Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Phosphorylation of caldesmon was assayed in canine colonic circular smooth muscle strips labelled with 32P and stimulated with 10 microM acetylcholine. Caldesmon was isolated by two-dimensional non-equilibrium pH gel electrophoresis. Stimulation with acetylcholine increased caldesmon phosphorylation significantly from a basal level of 0.6 +/- 0.07 to 1.1 +/- 0.15 mol P1 (mol caldesmon)-1 after 2 min. 2. MAP kinase activities were measured in SDS extracts of muscle by a gel reconstitution method using myelin basic protein. Myelin basic protein kinase activities were observed at 38, 44, 50 and 57 kDa by the gel reconstitution method. Endogenous caldesmon kinase activities were also identified by the gel reconstitution method at 38, 44 and 50 kDa. The 38 and 44 kDa kinases comigrated with proteins labelled by anti-ERK1 MAP kinase antibodies on Western blots. Both 38 and 44 kDa MBP kinase activities increased significantly during contractions induced by 10 microM acetylcholine, 0.1 microM neurokinin A and 70 mM potassium. 3. Phorbol dibutyrate (0.1 microM) potentiated activation of MAP kinases and contraction of depolarized muscles while producing a decrease in fura-2 fluorescence ratio. This suggests that protein kinase C activation is coupled to MAP kinase activity in colonic smooth muscle. 4. MAP kinases isolated form muscle homogenates by Mono Q chromatography were assayed using the specific MAP kinase substrate peptide APRTPGGRR. Stimulation of muscles for 2 min with 10 microM acetylcholine activated both ERK1 and ERK2 MAP kinase activities 2-fold. 5. To determine the effects of caldesmon phosphorylation by MAP kinase on the cross-bridge cycle, actin sliding velocity was measured with an in vitro motility assay. Unphosphorylated turkey gizzard caldesmon (3 microM) significantly reduced mean sliding velocity. Phosphorylation of caldesmon with sea star ERK1 MAP kinase reversed the inhibitory effect of caldesmon on sliding velocity. The results are consistent with a protein kinase cascade being activated by contractile agonists in gastrointestinal smooth muscle which activates ERK MAP kinases leading to phosphorylation of caldesmon. Phosphorylation of caldesmon in vivo may reverse inhibitory influences of caldesmon on cross-bridge cycling.
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PMID:Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle. 888 69

To test the hypothesis that mitogen-activated protein (MAP) kinases are activated by contractile agonists in intact nonproliferating airway smooth muscle, kinase activities were compared in resting and stimulated canine tracheal smooth muscle. Kinase activities in sodium dodecyl sulfate extracts were assayed by a gel renaturation method. Myelin basic protein kinase activities corresponding to ERK1 and ERK2 immunoreactive proteins were activated twofold above the basal level within 5 min by 1 microM carbachol. MAP kinase activity assayed in crude homogenates using a synthetic peptide substrate (APRTPGGRR) also increased twofold above basal in muscles stimulated with 1 microM carbachol. Two protein kinases separated by Mono-Q chromatography were identified on Western blots as ERK1 and ERK2 MAP kinases. Carbachol stimulation increased caldesmon phosphorylation in intact muscle, and purified caldesmon was a substrate for activated murine ERK2 MAP kinase. Activated ERK2 MAP kinase added to Triton X-100-permeabilized fibers potentiated Ca2+-induced contraction. The results show that ERK MAP kinases are activated after stimulation of muscarinic receptors in airway smooth muscle, which is consistent with coupling of MAP kinases to phosphorylation of caldesmon in vivo.
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PMID:Activation of MAP kinases in airway smooth muscle. 912 75

We have previously shown that p21-activated kinase, PAK, induces Ca(2+)-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J. E., Arrell, D. K., Foster, D. B., Strauss, J. D., Heinonen, T. Y., Furmaniak-Kazmierczak, E., Cote, G. P., and Mak, A. S. (1998) J. Biol. Chem. 273, 23433-23439). In this study, we provide biochemical evidence implicating a role for PAK in Ca(2+)-independent contraction of smooth muscle via phosphorylation of caldesmon. Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon. PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation. Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin. We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca(2+) sensitivity of smooth muscle contraction.
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PMID:Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction. 1063 98

