPKs. Several of the MKPs are themselves induced by the signaling pathways they regulate, forming negative feedback loops that attenuate the signals. This study shows that in mouse embryos, Fibroblast growth factor receptors (FGFRs) are required for transcription of Dusp6, which encodes MKP3, an extracellular signal-regulated kinase (ERK)-specific MKP. Targeted inactivation of Dusp6 increases levels of phosphorylated ERK, as well as the pERK target, Erm, and transcripts initiated from the Dusp6 promoter itself. Finally, the Dusp6 mutant allele causes variably penetrant, dominant postnatal lethality, skeletal dwarfism, coronal craniosynostosis and hearing loss; phenotypes that are also characteristic of mutations that activate FGFRs inappropriately. Taken together, these results show that DUSP6 serves in vivo as a negative feedback regulator of FGFR signaling and suggest that mutations in DUSP6 or related genes are candidates for causing or modifying unexplained cases of FGFR-like syndromes (Li, 2007).

MAP kinase nuclear entry

In response to extracellular stimuli, mitogen-activated protein kinase (MAPK, also known as ERK) translocates from the cytoplasm to the nucleus. MAP kinase kinase (MAPKK, also know as MEK), which possesses a nuclear export signal (NES), acts as a cytoplasmic anchor of MAPK. Tyrosine (Tyr190 in Xenopus MPK1/ERK2) phosphorylation of MAPK by MAPKK is necessary and sufficient for the dissociation of the MAPKK-MAPK complex, and the dissociation of the complex is required for the nuclear translocation of MAPK. Nuclear entry of MAPK through a nuclear pore occurs via two distinct mechanisms. Nuclear import of wild-type MAPK (mol. wt 42 kDa) is induced by activation of the MAPK pathway even in the presence of wheat germ agglutinin or dominant-negative Ran, whereas nuclear import of beta-galactosidase (beta-gal)-fused MAPK (mol. wt 160 kDa), which occurs in response to stimuli, is completely blocked by these inhibitors. Moreover, while a dimerization-deficient mutant of MAPK is able to translocate to the nucleus upon stimulation, this mutant MAPK, when fused to beta-gal, is unable to enter the nucleus. These results suggest that monomeric and dimeric forms of MAPK enter the nucleus by passive diffusion and active transport mechanisms, respectively (Adachi, 2000).

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. PEA-15, a protein variably expressed in multiple vertebrate cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase (Formstecher, 2001).

The death effector domain (DED) is one of several small protein recognition modules that mediate the assembly of complexes required for signal transduction in programmed cell death. DEDs found in the adaptor protein FADD and the proforms of the initiator caspases, caspase-8 (FLICE, MACH) and caspase-10, play a pivotal role in the initiation of death receptor-mediated apoptosis, whereas DEDs in viral or cellular FLICE-inhibitory proteins (FLIPs) have the ability to block apoptosis. The DED, together with the structurally related death domain (DD) and caspase recruitment domain (CARD), are members of the death motif superfamily characterized by a conserved six a-helix bundle structure. In addition to a common three-dimensional (3D) fold, these protein domains typically associate via homotypic interactions (DD-DD, DED- DED or CARD-CARD) with complementary domains in their binding partners. Surprisingly, no common protein interaction surface has been discernible for these structurally related motifs (Hill, 2002 and references therein).

DED-containing proteins are involved in other cellular signaling events besides the regulation of apoptosis. For example, the phosphoprotein enriched in astrocytes (PEA-15) activates the extracellular signal receptor-activated kinases (ERK1/2), members of the MAP kinase family. PEA-15 is a small protein (15 kDa) that was first identified as an abundant phosphoprotein in brain astrocytes and subsequently was shown to be widely expressed in human tissues and highly conserved among mammals. Several studies have shown that PEA-15 regulates multiple cellular functions including Fas- and TNF-a-induced apoptosis and phospholipase D expression, and promotes resistance to insulin in type II diabetes (Hill, 2002 and references therein).

PEA-15 has been recently demonstrated to regulate the subcellular localization of ERK1/2 and to control the biological outcomes of the ERK cascade. PEA-15 activates the ERK pathway in a Ras-dependent manner and binds specifically to ERK1/2 and not to the related MAP kinases p38 and JNK, or to other kinases in the ERK cascade. The expression of PEA-15 in cells blocks ERK-dependent transcription and proliferation by preventing the translocation of ERK1/2 into the nucleus. Within the N-terminus of PEA-15, there is a predicted nuclear export sequence that could mediate the relocation of ERK to the cytoplasm, reminiscent of the MEK-dependent nuclear export of ERK (Hill, 2002 and references therein).

Death motifs usually are regarded as modules specialized for the recognition of complementary motifs in proteins that regulate apoptosis. However, the observation of an interaction between PEA-15 DED and ERK1/2 suggested a greater functional versatility for this structural motif. To examine this unusual death motif function, the 3D structure of PEA-15 was determined using NMR spectroscopy and its interaction with ERK2 established from an in vitro and in vivo analysis of PEA-15 mutant proteins. The structure of PEA-15 is comprised of a canonical N-terminal DED and an irregularly structured C-terminal tail. NMR 'footprinting' and mutagenesis of PEA-15 identified distinct regions in both the DED and C-terminal tail that are required for ERK2 binding. Remarkably, the ERK-binding surface of the PEA-15 DED is topologically similar to the Pelle-binding surface of Drosophila Tube death domain. Thus, despite the absence of functional or sequence similarity between PEA-15 and Tube, these proteins appear to utilize a common epitope to recognize structurally and functionally diverse targets (Hill, 2002).

In response to retinoic acid, embryonic stem and carcinoma cells undergo differentiation to embryonic primitive endoderm cells, accompanied by a reduction in cell proliferation. Differentiation does not reduce the activation of cellular MAPK/Erk, but does uncouple mitogen-activated protein kinase (MAPK) activation from phosphorylation/activation of Elk-1 and results in inhibition of c-Fos expression, whereas phosphorylation of the cytoplasmic substrate p90RSK remains unaltered. Cell fractionation and confocal immunofluorescence microscopy has demonstrated that activated MAPK is restricted to the cytoplasmic compartment after differentiation. An intact actin and microtubule cytoskeleton appears to be required for the restriction of MAPK nuclear entry induced by retinoic acid treatment because the cytoskeletal disrupting agents nocodazole, colchicine, and cytochalasin D are able to revert the suppression of c-Fos expression. Thus, suppression of cell proliferation after retinoic acid-induced endoderm differentiation of embryonic stem and carcinoma cells is achieved by restricting nuclear entry of activated MAPK, and an intact cytoskeleton is required for the restraint (Smith, 2004).

The transition between the proliferation and differentiation of progenitor cells is a key step in organogenesis, and alterations in this process can lead to developmental disorders. The extracellular signal-regulated kinase 1/2 (ERK) signaling pathway is one of the most intensively studied signaling mechanisms that regulates both proliferation and differentiation. How a single molecule (e.g. ERK) can regulate two opposing cellular outcomes is still a mystery. Using both chick and mouse models, this study shed light on the mechanism responsible for the switch from proliferation to differentiation of head muscle progenitors and implicate ERK subcellular localization. Manipulation of the fibroblast growth factor (FGF)-ERK signaling pathway in chick embryos in vitro and in vivo demonstrated that blockage of this pathway accelerated myogenic differentiation, whereas its activation diminished it. Next, whether the spatial subcellular localization of ERK could act as a switch between proliferation (nuclear ERK) and differentiation (cytoplasmic ERK) of muscle progenitors was examined. A myristoylated peptide that blocks importin 7-mediated ERK nuclear translocation induced robust myogenic differentiation of muscle progenitor/stem cells in both head and trunk. In the mouse, analysis of Sprouty mutant embryos revealed that increased ERK signaling suppressed both head and trunk myogenesis. These findings, corroborated by mathematical modeling, suggest that ERK shuttling between the nucleus and the cytoplasm provides a switch-like transition between proliferation and differentiation of muscle progenitors (Michailovici, 2014).

Conserved docking motifs in MAP kinases

50 different proteins have been reported to be ERK substrates. These include signaling proteins likely to function upstream of ERK [such as Son-of-sevenless (Sos), guanine nucleotide exchange factor and MEK]; signaling proteins likely to function downstream of ERK (such as the protein kinase pp90rsk); transcription factors (such as c-Fos, GATA-2, c-Myc); ETS proteins (including Elk-1, LIN-1, and Aop/Yan), and proteins involved in a wide variety of other processes. These findings suggest that ERK plays a central role in signal propagation and feedback regulation. Furthermore, ERK is a transition point between signaling proteins and regulators of differentiation, suggesting it makes an important contribution to the specificity of RTK-Ras-ERK signaling pathways. Although a large number of ERK substrates have been identified, the understanding of ERK function remains fragmentary, since ERK probably phosphorylates different substrates in different cell types and the cellular context of most substrates has yet to be defined. In addition, many ERK substrates probably have not been identified. Little is known about how ERK recognizes such a diverse group of substrates. Although the structure of ERK was determined using X-ray crystallography, this approach has not revealed how ERK interacts with substrate proteins, because the structure of ERK bound to a substrate has yet to be determined. Studies of residues in substrate proteins that are phosphorylated by ERK and assays of peptide substrates have identified a serine or threonine followed by a proline (S/TP) as the minimal consensus sequence for phosphorylation by ERK. In addition, a proline at position 2 is favorable, whereas a proline at position 1 is unfavorable (the phosphoacceptor S/T is position 0). However, this information is not sufficient to explain how ERK recognizes specific proteins as substrates, because many proteins that contain S/TP sequences are not phosphorylated by ERK. Studies of the interaction of JNK with its substrate c-Jun have identified a sequence (that is, the delta domain) positioned amino-terminal to the S/TP sites that is required for efficient phosphorylation. This has led to the hypothesis that a docking site on the substrate protein that is separate from the phosphorylation sites mediates the interaction with JNK. Other protein kinases, such as cyclin-cdk2, also appear to interact with a docking site on substrate proteins . Although >50 proteins have been reported to be ERK substrates, only recently has one such docking site for ERK been identified. This docking site, a domain of Elk-1 called the D box, is similar in sequence to the delta domain of c-Jun, and these two domains are functionally interchangeable, suggesting that the delta domain/D box is a docking site for both ERK and JNK (Jacobs, 1999 and references).

The identification and characterization of a different docking site, the amino acid sequence FXFP, mediates interactions with ERK but not JNK. These two docking sites define three classes of substrates: proteins that contain only FXFP, only the delta domain/D box, or both. These findings suggest that a modular system of docking sites regulates interactions of the different MAP kinases with various substrates. In substrates that contain both docking sites, the sites function additively to create a high-affinity interaction with ERK. Thus, this system also modulates the affinity of substrates for ERK and may determine which residues are phosphorylated. This information is used to develop peptide inhibitors of ERK and identify new ERK substrates, including the kinase suppressor of the ras (KSR) family of protein kinases (Jacobs, 1999).

The Caenorhabidtis elegans LIN-1 protein contains an ETS DNA-binding domain and presumably regulates transcription. LIN-1 appears to be regulated directly by ERK, since LIN-1 is efficiently phosphorylated by Erk2 in vitro and lin-1 is regulated negatively by RTK-Ras-ERK pathways in vivo. Six gain-of-function mutations have been identified and characterized that impair the ability of lin-1 to be regulated negatively by RTK-Ras-ERK pathways and disrupt vulval development. Each mutation alters or eliminates FQFP, a sequence located in the carboxy-terminal region of LIN-1, suggesting that this motif is important for LIN-1 regulation. The sequences of other ETS proteins were analyzed and FQFP was found in vertebrate Elk-1, SAP-1a, and Net/ERP/SAP-2, all highly related proteins that comprise the Elk subfamily of ETS proteins. FQFP is positioned near the carboxyl terminus of a conserved region (named the C box), which contains multiple S/TP motifs that are phosphorylated by ERK. In addition, FQFHP was found in a comparable position of Drosophila Aop/Yan. Aop/Yan also appears to be regulated directly by ERK. This combination of sequence and functional similarities has led to a proposal that LIN-1 and Aop/Yan are members of the Elk subfamily of ETS proteins. Based on these observations, it is hypothesized that FQFP is an evolutionarily conserved docking site that mediates ERK binding to these ETS proteins. According to this model, the lin-1(gf) mutations diminish phosphorylation of LIN-1 by ERK because they alter or eliminate FQFP, resulting in constitutively active LIN-1 (Jacobs, 1999 and references).

It is proposed that this docking site motif be named DEF (docking site for ERK, FXFP). The second motif, the delta domain, the D box, and the docking site are all versions of the same motif, and this motif functions as a docking site for both ERK and JNK. It is proposed that this second motif be named DEJL (docking site for ERK and JNK, LXL). Evidence supports the model that the DEF and DEJL form a modular system that mediates recognition by ERK. A mutant Elk-1 protein lacking both motifs does not interact with ERK significantly. Restoring the DEF or DEJL decreases the Km 12- and 50-fold, respectively, showing that these motifs can independently mediate interactions with ERK. Restoring both sequences decreased the Km 170-fold, showing that in combination these motifs function additively rather than redundantly or synergistically. These findings, together with the observation that the sequences of the DEF and DEJL are not similar, suggest that the DEF and DEJL may interact with separate binding pockets of ERK. If ERK has separate binding pockets, then ERK may be capable of simultaneously interacting with the DEF and DEJL. The sequences of many proteins reported to be ERK substrates were examined to determine if they contained a candidate DEF or DEJL. This analysis has revealed four classes of substrate proteins. Class I substrates contain both a DEF and DEJL. Examples include the Elk subfamily of ETS proteins. In these proteins, the DEJL is amino-terminal to the DEF. Class II substrates contain only a DEF. Examples include GATA-2 and rat c-Fos. Class III substrates contain only a DEJL. Examples include MAP kinase kinases and c-Jun. Class IV substrates contain neither a DEF or DEJL. Examples include c-Myc, Sos, and pp90rsk. The existence of this class of reported substrates raises the possibility that ERK interacts with one or more yet-to-be-defined docking sites. Alternatively, these proteins may contain divergent DEF or DEJL motifs that do not match the current consensus sequences or they may not be physiological ERK substrates (Jacobs, 1999).

