rolled/MAPK
Map Kinase, GTPase activated pathways and stress
Heat shock decreases the electrophoretic mobility of the 40 and 43 kDa mitogen activated protein kinases (MAPKs) and increases MAPK activity. Heat shock activates Raf-1 kinase and causes an increase in phosphotyrosine content of the 52 kDa Shc protein accompanied by an increment in the amount of coimmunoprecipitated Grb2. These findings provide the first evidence that the Ras-Raf-MAPK pathway is activated in liver during heat shock in vivo (Bendinelli, 1995).
Exposure of NIH3T3 cells to elevated temperatures induces the phosphorylation and activation of mitogen-activated protein (MAP) kinases (or extracellular signal-regulated kinases [ERKs]). To investigate the significance of MAP kinase activation by heat shock, the effect of inhibiting the activity of MAP kinase on heat shock protein 70 (hsp 70) expression was examined. Overexpression of a dominant inhibitory mutant of ERK1, but not ERK2, in heat-shocked cells increases hsp70 reporter gene activity, suggesting that ERK1 acts as a repressor of hsp70 gene expression. Increases in ERK1 activity through treatment of cells with sodium vanadate (SV), an inhibitor of the dual-specificity MAP kinase phosphatase 1 (PAC1), results in increased phosphorylation of the heat shock transcription factor-1 (HSF-1) in unheated cells, delays the activation of HSF-1 by heat shock, and inhibits the induction of hsp 70 by heat shock. Furthermore, the induction of thermotolerance is reduced significantly in cells that increased ERK1 activity by SV pretreatment. HSF-1 is a potential in vivo substrate for ERK1 phosphorylation. Thus agents that modulate MAP kinase act as negative regulators of the heat shock response in mammalian cells by modulating HSF-1 activity and hsp 70 expression (Mivechi, 1995).
The activation of MAPKAP (MAP kinase-activated protein) kinase 2, an immediate downstream target of MAPK was investigated under heat-shock conditions in mouse Ehrlich ascites tumor (EAT) cells and after treatment of human MO7 cells with tumor necrosis factor-alpha (TNF-alpha). MAPKAP kinase 2 activity was determined using the small heat-shock proteins (sHsps) Hsp25 and Hsp27 as substrates. In both cell types, about a threefold increase in MAPKAP kinase 2 activity is detected in a time interval of about 10-15 min after stimulation either by heat shock or TNF-alpha. Phosphorylation of MAPKAP kinase 2, but not the level of MAPKAP kinase 2 mRNA, is increased after heat shock in EAT cells. Activation of MAPKAP kinase 2 in MO7 cells is accompanied by increased MAP kinase activity. These data strongly suggest that increased phosphorylation of the sHsps after either heat shock or TNF-alpha treatment results from phosphorylation by MAPKAP kinase 2, which itself is activated by phosphorylation through MAP kinases. Hence, MAPKAP kinase 2 is responsible not only for phosphorylation of sHsps in vitro but also in vivo. The findings link sHsp phosphorylation to the MAP kinase cascade, explaining the early phosphorylation of sHsp that is stimulated by a variety of inducers such as mitogens, phorbol esters, thrombin, calcium ionophores, and heat shock (Engel, 1995).
The currently known members of the MAP kinase family include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. Overexpression of p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 (see Drosophila Rac1)or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway (Frost, 1996).
MapK is activated downstream of integrins
Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. The nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK: Drosophila homolog Focal adhesion kinase-like) can be independently activated after fibronectin (FN) stimulation; their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction. The time course of FN-stimulated ERK2 activation parallels that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibits FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibits c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 result in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity is not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promotes Shc binding to FAK. In vitro, FAK directly phosphorylates Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc is observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promotes Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc is tyrosine phosphorylated after FN stimulation of human 293T cells. Its expression does not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also does not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 is inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. These studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation (Schlaepfer, 1998).
Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells were monitored using pharmacological and genetic approaches. Inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduces extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon is sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms are activated following adhesion. PKC is required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc (Miranti, 1999).
Tenascin-C is an extracellular matrix glycoprotein, the expression of which is upregulated in remodeling arteries. The presence of tenascin-C alters vascular smooth muscle cell shape and amplifies their proliferative response by promoting growth factor receptor clustering and phosphorylation. Denatured type I collagen induces smooth muscle cell tenascin-C protein production via beta3 integrins. The pathway by which beta3 integrins stimulate expression of tenascin-C has been examined, and a promoter sequence is defined that is critical for tenascin-C induction. On native collagen, A10 smooth muscle cells adopt a stellate morphology and produce low levels of tenascin-C mRNA and protein, whereas on denatured collagen they spread extensively and produce high levels of tenascin-C mRNA and protein, which is incorporated into an elaborate extracellular matrix. Increased tenascin-C synthesis on denatured collagen is associated with elevated protein tyrosine phosphorylation, including activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). beta3 integrin function-blocking antibodies attenuate ERK1/2 activation and tenascin-C protein synthesis. Consistent with these findings, treatment with the specific MEK inhibitor, PD 98059, results in suppression of tenascin-C protein synthesis. To investigate whether beta3 integrin-dependent activation of ERK1/2 regulates the tenascin-C promoter, A10 cells were transfected with a full-length (approx. 4 kb) mouse tenascin-C gene promoter-chloramphenicol acetyltransferse reporter construct. Relative to native collagen, the activity of the reporter construct is increased on denatured collagen. To identify regions of the promoter involved, a series of tenascin-C promoter constructs with 5' deletions were examined. Denatured collagen-dependent promoter activity is retained by a 122-base pair element, located -43 to -165 bp upstream of the RNA start site. Activation of this element is suppressed either by blocking beta3 integrins, or by preventing ERK1/2 activation. These observations demonstrate that smooth muscle cell binding to beta3 integrins activates the mitogen activated protein kinase pathway, which is required for the induction of tenascin-C gene expression via a potential extracellular matrix response element in the tenascin-C gene promoter. These data suggest a mechanism by which remodeling of type I collagen modulates tenascin-C gene expression via a beta3 integrin-mediated signaling pathway, and as such represents a paradigm for vascular development and disease whereby smooth muscle cells respond to perturbations in extracellular matrix composition by altering their phenotype and patterns of gene expression (Jones, 1999).
While previous reports have indicated that the temporal mitogen- and integrin-induced activation of ERK is linked to its spatial organization, by stimulation of nuclear translocation, this study shows that ERK can also be targeted to sites of cellular attachment where it is present in its active form. Integrin engagement generates cellular signals leading to the recruitment of structural and signalling molecules which, in concert with rearrangements of the actin cytoskeleton, leads to the formation of focal adhesion complexes. Using antisera reactive either with total ERK or with phosphorylated/activated forms of ERK, in rat embryo fibroblasts and embryonic avian cells that express v-Src, it has been found that active ERK is targeted to newly forming focal adhesions after integrin engagement or activation of v-Src. UO126, an inhibitor of MAP kinase kinase 1 (MEK1), suppresses focal adhesion targeting of active ERK and cell spreading. Also, integrin engagement and v-Src induces myosin light chain kinase (MLCK)-dependent phosphorylation of myosin light chain downstream of the MEK/ERK pathway, and MLCK and myosin activities are required for the focal adhesion targeting of ERK. The translocation of active ERK to newly forming focal adhesions may direct specificity towards appropriate downstream targets that influence adhesion assembly. These findings support a role for ERK in the regulation of the adhesion/cytoskeletal network and provide an explanation for the role of ERK in cell motility (Fincham, 2000).
FAK and its associated signaling pathways mediate cell cycle progression by integrins. The potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation was investigated by using tetracycline-regulated expression system as well as chimeric molecules. Induction of Pyk2 inhibits G(1) to S phase transition whereas comparable induction of FAK expression accelerates it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increases cell cycle progression as does FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibit cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicate that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 have little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibit Erk activation in cell adhesion whereas FAK and PFhy1 stimulate it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. While FAK and PFhy1 are present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) are tyrosine phosphorylated and associated with Src and Fyn. This suggests that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant DeltaC14 by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition (Zhao, 2000).
Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. These Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allow for the sustained activation of MEK, and this effect is mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with myosin light chain kinase inhibitor ML-7 leads to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase leads to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocks sustained ERK signaling, but only Rho kinase inhibition allows for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), are not affected by inhibition of MLCK or Rho kinase. Overall, these results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1 (Roovers, 2003).
The emerging evidence that stem cells develop in specialised niches highlights the potential role of environmental factors in their regulation. The role of ß1 integrin/extracellular matrix interactions has been examined in neural stem cells. High levels of ß1 integrin expression are found in the stem-cell containing regions of the embryonic CNS, with associated expression of the laminin alpha2 chain. Expression levels of laminin alpha2 are reduced in the postnatal CNS, but a population of cells expressing high levels of ß1 remains. Using neurospheres -- aggregate cultures, derived from single stem cells, that have a three-dimensional architecture that results in the localisation of the stem cell population around the edge of the sphere -- it has been shown directly that ß1 integrins are expressed at high levels on neural stem cells and can be used for their selection. MAPK (but not PI3K) signalling is required for neural stem cell maintenance, as assessed by neurosphere formation, and inhibition or genetic ablation of ß1 integrin using cre/lox technology reduces the level of MAPK activity. It is concluded that integrins are therefore an important part of the signalling mechanisms that control neural stem cell behaviour in specific areas of the CNS (Campos, 2004).
The Wnt signalling pathway regulates many developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility-group transcription factors. Wnt stabilizes cytosolic beta-catenin, which then binds to TCF and activates gene transcription. This signalling cascade is conserved in vertebrates, Drosophila and C. elegans. In C. elegans, the proteins MOM-4 and LIT-1 regulate Wnt signalling to polarize responding cells during embryogenesis. MOM-4 and LIT-1 are homologous to TAK1 (a kinase activated by transforming growth factor-beta) mitogen-activated protein-kinase-kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK), respectively, in mammalian cells. These results raise the possibility that TAK1 and NLK are also involved in Wnt signalling in mammalian cells. This study shows that TAK1 activation stimulates NLK activity and downregulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppresses the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK phosphorylates TCF/LEF factors and inhibits the interaction of the beta-catenin-TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway negatively regulates the Wnt signalling pathway (Ishitani, 1999).
Wnt signaling controls a variety of developmental processes. The canonical Wnt/beta-catenin pathway functions to stabilize beta-catenin, and the noncanonical Wnt/Ca(2+) pathway activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca(2+) pathway activated by Wnt-5a antagonizes the Wnt/beta-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/beta-catenin signaling pathway. Activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor [beta(2)AR-Rfz-2] containing the ligand-binding and transmembrane segments from the beta(2)-adrenergic receptor [beta(2)AR] and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the beta-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits beta-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca(2+) pathway and antagonizes canonical Wnt/beta-catenin signaling (Ishitani, 2003a; full text of article).
The Wnt/beta-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic beta-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. A specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. This study investigated the relationships among NLK, beta-catenin, and LEF-1/TCF. It was found that NLK interacts directly with LEF-1/TCF and indirectly with beta-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the beta-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity (Ishitani, 2003b; full text of article).
Genetic studies on endoderm-mesoderm specification in C. elegans have demonstrated a role for several Wnt cascade components as well as for a MAPK-like pathway in this process. The latter pathway includes the MAPK kinase kinase-like MOM-4/Tak1, its adaptor TAP-1/Tab1, and the MAPK-like LIT-1/Nemo-like kinase. A model has been proposed in which the Tak1 kinase cascade counteracts the Wnt cascade at the level of beta-catenin/TCF phosphorylation. In this model, the signal that activates the Tak1 kinase cascade is unknown. As an alternative explanation of these genetic data, whether Tak1 is directly activated by Wnt was explored. It was found that Wnt1 stimulation results in autophosphorylation and activation of MOM-4/Tak1 in a TAP-1/Tab1-dependent fashion. Wnt1-induced Tak1 stimulation activates Nemo-like kinase, resulting in the phosphorylation of TCF. These results combined with the genetic data from C. elegans imply a mechanism whereby Wnt directly activates the MOM-4/Tak1 kinase signaling pathway. Thus, Wnt signal transduction through the canonical pathway activates beta-catenin/TCF, whereas Wnt signal transduction through the Tak1 pathway phosphorylates and inhibits TCF, which might function as a feedback mechanism (Smit, 2004; full text of article).
Other pathways activating and inhibiting MapK
Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that interacts directly with MEK and MAPK. KSR1 translocates from the cytoplasm to the cell surface in response to growth factor treatment and this process is regulated by Cdc25C-associated kinase 1 (C-TAK1). C-TAK1 constitutively associates with mammalian KSR1 and phosphorylates serine 392 to confer 14-3-3 binding and cytoplasmic sequestration of KSR1 in unstimulated cells. In response to signal activation, the phosphorylation state of S392 is reduced, allowing the KSR1 complex to colocalize with activated Ras and Raf-1 at the plasma membrane, thereby facilitating the phosphorylation reactions required for the activation of MEK and MAPK (Muller, 2001).
