Coupling of M(2) muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle

Coupling of M(2) and M(3) muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M(3) receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser(789)), a putative downstream target of MAP kinases. Alkylation of M(3) receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 microM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M(2) receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M(2) receptor activation in smooth muscle.     

HIV-1 Tat activates dual Nox pathways leading to independent activation of ERK and JNK MAP kinases

Human immunodeficiency virus, type 1 Tat is known to exert pleiotropic effects on the vascular endothelium through mitogen-activated protein (MAP) kinases, although the signaling pathways leading to MAP kinase activation are incompletely understood. We focused on proximal pathways potentially governing downstream MAP kinase activity by Tat. Within 2 min, Tat activated both Ras and Rho GTPases in endothelial cells, leading to ERK phosphorylation by 10 min. Notably, Rac1 was necessary for downstream activation of RhoA and both Rac1 and RhoA acted upstream of the Ras/ERK cassette. Antioxidants and the oxidase inhibitor diphenylene iodonium blocked ERK phosphorylation, but specific interference with the canonical Nox2 oxidase had no effect on ERK. Instead, knock down of the novel oxidase Nox4 completely suppressed Tat-dependent Ras and ERK activation downstream of Rac1 and RhoA. Conversely, interference with Rac1, PAK1, and Nox2 blocked JNK phosphorylation, whereas RhoA(N19) and Nox4 knock down did not. Further, knock down of Nox2, but not Nox4, blocked Tat-induced cytoskeletal rearrangement, whereas knock down of Nox4, but not Nox2, blocked Tat-dependent proliferation. Rac1, therefore, bifurcates Tat signaling, leading to concurrent but separate Nox4-dependent Ras/ERK activation, and Nox2-dependent JNK activation. Tat signaling, therefore, provides an example of Nox-specific differential control of MAP kinase pathways.     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

The activity of the extracellular signal-regulated kinase 2 is regulated by differential phosphorylation in the activation loop

The mitogen-activated protein kinases (MAP kinases) play a central role in signaling pathways initiated by extracellular stimuli such as growth factors, cytokines, and various forms of environmental stress. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Interestingly, down-regulation of MAP kinase activity can be initiated by multiple Ser/Thr phosphatases, Tyr-specific phosphatases, and dual-specificity phosphatases. This would inevitable lead to the formation of monophosphorylated MAP kinases. However, in much of the literature investigating MAP kinase signaling, there has been the implicit assumption that the monophosphorylated forms are inactive. Thus, the significance for the need of multiple phosphatases in regulating MAP kinase activity is not clear, and the biological functions of these monophosphorylated MAP kinases are currently unknown. We have prepared extracellular signal-regulated protein kinase 2 (ERK2) in all phosphorylated forms and kinetically characterized them using two proteins (the myelin basic protein and Elk-1) and ATP as substrates. Our results revealed that a single phosphorylation in the activation loop of ERK2 produces an intermediate activity state. Thus, the catalytic efficiencies of the monophosphorylated ERK2/pY and ERK2/pT (ERK2 phosphorylated on Tyr-185 and Thr-183, respectively) are approximately 2-3 orders of magnitude higher than that of the unphosphorylated ERK2 and are only 1-2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY. This raises the possibility that the monophosphorylated ERK2s may have distinct biological roles in vivo. Different phosphorylation states in the activation loop could be linked to graded effects on a single ERK2 function. Alternatively, they could be linked to distinct ERK2 functions. Although less active than the bisphosphorylated species, the monophosphorylated ERK2s may differentially phosphorylate pathway components.     

Mitogen-activated protein kinases are in vivo transducers of osmosensory signals in fish gill cells

The abundance and activity of three subgroups of mitogen-activated protein (MAP) kinases, the extracellular signal regulated kinase 1 (ERK1), stress-activated protein kinase 1/ Jun N-terminal kinase (SAPK1), and stress-activated protein kinase 2/ p38 (SAPK2), were measured in gill epithelium of the euryhaline teleost Fundulus heteroclitus exposed for 1 h to 4 weeks to hyper- and hyposmotic stress. The abundance of ERK1, SAPK1 and SAPK2 was analyzed by standard Western immunodetection. MAP kinase activity is a function of phosphorylation and was measured using phospho-specific and MAP kinase subgroup-specific antibodies. The abundance of the 63 kDa fish isoform of SAPK2 increases significantly during hyper- but not hyposmotic stress while ERK1 and SAPK1 protein levels remain unchanged during both types of osmotic stress. In contrast to this small effect of osmotic stress on MAP kinase abundance, the activity of all MAP kinases decreases significantly in response to hyperosmotic stress and increases significantly during hyposmotic stress. These results demonstrate for the first time that the activity of all major MAP kinases is osmoregulated in gill epithelium of euryhaline fish. Based on these results we conclude that MAP kinases are important components of salinity adaptation and participate in osmosensory signaling pathways in gill epithelium of euryhaline fishes.     

Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells

All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.     

Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase

Activation of the bradykinin B2 receptor in endothelial cells initiates a complex array of cellular responses mediated by diverse signaling pathways, including stimulation of the mitogen-activated protein (MAP) kinase cascade and activation of the endothelial isoform of nitric-oxide synthase (eNOS). Several protein kinases have been implicated in eNOS regulation, but the role of MAP kinases remains less well understood. We explored the interactions between eNOS and components of the MAP kinase pathway in bovine aortic endothelial cells (BAEC). Using co-immunoprecipitation experiments, we isolated eNOS in a complex with the MAP kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as the protein kinases Raf-1 and Akt. Within minutes of adding bradykinin to BAEC, the eNOS-Raf-1-ERK-Akt heteromeric complex dissociated, and it subsequently reassociated following more prolonged agonist stimulation. Bradykinin treatment of BAEC led to the activation of ERK, associated with an increase in phosphorylation of eNOS; phosphorylation of eNOS by ERK in vitro significantly reduced eNOS enzyme activity. Evidence for the direct phosphorylation of eNOS by MAP kinase in BAEC came from "back-phosphorylation" experiments using [gamma-(32)P]ATP and ERK in vitro to phosphorylate eNOS isolated from cells previously treated with bradykinin or the MAP kinase inhibitor PD98059. The ERK-catalyzed in vitro (32)P phosphorylation of eNOS isolated from BAEC treated with bradykinin was significantly attenuated compared with untreated cells, indicating that bradykinin treatment led to the phosphorylation of ERK-sensitive sites in cells. Conversely, eNOS isolated from endothelial cells pretreated with the MAP kinase inhibitor PD98059 showed increased ERK-promoted phosphorylation in vitro. Taken together, our results suggest that bradykinin-induced activation of ERK leads to eNOS phosphorylation and enzyme inhibition, a process influenced by the reversible associations of members of the MAP kinase pathway with eNOS.     

TRAF6 and Src kinase activity regulates Cot activation by IL-1

Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.     

Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss.     

Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1

Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2 small middle dotMEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding alpha-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding alpha-helix G in the interaction of ERK2 with MEK1/2.     

The JNK and P38 MAP kinase signaling pathways in T cell-mediated immune responses

The mitogen-activated protein (MAP) kinase family members, which include the extracellular response kinases (ERK), p38, and c-Jun amino terminal kinases (JNK), play a role in mediating signals triggered by cytokines, growth factors, and environmental stress. JNK and p38 MAP kinases have been involved in inflammatory processes induced by a variety of stimuli, such as oxidative stress. Here, we describe the role of the JNK and p38 MAP kinase signaling pathways in the development of T cells in the thymus, and activation and differentiation of T cells in the peripheral immune system.     

Glutamate-stimulated activation of DNA synthesis via mitogen-activated protein kinase in primary astrocytes: involvement of protein kinase C and related adhesion focal tyrosine kinase

Glutamate is the major excitatory neurotransmitter in the CNS. Although its role in neurons has been studied extensively, little is known about its function in astrocytes. We studied the effects of glutamate on signaling pathways in primary astrocytes. We found that the tyrosine kinase related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated in response to glutamate in a time- and dose-dependent manner. This phosphorylation was pertussis toxin (PTX) sensitive and could be attenuated by the depletion of Ca2+ from intracellular stores. RAFTK tyrosine phosphorylation was mediated primarily by class I/II metabotropic glutamate receptors and depends on protein kinase C (PKC) activation. Glutamate treatment of primary astrocytes also results in a significant increase in the activity of the mitogen-activated protein kinases [extracellular signal-related kinases 1/2 (ERK1/2)]. Like RAFTK phosphorylation, ERK1/2 activation is PTX sensitive and can be attenuated by the depletion of intracellular Ca2+ and by PKC inhibition, suggesting that RAFTK might mediate the glutamate-dependent activation of ERK1/2. Furthermore, we demonstrated that glutamate stimulation of primary astrocytes leads to a significant increase in DNA synthesis. Glutamate-stimulated DNA synthesis is PTX sensitive and can be inhibited by the MAP kinase kinase inhibitor PD98059, suggesting that in primary astrocytes, glutamate might signal via RAFTK and MAP kinase to promote DNA synthesis and cell proliferation.     

Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway

To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase Cgamma signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation.