Table of contents
MAP kinase, growth factors and their receptors
Activation of the canonical mitogen-activated protein kinase (MAPK) cascade by soluble mitogens is blocked in non-adherent cells. It is also blocked in cells in which the cAMP-dependent protein kinase (PKA) is activated. Inhibition of PKA allows anchorage-independent stimulation of the MAPK cascade by growth factors. This effect is transient, and its duration correlates with sustained tyrosine phosphorylation of paxillin and focal-adhesion kinase (FAK) in non-adherent cells. The effect is sensitive to cytochalasin D, implicating the actin cytoskeleton as an important factor in mediating this anchorage-independent signaling. Interestingly, constitutively active p21-activated kinase (PAK) also allows anchorage-independent MAPK signaling. Furthermore, PKA negatively regulates PAK in vivo, and whereas the induction of anchorage-independent signaling resulting from PKA suppression is blocked by dominant negative PAK, it is markedly prolonged by constitutively active PAK. These observations indicate that PKA and PAK are important regulators of anchorage-dependent signal transduction (Howe, 2000).
Epidermal growth factor induces PC12 cell differentiation in the presence of the protein kinase inhibitor K-252a. K-252a blocks the actions of nerve growth factor and other neurotrophins and, at lower concentrations, selectively potentiates neurotrophin-3 actions. K-252a enhances epidermal growth factor (EGF)-and basic fibroblast growth factor (BFGF)-induced neurite outgrowth of PC12 cells at higher concentrations than required for neurotrophin inhibition. In parallel, tyrosine phosphorylation of extracellular signal-regulated kinases (ERKs) elicited by EGF or bFGF is also increased in the presence of K-252a. This signal was prolonged for 6 h. EGF- and bFGF-induced phosphorylation of phospholipase C-gamma 1 were not changed. The effect of K-252a on ERKs is resistant to chronic treatment with phorbol ester, indicating that protein kinase C is not involved in this potentiation. Although K-252a alone does not induce neurite outgrowth or tyrosine phosphorylation of ERKs or phospholipase C-gamma 1, this compound alone stimulates phosphatidylinositol hydrolysis. These findings identify activities of K-252a besides the direct interaction with neurotrophin receptors and suggest that a K-252a-sensitive protein kinase or phosphatase might be involved in signal transduction of EGF and bFGF. These results are also compatible with the hypothesis that sustained activation of ERKs may be important in PC12 differentiation (Isono, 1994).
A paradigm has been established whereby mutant tyrosine kinase receptors such as the v-erbB and v-fms gene products function as oncoproteins in the absence of ligand. A spontaneously occurring deletional mutant of the human epidermal growth factor receptor (EGFR-vIII) has been isolated from astrocytic neoplasms and transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is constitutively complexed with the majority of cellular GRB2, suggesting a link to the Ras-Mitogen-activated protein (MAP) kinase pathway. The presence of EGFRvIII suppresses activation of p42 and p44 MAP kinases by phorbol and serum; however, MEK activation by PMA is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase activity in EGFRvIII-transfected cells is augmented by the tyrosine phosphatase inhibitor sodium vanadate. EGFR-vIII is capable of transducing downstream signals through MAP kinase as evidenced by activation of cytoplasmic phospholipase A2 at levels similar to that induced by intact EGFR. These results suggest that EGFR-vIII constitutively activates downstream signal transduction through MAP kinase. This chronic stimulation of the MAP kinase pathway may represent one means by which mutant EGFR transduces an oncogenic signal (Montgomery, 1995).
A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these events are actuated by biochemical signaling pathways, an investigation was carried out as to whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. Known motility-associated pathways were examined to determine whether these contribute to EGF-induced effects. Phospholipase C(gamma) (PLC[gamma]) activation and mobilization of gelsolin from a plasma membrane-bound state have been shown to be required for EGFR-mediated cell motility. In contrast, this study found that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) that fails to activate PLC(gamma) or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLC(gamma). Because both WT and c'973 EGFR activate the erk MAP kinase pathway, additional exploration of this signaling pathway has been carried out. The pathway has not previously been associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, EGFR kinase activity to directly stimulates focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. A model of EGF-induced motogenic cell responses is proposed in which the PLC(gamma) pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion (Xie, 1998).