Recent studies have shown that Drosophila Dishevelled (Dsh), an essential component of the wingless signal transduction pathway, is also involved in planar polarity signaling through the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway in Drosophila. Expression of a mouse homolog of Dsh (mDvl-1) in NIH3T3 cells activates JNK/SAPK, and its activator MKK7. A C-terminal half of mDvl-1, which contains the DEP domain, is sufficient for the activation of JNK/SAPK, whereas an N-terminal half of mDvl-1 as well as the DEP domain is required for stimulation of TCF/LEF-1-dependent transcriptional activation; this is a beta-catenin-dependent process. A single amino acid substitution (Met for Lys) within the DEP domain [mDvl-1 (KM)] abolishes the JNK/SAPK-activation of mDvl-1, but does not affect the activation of LEF-1-dependent transcription. Ectopic expression of mDvl-1 (KM) or an N-terminal half of mDvl-1, but not the C-terminal half, is able to induce a secondary axis in Xenopus embryos. Because the secondary axis formation is dependent on the Wnt/beta-catenin signaling pathway, these results suggest that distinct domains of mDvl-1 are responsible for the two downstream signaling pathways: the beta-catenin pathway and the JNK/SAPK pathway in vertebrates (Moriguchi, 1999).
The mechanism of v-src-induced morphological transformation is still obscure. LA29 rat fibroblasts, which express a temperature-sensitive (ts) v-src mutant, were compared with D1025 rat fibroblasts, transfected with a ts mutant of v-fps. Upon transformation, LA29 cells adopt an elongated shape with reduced focal adhesions and loss of actin stress fibers. In contrast, activation of v-fps in D1025 cells has little effect on morphology. In both cells, paxillin is strongly tyrosine phosphorylated upon activation of the kinases. This indicates that paxillin phosphorylation is either not required, or not sufficient, for the v-src-induced disruption of focal adhesions. v-src activates the ras-MAP kinase (MAPK) pathway, as indicated by tyrosine phosphorylation of the rasGAP-associated proteins p62 and p190 and MAPK phosphorylation. Since MAPK affects transcription, this suggests that novel gene transcription is required. This notion was confirmed using actinomycin D and cycloheximide, which do not impair activation of v-src kinase activity, but completely block v-src-induced morphological changes, as demonstrated using image analysis. v-src-induced changes in cell shape occur before the reduction in number and size of focal adhesions. It is concluded that v-src-induced transformation of rat fibroblasts depends on synthesis of a protein, which induces rapid changes in cell shape that precede the loss of focal adhesions (Meijne, 1997).
Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (See Drosophila PKC), the major cellular receptor for this class of agents. This proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. Dominant-negative PKC-alpha inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (alpha, beta, delta, epsilon, eta, and zeta) were employed. Transient transfection of these PKC mutants into Cos-7 cells shows that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes alpha and eta) are potent activators of c-Raf1, atypical PKC-zeta cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-alpha and PKC-eta is abrogated for Raf CAAX, which is a membrane-localized, partially active form of c-Raf1. Activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, a novel Raf desensitization induced by PKC-alpha is described, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in phorbol ester induced mitogenesis, and provide evidence for multiple PKC controls acting on this MAPK cascade (Schonwasser, 1998).
Cyclic adenosine monophosphate (cAMP) produces tissue-specific effects involving growth, differentiation, and gene expression. cAMP can activate the transcription factor Elk-1 and induce neuronal differentiation of PC12 cells via its activation of the MAP kinase cascade. These cell type-specific actions of cAMP require the expression of the serine/threonine kinase B-Raf and activation of the small G protein Rap1. Rap1, activated by mutation or by the cAMP-dependent protein kinase PKA, is a selective activator of B-Raf and an inhibitor of Raf-1. In PC12 cells, Rap1 and B-Raf are localized to the cell membrane and cytosol, respectively. 8-CPT stimulates the association of B-Raf with Rap1 within membranes. This action is specific for both cAMP and Rap1; no association of B-Raf with Rap1 as detected within membranes following treatment with EGF or in untreated cells, nor is B-Raf detected in immunoprecipitates using Ras antibody Y13-238. The dependence on GTP of this interaction was examined in COS-7 cells transfected with B-Raf and histidine-tagged Rap1b (His-Rap) or His-RapV12. B-Raf and its kinase activity are detected in eluates. The small amount of B-Raf associating with His-Rap1 is increased in cells cotransfected with PKA. The highest level of B-Raf is detected in cells cotransfected with His-RapV12. Only eluates from B-Raf-transfected cells contain B-Raf activity, as measured by immune complex assay using B-Raf antisera. B-Raf activity associated with His-Rap1 is greatly stimulated by PKA, to a level similar to that associated with His-RapV12. The expression of equal amounts of His-Rap was confirmed by immunoblotting with Rap1 antisera. These data suggest that the association of activated B-Raf protein with Rap1 is increased upon GTP loading, stimulated by PKA or by a V12 mutation. Therefore, in B-Raf-expressing cells, the activation of Rap1 provides a mechanism for tissue-specific regulation of cell growth and differentiation via MAP kinase (Vossler, 1998).
