Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA₂ activation. Incubation with the membrane phospholipid-derived PLA₂ hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA₂ activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS.     

Mitogen-Activated Protein Kinase (MAPK) Pathway Regulates Branching by Remodeling Epithelial Cell Adhesion

Although the growth factor (GF) signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK) pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA) protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions.     

Potassium Chloride Pulse Enhances Mitogen-Activated Protein Kinase Activity in Rat Hippocampal Slices

Abstract: Mitogen-activated protein (MAP) kinases have been implicated in multiple responses to extracellular stimuli. In this study we show that MAP kinase activity is enhanced after a KCI pulse. This activation correlates with an increased tyrosine phosphorylation of a 42-kDa protein as determined by antiphosphotyrosine immunoblot. The same band is found in an anti-MAP kinase immunoblot. Activity is enhanced within 1 min, reaches a maximum at 2 min, and returns to basal level after 10 min. A second peak of activity is observed between 12 and 30 min. The activation is completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing the involvement of the AMPA type of glutamate receptor. Partial inhibition of MAP kinase activation by 2-amino-5-phosphonovalerate (APV) also shows the involvement of the NMDA receptor. Because the KCI pulse used induces long-term potentiation (LTP) in rat hippocampal slice, we conclude that MAP kinase may be involved in neuronal transduction events leading to LTP.     

Coordinated Regulation of Radioadaptive Response by Protein Kinase C and p38 Mitogen-Activated Protein Kinase

Eukaryotic cells are known to have an inducible or adaptive response that enhances radioresistance after a low priming dose of radiation. This radioadaptive response seems to present a novel cellular defense mechanism. However, its molecular processing and signaling mechanisms are largely unknown. Here, we studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the expression of radioadaptive response in cultured mouse cells. Protein immunoblot analysis using isoform-specific antibodies showed an immediate activation of PKC-alpha upon X-irradiation as indicated by a translocation from cytosol to membrane. A low priming dose caused a prolonged translocation, while a nonadaptive high dose dramatically downregulated the total PKC level. Low-dose X-rays also activated the p38 MAPK. The activation of p38 MAPK and resistance to chromosome aberration formation were blocked by SB203580, an inhibitor of p38 MAPK, and Calphostin C, an inhibitor of PKC. Furthermore, it was demonstrated that p38 MAPK was physically associated with delta1 isoform of phospholipase C (PLC-delta1), which hydrolyzed phosphatidylinositol bisphosphate into diacylglycerol, an activator of PKC, and that SB203580 also blocked the activation of PKC-alpha. These results indicate the presence of a novel mechanism for coordinated regulation of adaptive response to low-dose X-rays by a nexus of PKC-alpha/p38 MAPK/PLC-delta1 circuitry feedback signaling pathway with its breakage operated by downregulation of labile PKC-alpha at high doses or excess stimuli.     

The Colletotrichum orbiculare ssd1 Mutant Enhances Nicotiana benthamiana Basal Resistance by Activating a Mitogen-Activated Protein Kinase Pathway

Plant basal resistance is activated by virulent pathogens in susceptible host plants. A Colletotrichum orbiculare fungal mutant defective in the SSD1 gene, which regulates cell wall composition, is restricted by host basal resistance responses. Here, we identified the Nicotiana benthamiana signaling pathway involved in basal resistance by silencing the defense-related genes required for restricting the growth of the C. orbiculare mutant. Only silencing of MAP Kinase Kinase2 or of both Salicylic Acid Induced Protein Kinase (SIPK) and Wound Induced Protein Kinase (WIPK), two mitogen-activated protein (MAP) kinases, allowed the mutant to infect and produce necrotic lesions similar to those of the wild type on inoculated leaves. The fungal mutant penetrated host cells to produce infection hyphae at a higher frequency in SIPK WIPK-silenced plants than in nonsilenced plants, without inducing host cellular defense responses. Immunocomplex kinase assays revealed that SIPK and WIPK were more active in leaves inoculated with mutant fungus than with the wild type, suggesting that induced resistance correlates with MAP kinase activity. Infiltration of heat-inactivated mutant conidia induced both SIPK and WIPK more strongly than did those of the wild type, while conidial exudates of the wild type did not suppress MAP kinase induction by mutant conidia. Therefore, activation of a specific MAP kinase pathway by fungal cell surface components determines the effective level of basal plant resistance.     

Activation of Mitogen-Activated Protein Kinase by Epidermal Growth Factor in Hippocampal Neurons and Neuronal Cell Lines

Abstract: Epidermal growth factor (EGF) functions in a bimodal capacity in the nervous system, acting as a mitogen in neuronal stem cells and a neurotrophic factor in differentiated adult neurons. Thus, it is likely that EGF signal transduction, as well as receptor expression, differs among various cell types and possibly in the same cell type at different stages of development. We used hippocampal neuronal cell lines capable of terminal differentiation to investigate changes in EGF receptor expression, DNA synthesis, and stimulation of mitogen-activated protein (MAP) kinase by EGF before and after differentiation. H19-7, the line that was most representative of hippocampal neurons, was mitogenically responsive to EGF only before differentiation and increased in EGF binding after differentiation. MAP kinase was stimulated by EGF in both undifferentiated and differentiated cells, as well as in primary hippocampal cultures treated with either EGF or glutamate. These results indicate that the activation of MAP kinase by EGF is an early signaling event in both mitotic and postmitotic neuronal cells. Furthermore, these studies demonstrate the usefulness of hippocampal cell lines as a homogeneous neuronal system for studies of EGF signaling or other receptor signaling mechanisms in the brain.     

Calcium/Ganglioside-Dependent Protein Kinase Activity in Rat Brain Membrane

The effects of gangliosides on phosphorylation were studied in rat brain membrane. Gangliosides stimulated phosphorylation only in the presence of Ca2+ with major phosphoproteins of 45,000, 50,000, 60,000, and 80,000 daltons and high-molecular-weight species. In addition, gangliosides inhibited the phosphorylation of three proteins with molecular weights of 15,000, 20,000, and 78,000 daltons. The two low-molecular-weight proteins comigrated with rat myelin basic proteins. Ganglioside stimulation was dependent on the formation of a Ca2+-ganglioside complex since the calcium salt of gangliosides stimulated phosphorylation maximally. Disialo and trisialo gangliosides were more potent stimulators of kinase activity than the monosialo GM1 X GD1a was the most potent activator tested. Asialo-GM1, cerebroside, sialic acid, neuraminyllactose, sulfatide, and the acidic phospholipids phosphatidylserine and phosphatidylinositol did not stimulate kinase activity. The Ca2+-dependent, ganglioside-stimulated phosphorylation was qualitatively similar to the pattern for calmodulin-dependent phosphorylation. However, while calmodulin-dependent kinase activity was inhibited with an IC50 of 10 microM trifluoperazine, ganglioside-stimulated kinase was inhibited with an IC50 of 200 microM trifluoperazine. These results indicate that gangliosides have complex effects on membrane-associated kinase activities and suggest that Ca2+-ganglioside complexes are potent stimulators of membrane kinase activity.     

Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen-Activated Protein Kinase Cascade

Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β-peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.     

Fibroblast Growth Factors Stimulate Protein Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase Activity in Primary Cultures of Hippocampal Neurons

Abstract: Fibroblast growth factors (FGFs) are not only mitogens, but they also promote the differentiation of various cell types. For instance, basic FGF (bFGF) provides a critical trophic support for hippocampal neurons in culture. To elicit their biological effects, FGFs interact with high-affinity receptors that are transmembrane proteins with a cytoplasmic portion containing a tyrosine kinase activity. The tyrosine phosphorylation pattern was examined in primary cultures of hippocampal neurons derived from rat embryos. In these cultures grown for 3 days in the absence of serum, the addition of bFGF causes a rapid increase of tyrosine phosphorylation for various proteins with an optimal level after 5 min of bFGF exposure. Concomitantly, bFGF activates mitogen-activated protein kinase (MAP kinase) activity measured with a selective MAP kinase peptide. The activity increased rapidly after the addition of bFGF and remained elevated even when cultures were treated for 1 h with bFGF. Both acidic and basic FGF were able to enhance protein tyrosine phosphorylation and MAP kinase activity, whereas nerve growth factor and epidermal growth factor did not elicit any of these responses. These data indicate that some of the transduction signals (i.e., tyrosine phosphorylation and activation of MAP kinase) that have been described for the proliferative effect of FGFs are also involved when FGFs act as trophic factors for postmitotic neurons in culture.     

Direct Activation of Mitogen-Activated Protein Kinase Kinase Kinase MEKK1 by the Ste20p Homologue GCK and the Adapter Protein TRAF2

Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues. The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood. Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive. Here we show that endogenous GCK and MEKK1 associate in vivo. In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1. The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not. Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation. Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo. These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins.     

The Expression Pattern of the Gene for NPK1 Protein Kinase Related to Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) in a Tobacco Plant: Correlation with Cell Proliferation

Mitogen-activated protein kinase (MAPK) cascades consist ofmembers of three families of protein kinases: the MAPK family,the MAPK kinase family, and the MAPK kinase kinase (MAPKKK)family. Some of these cascades have been shown to play centralroles in the transmission of signals that control various cellularprocesses including cell proliferation. Protein kinase NPK1is a structural and functional tobacco homologue of MAPKKK,but its physiological function is yet unknown. In the presentstudy, we have investigated sites of expression of the NPK1gene in a tobacco plant and developmental and physiologicalcontrols of this expression. After germination, expression ofNPK1 was first detected in tips of a radicle and cotyledons,then in shoot and root apical meristems, surrounding tissuesof the apical meristems, primordia of lateral roots, and youngdeveloping organs. No expression was, however, observed in matureorgans. Incubation of discs from mature leaves of tobacco withboth auxin and cytokinin induced NPK1 expression before thedivision of cells. It was also induced at early stages of thedevelopment of primordia of lateral roots and adventitious roots.Thus, NPK1 expression appears to be tightly correlated withcell division or division competence. Even when an inhibitorof DNA synthesis was added during the germination or the inductionof lateral roots by auxin, NPK1 expression was detected. Theseresults showed that the NPK1 expression precedes DNA replication.We propose that NPK1 participates in a process involving thedivision of plant cells. (Received January 26, 1998; Accepted April 9, 1998)     