Mitogen-activated protein kinases (MAPKs) are specifically phosphorylated and activated by the MAPK kinases; they phosphorylate various targets such as MAPK-activated protein kinases and transcription factors, and are inactivated by specific phosphatases. Recently, docking interactions via the non-catalytic regions of MAPKs have been suggested to be important in regulating these reactions. Docking sites have been identified in MAPKs and in MAPK-interacting enzymes. A docking domain in extracellular-signal-regulated kinase (ERK), a MAPK, serves as a common site for binding to the MAPK kinase MEK1, the MAPK-activated protein kinase MNK1 and the MAPK phosphatase MKP3. Two aspartic acids in this domain are essential for docking, one of which is mutated in the sevenmaker mutant of Drosophila ERK/Rolled. A corresponding domain in the MAPKs p38 and JNK/SAPK also serves as a common docking site for their MEKs, MAPK-activated protein kinases and MKPs. These docking interactions increase the efficiency of the enzymatic reactions. These findings reveal a hitherto unidentified docking motif in MAPKs that is used in common for recognition of their activators, substrates and regulators (Tanoue, 2000).

Do other members of the MAPK family, specifically p38 and JNK, have a similar docking domain? An alignment of the sequences corresponding to the common domain (CD) of ERK2 reveals a conserved aspartic acid (corresponding to Asp 321 in Xenopus ERK2 and Asp 316 in rat ERK2) just after the kinase domain in the C-terminal region of all the members of the MAPK family. The sequences around this aspartic acid show similarity to one another, such as the presence of acidic amino acids in the C-terminal side. Some of these acidic amino acids, including the conserved aspartic acid, appear to be exposed to the surface of the molecule and are close to one another in the steric structure, as deduced from the crystal structure of p38 and JNK3. For example, in the case of p38, three acidic amino-acid residues (Asp 313, Asp 315 and Asp 316) are close to one another on the surface of the molecule. Thus, it is possible that this domain may serve as a docking site on p38 and JNK, like the CD domain on ERK2 (Tanoue, 2000).

To test this possibility, binding assays were performed by constructing various mutant enzymes. Wild-type p38 binds to MKK6 (a direct activator of p38); to wild-type MKP5 (a dual-specificity MKP acting on p38 and JNK), and to wild-type MNK1 (a MAPKAPK). However, a mutant p38 (mut2), in which the above three aspartic acids were replaced by asparagines, does not bind to any of these. The mutation of two aspartic acids instead of the three in p38 induces a less severe, but significant, defect in the binding ability, indicating the importance of the three acidic amino acids in docking. When basic amino acids in the putative p38-docking sites of MKK6, MKP5 and MNK1 are replaced by methionines, their ability to bind to p38 is almost completely lost. As for JNK, whereas wild-type JNK2 binds to wild-type SEK1/MKK4 (a direct activator of JNK) and wild-type MKP5, a mutant JNK2 in which Asp 326 is replaced by asparagine does not. The mutant MKP5 shows a decreased ability to bind to wild-type JNK2. These results suggest that p38 and JNK also have a CD domain (Tanoue, 2000).

The sevenmaker mutant of Drosophila shows a gain-of-function phenotype, which could be explained as follows in terms of the present findings. The phenotype might depend on a balance in the intracellular amounts of activators, inactivators and substrates of ERK2 and on the properties of their enzymatic reactions. Although there are only two activators of ERK2 -- MEK1 and MEK2 -- there are many inactivators (tyrosine phosphatases and dual-specificity phosphatases). There are also a number of phosphatases whose catalytic activity is enhanced (up to 35-fold for MKP3) by binding to ERK2. The inactivation of ERK2 might, therefore, be more severely impaired than its activation by the partial disruption of the CD domain. It should be noted that the docking interactions are not completely disrupted in the sevenmaker mutant of ERK/Rolled because only one of the two aspartic acids of the CD domain is replaced by asparagine. The result, however, also shows the decreased ability of the sevenmaker ERK2 to phosphorylate MNK1 in vitro. This result is apparently contradictory to the gain-of-function phenotype of the sevenmaker mutant. It should be taken into consideration that there are many parameters that affect ERK2 activity in vivo. The overall balance of the activity of the interacting proteins may define the final efficiency of the signal transduction. Thus, a complete understanding of the sevenmaker phenotype requires further thorough quantitative analyses (Tanoue, 2000).

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).

Structure of MAP kinases

Functional divergence caused by mutations in an energetic hotspot in ERK2

The most frequent extracellular signal-regulated kinase 2 (ERK2; see Drosophila Rolled) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila, only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. These results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions (Taylor, 2019).

Table of contents

rolled/MAPK: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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