Activation of MAP kinase by nerve growth factor involves two distinct pathways: the initial activation of MAP kinase requires the small G protein Ras, but its activation is sustained by the small G protein Rap1. Rap1 is activated by CRK adaptor proteins and the guanine-nucleotide-exchange factor C3G, and forms a stable complex with B-Raf, an activator of MAP kinase. Rap1 is required for at least two indices of neuronal differentiation by nerve growth factor: electrical excitability and the induction of neuron-specific genes. It is proposed that the activation of Rap1 by C3G represents a common mechanism to induce sustained activation of the MAP kinase cascade in cells that express B-Raf (York, 1998).
A sensitive assay for MAP kinase activity was used to investigate the role of endogenous fibroblast growth factor (FGF)-activated MAP kinase in early Xenopus embryonic patterning. MAP kinase activity is low during cleavage stages and increases significantly during gastrulation. The temporal profile of this activity correlates well with the expression pattern of Xenopus eFGF. Spatially, MAP kinase activity is lowest in animal pole tissue and higher in vegetal pole cells and the marginal zone. Endogenous MAP kinase activity is FGF receptor-dependent, demonstrating that FGF signaling is active in all three germ layers of the early embryo. This activity is necessary for normal expression of Mix.1, a mesoendodermal marker, in the endoderm as well as in the mesoderm, indicating that MAP kinase plays a functional role in patterning both of these germ layers. Spatial and temporal changes in MAP kinase activation during gastrulation also suggest a role for FGF signaling in this process. Embryonic wounding during dissection results in significant stimulation of this pathway, providing a possible explanation for earlier observations of effects of surgical manipulation on cell fate in early embryos (LaBonne, 1997).
Epidermal growth factor (EGF) induces cell proliferation in a variety of cell types by binding to a prototype transmembrane tyrosine kinase receptor. Ligation of this receptor by EGF activates Erk1 and Erk2, members of the mitogen-activated protein (MAP) kinase family, through a Ras-dependent signal transduction pathway. Despite what is currently a detailed understanding of these events, the exact mechanism by which EGF causes cells to proliferate remains unclear. Big MAP kinase (Bmk1), also known as Erk5, is a member of the MAP kinase family that is activated in cells in response to oxidative stress, hyperosmolarity and treatment with serum. EGF is a potent activator of Bmk1. In contrast to Erk1/2, EGF-mediated activation of Bmk1 occurs independent of Ras and requires the MAP-kinase kinase Mek5. Expression of a dominant-negative form of Bmk1 blocks EGF-induced cell proliferation and prevents cells from entering the S phase of the cell cycle. These results demonstrate that Bmk1 is part of a distinct MAP-kinase signaling pathway that is required for EGF-induced cell proliferation and progression through the cell cycle (Kato, 1998).
Integrin-mediated anchorage of NIH3T3 fibroblasts to the extracellular matrix component fibronectin permits efficient growth factor signaling to the p42 and p44 forms of mitogen-activated protein kinase (MAPK). Since integrins bridge the extracellular matrix to focal adhesion sites and to the actin cytoskeleton, the role of these integrin-associated structures in efficient growth factor activation of p42 and p44-MAPKs were analyzed. Use of specific reagents that disrupt actin stress fiber and focal adhesion formation demonstrate that upon readhesion of NIH3T3 cells to fibronectin, cells that are poorly spread and lack prominent focal adhesions but that form cortical actin structures, efficiently signal to p42 and p44-MAPKs upon EGF stimulation. In contrast, failure to form the cortical actin structures, despite attachment to fibronectin, precludes effective EGF signaling to p42 and p44-MAPKs. Actin cytoskeletal changes induced by expression of dominant-negative and constitutively active forms of Rho GTPases do not alter EGF activation of MAPK in adherent cells. However, active Cdc42, but not active Rac1 or RhoA, partially rescue EGF signaling to p44-MAPK in cells maintained in suspension. These data indicate that a limited degree of adhesion-mediated cytoskeletal organization and focal adhesion complex formation are required for efficient EGF activation of p42 and p44-MAPKs. These studies exclude a major role for the GTPases RhoA and Rac1 in the formation of cytoskeletal structures relevant for signaling, but indicate that structures regulated by Cdc42 enhance the ability of suspension cells to activate MAPK in response to growth factors (Aplin, 1999).
Fibroblast growth factor (FGF) stimulates proliferation and represses differentiation of a myoblast cell line. The predominant FGF receptor present on MM14 cells, FGFR1, is a receptor tyrosine kinase capable of activating the mitogen-activated protein kinase (MAPK) cascade in fibroblast and neuronal cell lines. To determine whether the FGF signal is mediated via the MAPK cascade in myoblasts, MM14 cells were stimulated with basic FGF (bFGF) and activities of the various kinases were measured. After withdrawal from serum and bFGF for 3 hr, bFGF stimulates MAPK kinase (MAPKK) activity, but MAPK and S6 peptide kinase activities are not detected. In contrast, when serum and bFGF are withdrawn for 10 hr, the activities of MAPKK, MAPK, and S6 peptide kinase are all stimulated by bFGF treatment. The inability of bFGF to stimulate MAPK after 3 hr of withdrawal may be due, in part, to the presence of a MAPK phosphatase activity that is detected in MM14 cell extracts. This dephosphorylating activity diminishes during commitment to terminal differentiation and is inhibited by sodium orthovanadate. Thus, the ability of bFGF to stimulate MAPK in MM14 cells is correlated with the loss of a MAPK phosphatase activity. These results show that although bFGF activates MAPKK in proliferating myoblasts, the mitogenic signal does not progress to the downstream kinases, providing a physiological example of an uncoupling of the MAPK cascade (Campbell, 1995).
Basic fibroblast growth factor (bFGF) plays an important role in development of the central nervous system and is neurotropic for a variety of neurons. bFGF is neurotropic for GT1 GnRH neuronal cells that express functional FGF receptors (FGFRs). The GT1 cell lines generated by genetically targeted tumorigenesis display highly differentiated properties of GnRH neurons. Addition of 2 and 10 ng/ml bFGF increases neurite outgrowth of GT1-7 cells and results in a significant increase of GT1 cell survival in serum-free medium. GT1 cells express FGFRs 1 and 3 but not 2. Occupancy of FGFRs with 10 ng/ml bFGF stimulates the sustained tyrosine phosphorylation of both the 42- and 44-kilodalton mitogen-activated protein kinases (MAPKs) for up to 6 h. GT1-1 and GT1-7 cells also express messenger RNA for bFGF, although the level of bioactive bFGF synthesized by GT1 cells appears suboptimal because GT1 cells can further respond to exogenously added bFGF. Thus, bFGF is a neurotropic factor in GT1 GnRh neuronal cell lines, raising the possibility that bFGF may play a role in the neurobiology of GnRH neurons (Tsai, 1995).
In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Using a dominant negative mutant approach, it has been shown that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, novel, activated mutants of SHP-2 have been generated. Expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. These results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. These activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems (O'Reilly, 2000).
LAR is a widely expressed receptor-like protein tyrosine phosphatase that is implicated in regulation of intracellular signaling triggered by both cell adhesion and peptide growth factors. Genetic studies have revealed that LAR regulates neuron axon path finding in Drosophila and mammary gland epithelial cell differentiation in mice. The molecular mechanism underlying the tissue specific function of LAR has not been clearly understood. The role and mechanism of LAR has been studied in peptide growth factors EGF and FGF signaling in human tissue culture cells in which the expression of LAR is under the control of an inducible promoter. Although both EGF and FGF induce activation of mitogen-activated protein kinase (MAPK), LAR only inhibits FGF-induced MAPK activation. LAR does not interact directly with the peptide growth factor receptors, since the ligand-induced autophosphorylation of growth factor receptors was not affected by induction of LAR. The specific effect of LAR on FGF-induced MAPK activation appears to be mediated by specific inhibition of the phosphorylation of two signal transducers that act downstream of the FGF receptor, FRS2 and a 180 kDa protein, and by prevention of their interaction with the adaptor protein GRB2. In contrast, LAR selectively inhibits the epidermal growth factor (EGF)-induced phosphorylation of p130CAS (see CAS/CSE1 segregation protein) and the formation of the complex between p130CAS and GRB2 but this effect does not influence the activation of MAPK by EGF. These data suggest that LAR and similar receptor-like protein tyrosine phosphatases may contribute to the regulation of transmembrane signaling by selectively inhibiting the tyrosine phosphorylation of specific signal transducers that act downstream of the plasma membrane-associated tyrosine kinases. The consequent inhibition of the formation of signaling complexes by these proteins may contrbute to the specificity of the signals generated by specific peptide growth factors as well as extracellular matrix proteins (Wang, 2000).
Phosphoinositide (PI) 3-kinase and the mitogen-activated protein (MAP) kinase cascades are activated by many of the same ligands. Several groups have reported involvement of PI 3-kinase in the activation of Erk1 and Erk2, whereas many other groups have shown that activation of Erk1 and Erk2 is not sensitive to inhibitors of PI 3-kinase, such as wortmannin. Wortmannin inhibition of the MAP kinase pathway is cell type- and ligand-specific. Wortmannin blocks platelet-derived growth factor (PDGF)-dependent activation of Raf-1 and the MAP kinase cascade in Chinese hamster ovary cells, which have few PDGF receptors, but has no significant effect on Erk activation in Swiss 3T3 cells, which have high levels of PDGF receptors. However, wortmannin blocks activation of Erk proteins if Swiss 3T3 cells are stimulated with lower, physiological levels of PDGF. These results suggest that PI 3-kinase is in an efficient pathway for activation of MAP kinase, but that MAP kinase can be stimulated by a redundant pathway when a large number of receptors are activated. Evidence is presented that a protein kinase C family member downstream of phospholipase Cgamma is involved in the redundant pathway (Duckworth, 1997).
When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF-R indicates that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone is insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma is also required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, is not required for PC12 cell differentiation. Thus growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway (Vaillancourt, 1995).
In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) [See Drosophila Hopscotch and Marelle respectively] and the Ras/Raf-1/mitogen-activated protein kinase cascades. Electroporation of antibodies against MEK1 or ERK1 abolishes vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolishes proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevents proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 has no effect on angiotensin II- or platelet-derived growth factor-induced Ras/Raf-1 complex formation. These results indicate that (1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, (2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and (3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors (Marrero, 1997).
Insulin-like growth factor-I (IGF-I) induces neuronal differentiation in vitro. In SH-SY5Y human neuroblastoma cells, treatment with IGF-I induces concentration- and time-dependent tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and extracellular signal-regulated protein kinases (ERK) 1 and 2. These effects of IGF-I are blocked by a neutralizing antibody against IGF-IR. Whereas IGF-IR phosphorylation is observed within 1 min, maximal phosphorylation of ERKs is not reached for 30 min. Both IGF-IR and ERK phosphorylation are maintained for at least 24 h. The concentration dependence of IGF-I-stimulated IGF-IR and ERK tyrosine phosphorylation parallels that of IGF-I-mediated neurite outgrowth. Mitogen-activated protein kinase inhibitor PD98059 has no effect on IGF-IR phosphorylation, but PD98059 reduces IGF-I-mediated ERK tyrosine phosphorylation and ERK phosphorylation of the substrate Elk-1. PD98059 also produces a parallel reduction of IGF-I-stimulated neurite outgrowth. Consistent with its ability to block neuronal differentiation, PD98059 inhibits IGF-I-dependent changes of GAP-43 and c-myc gene expression. Together these results suggest that activation of ERKs is essential for IGF-I-stimulated neuronal differentiation (Kim, 1997).