Activation of p42 Mitogen-Activated Protein Kinase by Glutamate in Cultured Radial Glia

The effect of L-glutamate (Glu) and its structural analogs N-methyl-D-aspartate (NMDA), kainate (KA) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), on the activation of p42 mitogen activated protein kinase (MAPK) was examined in cultured chick radial glia cells, namely retinal Müller cells and cerebellar Bergmann cells. Glu, NMDA, AMPA and KA evoked a dose and time dependent increase in MAPK activity. AMPA and KA responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) whereas NMDA responses were sensitive to 3-[(RS)-2-carboxypiperazin-4-yl)]-propyl-1-phosphonate (CPP) indicating that the increase in MAPK activity is mediated by AMPA/low affinity KA and NMDA subtypes of Glu receptors. The present findings open the possibility of a MAPK cascade involvement in the regulation of Glu-induced gene expression in radial glia.     

Nucleus-Specific and Cell Cycle-Regulated Degradation of Mitogen-Activated Protein Kinase Scaffold Protein Ste5 Contributes to the Control of Signaling Competence

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G₁ phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF^Cdc4 ubiquitin ligase and requires phosphorylation by the G₁ cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G₁ Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.Scaffold proteins play a pivotal role in spatial and temporal regulation of multitiered mitogen-activated protein kinase (MAPK) cascades (8, 30, 107). Scaffold protein function can be controlled at several different levels, including phosphorylation, oligomerization, and subcellular localization, which can dramatically influence signaling (5, 21, 61).A well-characterized scaffold-dependent MAPK pathway drives the mating pheromone response in budding yeast Saccharomyces cerevisiae (15). The occupancy of a heterotrimeric G-protein-coupled receptor by pheromone results in release of its associated membrane-tethered Gβγ (Ste4-Ste18) complex. Ste5 scaffold protein (917 residues) is recruited to the plasma membrane via its association with this freed Gβγ (106) and by additional multivalent contacts with membrane phospholipids mediated by an N-terminal amphipathic α-helix (PM motif) (111) and an internal PH domain (34). Because Ste5 is also able to bind a MAPK kinase kinase (Ste11), a MAPK kinase (Ste7), and two MAPKs (Fus3 and Kss1) (102), membrane recruitment of Ste5 delivers these components to the plasma membrane. Membrane localization of Ste5 juxtaposes its passenger kinases to Ste20, a p21-activated protein kinase that also interacts with membrane phospholipids (94) and requires plasma membrane-tethered and GTP-loaded Cdc42 for its activation (56, 58, 60). GTP-bound Cdc42 is generated in this vicinity via other Gβγ-recruited effectors, especially Far1, which binds the Cdc42 guanine nucleotide exchange factor, Cdc24 (14, 98). Once activated, Ste20 directly phosphorylates and activates the Ste11 MAPK kinase kinase, triggering the MAPK cascade (24, 114).In naïve haploid cells, Ste5 undergoes continuous nucleocytoplasmic shuttling but is located predominantly in the nucleus (53, 66). In response to pheromone, this flux is dramatically shifted in favor of export, elevating the cytosolic pool of Ste5, thereby raising the number of molecules available for membrane recruitment (66, 79). Pheromone-induced nuclear export of Ste5 requires the exportin, Msn5/Ste21 (66).Little is known about why Ste5 is located in the nucleus in unstimulated cells. It has been suggested that passage of Ste5 through the nucleus modifies it in an as yet undefined manner to make it “competent” to subsequently promote signaling at the membrane (66, 103). However, other evidence indicates that nuclear shuttling of Ste5 is not necessary for its translocation to the plasma membrane or its function (34, 79, 111) and that reimport into the nucleus contributes to pathway downregulation following initial stimulation (53). It has remained obscure, mechanistically speaking, how nuclear localization of Ste5 contributes to the regulation of pathway activation and signal flux.Given that Ste5 is the least abundant component of this entire signaling system (≤500 molecules per haploid cell) (38), we suspected that dynamic regulation of the location and level of this scaffold protein provides a critically important control point for influencing the timing, potency, duration, and specificity of signaling in this pathway. Indeed, as described here, we found that the subcellular localization of Ste5 and cell cycle progression have dramatic effects in controlling the stability of Ste5. Our findings provide new insights about the physiological importance of Ste5 nuclear localization and G₁ cyclin-dependent protein kinase 1 (CDK1) action in establishment and maintenance of the conditions that preserve signaling fidelity in this system.