The neural cell adhesion molecule (See Drosophila Fas 2) is a homophilic cell adhesion protein that influences neurite outgrowth. A model for N-CAM homophilic binding has been proposed in which the Ig domains bind in a pairwise antiparallel manner such that Ig I binds Ig V, Ig II binds Ig IV, and Ig III binds Ig III. Astrocyte proliferation induced by growth factors such as basic fibroblast growth factor (bFGF) is inhibited by N-CAM. This inhibition is partially reversed by the glucocorticoid antagonist RU-486, suggesting that N-CAM signaling might activate the glucocorticoid receptor. Signaling after N-CAM binding in neurons has been examined in neurite outgrowth assays. It has been proposed that N-CAM signaling occurs through the cis interaction of N-CAM with the FGF receptor and intracellular pathways stimulated by the FGF receptor. It is therefore important to assess whether signaling after N-CAM binding involves both the glucocorticoid receptor and FGF receptor pathways or whether there may be different pathways involved, depending on cell type or specific cellular events (Krushel, 1998).
N-CAM inhibits astrocyte proliferation in vitro and in vivo, and this effect is partially reversed by the glucocorticoid antagonist RU-486. The present studies have tested the hypothesis that N-CAM-mediated inhibition of astrocyte proliferation is caused by homophilic binding and involves the activation of glucocorticoid receptors. It was observed that all N-CAM Ig domains inhibit astrocyte proliferation in parallel with their ability to influence N-CAM binding. At an intermediate concentration of the Ig domains there is a distinct difference in the ability of Ig domain fragments to inhibit proliferation (Ig III > Ig I > Ig V > Ig II > Ig IV). The proliferation of other N-CAM-expressing cells also is inhibited by the addition of affinity chromatography purified N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM is not inhibited by added N-CAM. These findings support the hypothesis that it is binding of soluble N-CAM to N-CAM on the astrocyte surface that leads to decreased proliferation. Signaling pathways stimulated by growth factors include activation of mitogen-activated protein (MAP) kinase. Addition of N-CAM inhibits MAP kinase activity induced by basic fibroblast growth factor in astrocytes. In accord with previous findings, that RU-486 can partially prevent the proliferative effects of N-CAM, inhibition of MAP kinase activity by N-CAM is reversed by RU-486. MAP kinase activity in astrocytes is increased over 4-fold after bFGF treatment. However, when N-CAM is added simultaneously with bFGF, MAP kinase activity is reduced to 46% of the value stimulated by bFGF alone. This inhibitory effect on MAP kinase activity is completely reversed if the glucocorticoid antagonist RU486 is included with bFGF and N-CAM. The addition of N-CAM alone produces a small, but reproducible, decrease in basal MAP kinase activity, whereas the addition of RU486 alone has little or no effect. These results suggest that soluble N-CAM inhibits growth factor-induced MAP kinase activity and that this inhibition requires activation of the glucocorticoid receptor (Krushel, 1998).
The ability of N-CAM to promote neurite outgrowth in neurons has been postulated to occur through cis binding of N-CAM to a segment of the FGF receptor called the CAM homology domain. This conclusion was based on the observation that the presence of a 20-aa synthetic peptide corresponding to this domain could inhibit N-CAM-dependent neurite outgrowth. Proliferation stimulated by bFGF in astrocytes is reduced in the presence of N-CAM. The effects of the FGF receptor peptide was tested on the ability of N-CAM to affect astrocyte proliferation. Inclusion of N-CAM with either the FGF receptor peptide or a control peptide with the same amino acids in a random order reduces the amount of [3H]thymidine incorporation to levels equivalent to those of N-CAM alone, although addition of either peptide alone results in a slight decrease in proliferation. These results suggest that the ability of N-CAM to inhibit astrocyte proliferation is not likely to occur through direct interactions of N-CAM with the FGF receptor. Together, these findings indicate that homophilic N-CAM binding leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor and that the ability of N-CAM to influence astrocyte proliferation and neurite outgrowth involves different signal pathways (Krushel, 1998).