Activation of the MAP kinase ERK2 by serum or purified growth factors is strongly dependent on cell adhesion to extracellular matrix proteins. This effect is specific to soluble growth factors, since suspended cells still activate ERK2 in response to plating on fibronectin, and is reversible. Analysis of endogenous Ras and Raf shows that these proteins are still activated by serum in suspended cells, whereas MEK activity is inhibited. Conversely, activation of ERK2 by activated mutants of Ras and Raf is still adhesion-dependent but activation by MEK is not. Consistent with these results, activated MEK enhances growth of ras-transformed cells in suspension, but not when the cells are adherent. These results identify a novel synergism between cell adhesion- and growth factor-regulated pathways, and explain how the oncogenic activation of MAP kinases induces both serum- and anchorage-independent growth (Renshaw, 1997).
Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell-cell junctions and subsequent cell scattering. In Madin-Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induce the loss of the adherens junction proteins E-cadherin and beta-catenin from intercellular junctions during cell spreading, and the HGF/SF response is blocked by dominant-negative N17Ras. Desmosomes and tight junctions are regulated separately from adherens junctions, because the adherens junctions are not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac are required downstream of Ras, because loss of adherens junctions is blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of either or both MAP kinase kinase 1 or Rac1. All of these inhibitors also prevent HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110alpha subunit of PI 3-kinase alone does not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras are required for adherens junction disassembly and that the disassembly process is essential for the motile response to HGF/SF (Potempa, 1998).
A mechanism by which the Ras-mitogen-activated protein kinase (MAPK) signaling pathway mediates growth factor-dependent cell survival has been characterized. The neurotrophin BDNF (brain-derived neurotrophic factor) and its receptor TrkB regulate the survival of newly generated granule neurons within the developing cerebellum. BDNF promotes the survival of cultured rat cerebellar granule neurons; upon BDNF withdrawal, these neurons die by apoptosis. BDNF induces phosphorylation of MAPK. Inhibition of MAPK activity by PD098059, a pharmacological agent that blocks MEK activity, diminishes the effect of BDNF on the survival of cerebellar granule cells. Likewise, the introduction of a dominant interfering form of MEK (MEK-KA97) blocks BDNF-enhancement of neuronal survival. These results indicate that activation of MAPK is required for BDNF-induced survival of cerebellar granule neurons (Bonni, 1999).
Like BDNF, insulin-like growth factor 1 (IGF-1) (or a high concentration of insulin that stimulates the IGF-1 receptor) promotes the survival of cerebellar granule neurons. Both BDNF and IGF-1 activate phosphatidylinositol 3-kinase (PI-3K) and the protein kinase Akt (PKB: Drosophila homolog Akt1) cascade in cerebellar granule neurons. Although the PI-3K-Akt signaling pathway mediates the survival-promoting effects of BDNF and IGF-1, inhibition of MAPK in cerebellar neurons has no effect on IGF-1 receptor-mediated cell survival. These results suggest that BDNF and IGF-1 promote cell survival at least in part by distinct mechanisms (Bonni, 1999).
The MAPK-activated kinases, the Rsks (see Drosophila S6kII), catalyze the phosphorylation of the pro-apoptotic protein BAD at serine 112 both in vitro and in vivo. The Rsk-induced phosphorylation of BAD at serine 112 suppresses BAD-mediated apoptosis in neurons. Rsks also are known to phosphorylate the transcription factor CREB (cAMP response element-binding protein) at serine 133. Activated CREB promotes cell survival, and inhibition of CREB phosphorylation at serine 133 triggers apoptosis. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification and inactivation of a component of the cell death machinery and the increased transcription of pro-survival genes (Bonni, 1999).
To determine whether CREB contributes to BDNF's ability to enhance cerebellar granule cell survival, the effects of two distinct dominant interfering forms of CREB on the BDNF survival response were tested. K-CREB, in which Arg287 is converted to Leu, forms dimers with endogenous CREB proteins via its leucine zipper domain. K-CREB inhibits the binding of endogenous CREB to the promoters of CREB-responsive genes. M1-CREB, in which Ser133 is converted to Ala, competes with endogenous CREB proteins for binding to the promoters of CREB-responsive genes. However, once bound to DNA, M1-CREB does not activate transcription. When transfected into cerebellar granule neurons, either K-CREB or M1-CREB inhibits the effect of BDNF on cell survival. However, the dominant interfering forms of CREB do not inhibit IGF-1-mediated cerebellar granule cell survival; this finding suggests that these proteins act specifically to block the BDNF response. In addition, M1-CREB does not lead to inhibition of Rsk function because its expression in 293T cells does not inhibit the MEK-induced phosphorylation of BAD at Ser112 (Bonni, 1999).
CREB has been implicated in mediating adaptive responses of neurons to trans-synaptic stimuli. These findings indicate that CREB may also have a function in the regulation of neuronal survival in the developing central nervous system. Mice in which the CREB gene has been disrupted die perinatally before the majority of cerebellar granule neurons are generated However, analysis of the CREB-/- mouse embryos has revealed a number of abnormalities in brain development that may reflect the contribution of CREB to the regulation of the survival of neurons. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism that modulates the cell death machinery directly by phosphorylating and thereby inhibiting the pro-apoptotic protein BAD, and by inducing the expression of pro-survival genes in a CREB-dependent manner. Suppression of BAD-mediated cell death by Rsk occurs relatively early after the removal of extracellular survival factors, whereas the contribution of CREB-mediated cell survival is detected significantly later. Therefore, the two arms of the MAPK-Rsk-regulated mechanism might act with different kinetics or at different times in developing neurons (Bonni, 1999).
Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators: MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. Identified here is a pro-survival role for the serine/threonine kinase S6 kinase p90 ribosomal S6 kinase Rsk1, a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonize the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminates the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restores the MEK-MAP kinase survival signal. Experiments in vitro and in vivo have demonstrated that Rsk1 directly phosphorylates the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bad's pro-apoptotic function. Constitutively active Rsk1 causes constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevent phosphorylation by Rsk1 also inhibit Rsk1-mediated cell survival. These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad (Shimamura, 2000).
Bone morphogenetic protein-4 (BMP-4) induces epidermis and represses neural fate in Xenopus ectoderm. p42 Erk MAP kinase (MAPK) is implicated in the response to neural induction. The effects of BMP-4 on MAPK activity were examined in gastrula ectoderm. Expression of a dominant negative BMP-4 receptor results in a 4.5-fold elevation in MAPK activity in midgastrula ectoderm. MAPK activity is reduced in ectoderm expressing a constitutively active BMP-4 receptor, or ectoderm treated with BMP-4 protein in the presence or absence of cycloheximide. Overexpression of TAK1 leads to a reduction in MAPK activity in early gastrula ectoderm. The inhibitory effects of TAK1 can be reversed by 1 mM SB 203580, a p38 inhibitor. Treatment of isolated ectoderm with SB 203580 leads to expression of otx2, NCAM, and noggin. Western blot analyses indicate that the BMP-4 pathway does not activate JNKs in ectoderm. These findings indicate that BMP-4 inhibits ectodermal MAPK activity through a TAK1/p38-type pathway. MAPK has been shown to inactivate Smad1. Thus, these results suggest that BMP-4 and MAPK pathways are mutually antagonistic in Xenopus ectoderm, and that interactions between these pathways may govern the choice between epidermal and neural fate (Goswami, 2001).
Two signaling pathways are activated by signaling through the BMP-4 receptor complex. The first involves phosphorylation of the BMP-4 effector Smad1 at the C terminus; thus phosphorylated, Smad1 binds Smad4, translocates to the nucleus, and participates in transcriptional regulation. The second pathway is mediated by TGF-beta-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. In mammalian cells, TAK1 has been shown to act via either Jun N-terminal kinases (JNKs), both members of the MAP kinase family. In C. elegans, TAK1 activates lit-1, a homolog of the distantly related MAP kinase family member Nemo-like kinase; this pathway culminates in the inhibitory phosphorylation of TCF-1/Lef-1 and the down-regulation of wnt/beta-catenin-inducible transcription. The TAK1/NLK pathway has also been shown to inhibit wnt/beta-catenin-dependent signaling in vertebrates. In Xenopus embryos, TAK1 has been implicated in the establishment of ventral mesoderm in response to BMP-4 (Goswami, 2001 and references therein).
The telencephalon is formed in the most anterior part of the central nervous system (CNS) and is organized into ventral subpallial and dorsal pallial domains. In mice, it has been demonstrated that Fgf signaling has an important role in induction and patterning of the telencephalon. However, the precise role of Fgf signaling is still unclear, owing to overlapping functions of Fgf family genes. To address this in zebrafish embryos, the activation of Ras/mitogen-activated protein kinase (MAPK), one of the major downstream targets of Fgf signaling, has been examined. Immunohistochemical analysis reveals that an extracellular signal-regulated kinase (ERK), a vertebrate MAPK, is activated in the anterior neural boundary (ANB) of the developing CNS at early segmentation stages. Experiments with Fgf inhibitors reveal that ERK activation at this stage is totally dependent on Fgf signaling. Interestingly, a substantial amount of ERK activation is observed in ace mutants in which fgf8 gene is mutated. The function of Fgf signaling in telencephalic development was analyzed by use of several inhibitors to Fgf signaling cascade, including dominant-negative forms of Ras (RasN17) and the Fgf receptor (Fgfr), and a chemical inhibitor of Fgfr, SU5402. In treated embryos, the induction of telencephalic territory normally proceeds but the development of the subpallial telencephalon is suppressed, indicating that Fgf signaling is required for the regionalization within the telencephalon. Finally, antisense experiments with morpholino-modified oligonucleotides suggest that zebrafish fgf3, which is also expressed in the ANB, co-operates with fgf8 in subpallial development (Shinya, 2001).
Members of the fibroblast growth factor (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibers; a low dose of FGF can stimulate cell proliferation (but not fiber differentiation), whereas higher doses of FGF are required to induce fiber differentiation. To determine if these cellular events are regulated by the same signaling pathways, the role of MAP kinase signaling in FGF-induced lens cell proliferation and differentiation was examined. FGF induces a dose-dependent activation of ERK1/2 as early as 15 minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fiber differentiation. Interestingly, inhibition of ERK signaling can block the morphological changes associated with FGF-induced lens fiber differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, ß-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, these data indicate that different levels of ERK signaling may be important for the regulation of lens cell proliferation and early morphological events associated with fiber differentiation; however, multiple signaling pathways are likely to be required for the process of lens fiber differentiation and maturation (Lovicu, 2001).
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).
FGFs mediate their pleiotropic responses by binding to and activating a family of receptor tyrosine kinases (RTKs) designated FGF receptors (FGFR) 1-4. Many of the cellular responses of FGFs are mediated by the membrane-linked docking proteins, FRS2alpha and FRS2beta, that have no closely related Drosophila homologs. Both FRS2alpha and FRS2beta contain myristyl anchors and phosphotyrosine binding (PTB) domains in their N termini and multiple tyrosine phosphorylation sites in their C termini that serve as binding sites for the adaptor protein Grb2 and the protein tyrosine phosphatase Shp2. The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. In addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2alpha and a mechanism for heterologous control of signaling via FGF receptors (Lax, 2002).
STAT3 is the key mediator of apoptosis in mammary gland. Leukemia inhibitory factor (LIF) is the physiological activator of STAT3, because in involuting mammary glands of Lif-/- mice, pSTAT3 is absent and the STAT3 target, C/EBPdelta, is not upregulated. Similar to Stat3 knockouts, Lif-/- mammary glands exhibit delayed involution, reduced apoptosis and elevated levels of p53. Significantly, Lif-/- glands display precocious development during pregnancy, when pSTAT3 is not normally detected. pERK1/2 is significantly reduced in Lif-/- glands at this time, suggesting that at this stage LIF mediates its effects through pERK1/2. Inhibition of LIF-mediated ERK1/2 phosphorylation potentiates the proapoptotic effects of STAT3. LIF therefore signals alternately through ERK1/2, then STAT3, to regulate mammary growth and apoptosis (Kritikou, 2003).
Epithelial cells undergo tubulogenesis in response to morphogens such as hepatocyte growth factor (HGF). To organize into tubules, cells must execute a complex series of morphogenetic events; however, the mechanisms that underlie the timing and sequence of these events are poorly understood. Downstream effectors of HGF coordinately regulate successive stages of tubulogenesis. Activation of extracellular-regulated kinase (ERK) is necessary and sufficient for the initial stage, during which cells depolarize and migrate. The Raf-MEK-ERK transduces signals from the HGF receptor Met to the nucleus. Constitutive extracellular-regulated kinase (ERK) activation is essential for complete epithelial-mesenchymal in both MDCK cells and in vivo models of epithelial tumor metastasis. ERK becomes dispensable for the latter stage, during which cells repolarize and differentiate. Conversely, the activity of matrix metalloproteases (MMPs) is essential for the late stage but not the initial stage. Thus, ERK and MMPs define two regulatory subprograms that act in sequence. By inducing these reciprocal signals, HGF directs the morphogenetic progression of tubule development (O'Brien, 2004).
Neuronal precursor cells have the capacity to engage the Raf-MEK-ERK signal module to drive either of two distinctly different regulatory programs, proliferation and differentiation. This is, at least in part, a consequence of stimulus-specific shaping of the kinase cascade response. For example, the mitogen EGF induces a transient ERK activation, whereas the neurotrophin NGF induces prolonged ERK activation. A novel component has been defined of the regulatory machinery contributing to the selective integration of MAP kinase signaling with discrete biological responses. The scaffold/adaptor protein CNK2/MAGUIN-1 is required for NGF- but not EGF-induced ERK activation. In addition, CNK2 makes a separate, essential contribution to the coupling of NGF signaling to membrane/cytoskeletal remodeling. It is proposed that CNK2 integrates multiple regulatory pathways that must function in concert to drive an appropriate biological response to external stimuli (Bumeister, 2004).
ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. This study shows that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. These results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis (Nie, 2007).
Signaling between tissues is essential to form the complex, three-dimensional organization of an embryo. Because many receptor tyrosine kinases signal through the RAS-MAPK pathway, phosphorylated ERK can be used as an indicator of when and where signaling is active during development. Using whole-mount immunohistochemistry with antibodies specific to phosphorylated ERK1 and ERK2, the location, timing, distribution, duration and intensity of ERK signaling was analyzed during mouse embryogenesis (5-10.5 days postcoitum). Spatial and temporal domains of ERK activation were discrete with well-defined boundaries, indicating specific regulation of signaling in vivo. Prominent, sustained domains of ERK activation were seen in the ectoplacental cone, extra-embryonic ectoderm, limb buds, branchial arches, frontonasal process, forebrain, midbrain-hindbrain boundary, tailbud, foregut and liver. Transient activation was seen in neural crest, peripheral nervous system, nascent blood vessels, and anlagen of the eye, ear and heart. In the contiguous domains of ERK signaling, phospho-ERK staining was cytoplasmic with no sign of nuclear translocation. With few exceptions, the strongest domains of ERK activation correlate with regions of known or suspected fibroblast growth factor (FGF) signaling, and brief incubation with an inhibitor of the fibroblast growth factor receptor (FGFR) specifically diminishes the phospho-ERK staining in these regions. Although many domains of ERK activation are FGFR-dependent, not all domains of FGF signaling were phospho-ERK positive. These studies identify key domains of sustained ERK signaling in the intact mouse embryo, give significant insight into the regulation of this signaling in vivo and pinpoint regions where downstream target genes can be sought (Corson, 2003).
Vertebrate segments called somites are generated by periodic segmentation of the anterior extremity of the presomitic mesoderm (PSM). During somite segmentation in zebrafish, mesp-b determines a future somite boundary at position B-2 within the PSM. Heat-shock experiments, however, suggest that an earlier future somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. This study characterized fibroblast growth factor (FGF) signal activity within the PSM, and demonstrated that an anterior limit of downstream Erk activity corresponds to the future B-5 somite boundary. Moreover, the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. These results provide the first molecular evidence of the future somite boundary at B-5, and it is proposed that clock-dependent cyclic inhibition of the FGF/Erk signal is a key mechanism in the generation of perfect repetitive structures in zebrafish development (Akiyama, 2014).
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