In cell culture systems, the TCF Elk-1 represents a convergence point for extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) subclasses of mitogen-activated protein kinase (MAPK) cascades. Its phosphorylation strongly potentiates its ability to activate transcription of the c-fos promoter through a ternary complex assembled on the c-fos serum response element. In rat brain postmitotic neurons, Elk-1 is strongly expressed. However, its physiological role in these postmitotic neurons remains to be established. To investigate biochemically the signaling pathways targeting Elk-1 and c-fos in mature neurons, a semi-in vivo system was used, composed of brain slices stimulated with the excitatory neurotransmitter glutamate. Glutamate treatment leads to a robust, progressive activation of the ERK and JNK/SAPK MAPK cascades. This corresponds kinetically to a significant increase in Ser383-phosphorylated Elk-1 and the appearance of c-fos mRNA. Glutamate also causes increased levels of Ser133-phosphorylated cyclic AMP-responsive element-binding protein (CREB) but only transiently relative to Elk-1 and c-fos. ERK and Elk-1 phosphorylation are blocked by the MAPK kinase inhibitor PD98059, indicating the primary role of the ERK cascade in mediating glutamate signaling to Elk-1 in the rat striatum in vivo. Glutamate-mediated CREB phosphorylation is also inhibited by PD98059 treatment. Interestingly, KN62, which interferes with calcium-calmodulin kinase (CaM-K) activity, leads to a reduction of glutamate-induced ERK activation and of CREB phosphorylation. These data indicate that ERK functions as a common component in two signaling pathways (ERK/Elk-1 and ERK/?/CREB) converging on the c-fos promoter in postmitotic neuronal cells and that CaM-Ks act as positive regulators of these pathways (Vanhoutte, 1999)
Activation of the mitogen-activated protein kinase (MAPK) cascade plays an important role in synaptic plasticity in area CA1 of rat hippocampus. However, the upstream mechanisms regulating MAPK activity and the downstream effectors of MAPK in the hippocampus are uncharacterized. Hippocampal MAPK activation is regulated by both the PKA and PKC systems; moreover, a wide variety of neuromodulatory neurotransmitter receptors (metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, and beta-adrenergic receptors) couple to MAPK activation via these two cascades. PKC is a powerful regulator of CREB phosphorylation in area CA1. MAPK plays a critical role in transcriptional regulation by PKC, because MAPK activation is a necessary component for increased CREB phosphorylation in response to the activation of this kinase. Surprisingly, MAPK activation is necessary for PKA coupling to CREB phosphorylation in area CA1. Overall, these studies indicate an unexpected richness of diversity in the regulation of MAPK in the hippocampus and suggest the possibility of a broad role for the MAPK cascade in regulating gene expression in long-term forms of hippocampal synaptic plasticity (Roberson, 1999).
Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways exert opposing effects on muscle cell hypertrophy. Manipulation of these pathways during muscle differentiation indicates that inhibition of the Ras-Raf-MEK-ERK pathway promotes differentiation, whereas inhibition of PI3K blocks differentiation. However, the roles of these two pathways in the process of skeletal muscle hypertrophy has not previously been evaluated. C2C12 myoblasts normally proliferate and are mononucleated. When deprived of serum at confluence, they fuse and differentiate into postmitotic, elongated, and multinucleated myotubes. The hypertrophic action of insulin-like growth factor-1 (IGF-1) on muscle cells in vivo is mimicked by the addition of IGF-1 during the differentiation of C2C12 myotubes in vitro, resulting in the generation of thicker myotubes. In addition to inducing hypertrophy of myotubes in vivo, IGF-1 has been shown to activate both the Raf-MEK-ERK pathway and the PI3K-Akt pathway. The roles of these two pathways in the differentiation and hypertrophy of C2C12 myotubes were examined by genetic manipulation. Expression of a constitutively active form of Raf (c.a.-Raf) results in the generation of smaller and thinner myotubes, whereas expression of a dominant negative form of Raf (d.n.-Raf) results in markedly thicker myotubes. Thus, inhibition of the Raf-MEK-ERK pathway induced a hypertrophic phenotype similar to that elicited by IGF-1 treatment. In contrast, activation of the Akt pathway by expression of a constitutively active form of Akt (c.a.-Akt) results in a hypertrophic phenotype more pronounced than that observed with d.n.-Raf and characterized by multinucleated myotubes that are both thickened and shortened. Thus, genetic manipulation of the Raf-MEK-ERK and PI3K-Akt pathways reveals opposing phenotypic effects of these pathways during muscle differentiation, with the Raf-MEK-ERK pathway inhibiting development of the hypertrophic phenotype and the PI3K-Akt pathway promoting it. The PI3K-Akt pathway inhibits the Raf-MEK-ERK pathway; this cross-regulation depends on the differentiation state of the cell: Akt activation inhibits the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlates with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex (Rommel, 1999).
In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism. B-Raf has been shown in this study to be activated by cAMP in melanocytes. A dominant-negative mutant of B-Raf, but not of Raf-1, blocks the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect. Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action. Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes. These results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP. Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation. Neither the Ras exchange factor [Son of sevenless (SOS)] nor the cAMP-responsive Rap-1 exchange factor (Epac), participates in the cAMP-dependent activation of Ras. These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP (Busca, 2000).
Because nitric oxide (NO) is a highly reactive signaling molecule, chemical inactivation by reaction with oxygen, superoxide, and glutathione competes with specific interactions with target proteins. NO signaling may be enhanced by adaptor proteins that couple neuronal NO synthase (nNOS) to specific target proteins (Fang, 2000).
A selective interaction has been identified for the nNOS adaptor protein CAPON with Dexras1, a brain-enriched member of the Ras family of small monomeric G proteins. CAPON directly interacts with the nNOS PDZ domain through its C terminus. Dexras1 is activated by NO donors as well as by NMDA receptor-stimulated NO synthesis in cortical neurons. The importance of Dexras1 as a physiologic target of nNOS is established by the selective decrease of Dexras1 activation, but not H-Ras or four other Ras family members, in the brains of mice harboring a targeted genomic deletion of nNOS (nNOS-/-). nNOS, CAPON, and Dexras1 form a ternary complex that enhances the ability of nNOS to activate Dexras1. These findings identify Dexras1 as a novel physiologic NO effector and suggest that anchoring of nNOS to specific targets is a mechanism by which NO signaling is enhanced. There has been little characterization of Dexras1, and its downstream targets are not definitively established. Members of the Rap subfamily of Ras-like G proteins transmit growth factor signals to MAP kinase; signaling cascades through B-Raf preferentially over A-Raf. Dexras1 may have similar effectors as other Rap subfamily members, since its effector loop (residues 53-61) is 78% identical to the effector loop in Rap2b (residues 32-40). Indeed, in preliminary experiments, Dexras1 has been transfected into HEK293 cells and activation of MAP kinase activity has been detected, monitored by the phosphorylation of myelin basic protein. Conceivably, Dexras1-specific effectors may exist that mediate the effects of NO in the central nervous system (Fang, 2000).
Neuronal growth cones are guided to their targets by attractive and repulsive guidance cues. In mammals, netrin-1 is a bifunctional cue, attracting some axons and repelling others. Deleted in colorectal cancer (Dcc) is a receptor for netrin-1 that mediates its chemoattractive effect on commissural axons, but the signalling mechanisms that transduce this effect are poorly understood. Dcc is shown to activate mitogen-activated protein kinase (MAPK) signalling, by means of extracellular signal-regulated kinase (ERK)-1 and -2, upon netrin-1 binding in both transfected cells and commissural neurons. This activation is associated with recruitment of ERK-1/2 to a Dcc receptor complex. Inhibition of ERK-1/2 antagonizes netrin-dependent axon outgrowth and orientation. Thus, activation of MAPK signalling through Dcc contributes to netrin signalling in axon growth and guidance (Forcet, 2002).
These results support a role for the MAPK pathway in responses to the chemoattractant netrin-1. This pathway has been implicated in neurite extension stimulated by neurotrophic factors activating Trk receptors and by cell adhesion molecules like N-cadherin, laminin and L1. Conversely, activation of some repulsive receptors of the Eph family can repress MAPK signalling, although a causal role in repulsion has not been established. How does ERK activation affect axonal growth and guidance? Some effects probably result from phosphorylation of cytoskeletal ERK targets such as microtubule-associated proteins and neurofilaments. Interestingly, a MAPK-dependent mechanism drives internalization of the cell adhesion molecules ApCAM and L1 at the rear of the growth cone, which may allow protein cycling from the rear to the leading edge necessary for growth cone advance. Dcc-stimulated MAPK activation leads to activation of the transcription factor Elk-1 and SRE-regulated gene expression, providing a mechanism for transcriptional control by netrin-1. Elk-1 is present not just in neuronal cell bodies but also in axon terminals, but whether axonal Elk-1 participates in axon guidance is unknown. Other targets of ERK-1/2 involved in axon guidance may be translation regulators. New protein translation is stimulated by netrin-1 and required for netrin-mediated attraction of Xenopus retinal growth cones in vitro. Principal factors for translation initiation like eIF4E and eIF4E-BP1 are phosphorylated by an ERK-1/2-dependent pathway, providing a potential mechanism linking netrin-1 to protein synthesis for axon growth and guidance (Forcet, 2002).
Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. FRL-1, a Xenopus EGF-CFC gene isolated as a ligand of the FGF receptor and homolog of zebrafish One-eyed pinhead, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induces the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppresses the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) shows that FRL-1 is required for neural development. FRL-1MO-injected embryos lack neural structures but contained mesodermal tissue. It has been suggested that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibits the expression of neural markers that are induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response, and suggest that antagonistic effects between the FRL-1/FGFR/MAPK pathway and BMP signaling are involved in the establishment of neural versus epidermal cell fate (Yabe, 2003).
The MEK5-extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. The aPKCs are shown to interact in an EGF-inducible manner with MEK5; this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, zetaPKC and lambda/iotaPKC are shown to activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, it is concluded that MEK5 is a critical target of the aPKCs during mitogenic signaling (Diaz-Meco, 2001).
The relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway has been studied. cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 results in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) -/- mice, which lack appreciable cdk5 activity, an increase is observed in the phosphorylation of NF-M subunit of neurofilament proteins that correlate with an up-regulation of MEK1 and ERK1/2 activity. The activity of a constitutively active MEK1 with threonine 286 mutated to alanine (within a TPXK cdk5 phosphorylation motif in the proline-rich domain) is not affected by cdk5 phosphorylation, suggesting that Thr286 might be the cdk5/p35 phosphorylation-dependent regulatory site. These findings support the hypothesis that cdk5 and the MAP kinase pathway cross-talk in the regulation of neuronal functions. Moreover, these data have prompted the proposal of a model for feedback down-regulation of the MAP kinase signal cascade by cdk5 inactivation of MEK1 (Sharma, 2002).
The ERK1/ERK2 MAP kinases (MAPKs) are transiently activated during mitosis, and MAPK activation has been implicated in the spindle assembly checkpoint and in establishing the timing of an unperturbed mitosis. The MAPK activator MEK1 is required for mitotic activation of p42 MAPK in Xenopus egg extracts; however, the identity of the kinase that activates MEK1 is unknown. A Cdc2-cyclin B-induced MEK-activating protein kinase has been partially purified from mitotic Xenopus egg extracts, and it has been identified as the Mos protooncoprotein, a MAP kinase kinase kinase present at low levels in mitotic egg extracts, early embryos, and somatic cells. Immunodepletion of Mos from interphase egg extracts was found to abolish Delta90 cyclin B-Cdc2-stimulated p42 MAPK activation. In contrast, immunodepletion of Raf-1 and B-Raf, two other MEK-activating kinases present in Xenopus egg extracts, had little effect on cyclin-stimulated p42 MAPK activation. Immunodepletion of Mos also abolished the transient activation of p42 MAPK in cycling egg extracts. Taken together, these data demonstrate that Mos is responsible for the mitotic activation of the p42 MAPK pathway in Xenopus egg extracts (Yue, 2004).
So far, most of the work on Mos function has focused on meiosis because during meiosis the Mos protein is expressed at relatively high levels. The most obvious defect in Mos knockout mice is a diminished ability of their oocytes to arrest properly in meiosis II; this phenotype is consistent with the idea that Mos functions primarily or exclusively during oocyte meiosis. However, the present results suggest that the low levels of Mos protein that remain after fertilization might be functionally significant as well; without this Mos, Xenopus egg extracts fail to activate their p42 MAPK during mitosis. This raises the possibility that low concentrations of Mos might contribute to the transient activation of MAPK and MEK seen at the spindle poles and kinetochores of somatic cells during mitosis. In a manner consistent with this idea, it has been shown that microinjected Mos localizes to kinetochores in fibroblasts. If, as seems plausible, endogenous Mos also localizes to kinetochores, it would put Mos in the appropriate location to be responsible for the mitotic activation of kinetochore-associated MEK and ERKs in somatic cells. Mos mRNA and protein have also been reported to be present in other somatic cells, albeit at low levels. It will be of interest to reopen the question of whether Mos is involved in M phase regulation in somatic cells as well as in oocytes and embryos (Yue, 2004).
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