Bordetella pertussis is an important cause of infection in humans worldwide, with full expression of the syndrome associated with characteristic increases in lung permeability and airway edema. The exact cellular mechanisms by which pertussis toxin (PTX) exerts pulmonary toxicity remain unknown, but may involve its ability to ADP-ribosylate-specific G-proteins. We determined that PTX directly and reproducibly reduced lung endothelial and epithelial cell barrier function in vitro and in vivo assessed by decreases in transmonolayer electrical resistance (TER) and isolated perfused lung preparations. Alterations in lung permeability began approximately 30 min after PTX and were dependent on intrinsic ADP-ribosyltransferase activity, as neither the cell binding beta-oligomer subunit or a genetically engineered PTX mutant (devoid of ADP-ribosyltransferase activity) altered TER. PTX-induced barrier dysfunction was associated with mild increases in F-actin stress fiber formation and causally linked to p38 MAP kinase activities. PTX-mediated p38 MAP kinase activation did not involve either p42/p44 ERK, p60src, Rho family of GTPases, or phosphatidylinositol-3' kinase pathways. PTX-mediated decreases in TER were temporally linked to phosphorylation of the actin binding proteins Hsp27 and caldesmon, known substrates for the Ser/Thr kinase MAPKAP2, whose activity is regulated by p38 MAP kinase. In addition to defining novel signaling pathways involved in PTX-induced respiratory pathophysiology, these data suggest that the direct cell-activating effects of PTX be carefully considered as a potential limitation to its use as a tool in signal transduction analysis.
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PMID:Critical involvement of p38 MAP kinase in pertussis toxin-induced cytoskeletal reorganization and lung permeability. 1208 68

In Triton-skinned phasic ileal smooth muscle, constitutively active recombinant p21-activated kinase (PAK3) has been shown to induce Ca(2+)-independent contraction, which is accompanied by phosphorylation of caldesmon and desmin (Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, Furmaniak-Kazmierczak E, Cote GP, and Mak AS. J Biol Chem 273: 23433-23439, 1998). In the present study, we investigated whether PAK has a broad impact on smooth muscle in general by testing the hypothesis that PAK induces Ca(2+)-independent contractions and/or Ca(2+) sensitization in tonic airway smooth muscle and that the process is mediated via phosphorylation of caldesmon. In the absence of Ca(2+) (pCa > 9), constitutively active glutathione-S-transferase-murine PAK3 (GST-mPAK3) caused force generation of Triton-skinned canine tracheal smooth muscle (TSM) fibers to approximately 40% of the maximal force generated by Ca(2+) at pCa 4.4. In addition, GST-mPAK3 enhanced Ca(2+) sensitivity of contraction by increasing force generation by 80% at intermediate Ca(2+) concentrations (pCa 6.2), whereas it had no effect at pCa 4.4. Catalytically inactive GST-mPAK3(K297R) had no effect on force production. Using antibody against one of the PAK-phosphorylated sites (Ser(657)) on caldesmon, we showed that a basal level of phosphorylation of caldesmon occurs at this site in skinned TSM and that PAK-induced contraction was accompanied by a significant increase in the level of phosphorylation. Western blot analyses show that PAK1 is the predominant PAK isoform expressed in murine, rat, canine, and porcine TSM. We conclude that PAK causes Ca(2+)-independent contractions and produces Ca(2+) sensitization of skinned phasic and tonic smooth muscle, which involves an incremental increase in caldesmon phosphorylation.
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PMID:Calcium-independent contraction and sensitization of airway smooth muscle by p21-activated protein kinase. 1251 68

Gastrointestinal stromal tumor (GIST) is now defined as a specific, KIT-expressing and KIT-signaling driven mesenchymal tumor of the gastrointestinal (GI) tract. The specific identification of GIST has become more important after the availability of KIT-selective tyrosine kinase inhibitor Imatinib mesylate, STI571, commercially known as Gleevec/Glivec (Novartis Pharma, Basel, Switzerland) in the treatment of unresectable and metastatic tumors. GISTs are the most common mesenchymal neoplasms of the GI tract, and encompass most tumors previously classified as gastric and intestinal smooth muscle tumors. GISTs typically present in adults over 40 years (median age 55-60 years) and only exceptionally in children. They can present anywhere in the GI-tract from the lower esophagus to the anus. A great majority of GISTs occur in the stomach (60-70%) or small intestine (25-35%). Colon, rectum, appendix (together 5%) and esophagus (2-3%) are rare sites. Some GISTs are primary in the omentum, mesentery or retroperitoneum, unrelated to the tubular GI-tract, but most GISTs in these sites are metastases from gastric or intestinal primary. Histologically GISTs vary from cellular spindle cell tumors to epithelioid and pleomorphic ones, and morphology differs somewhat by site. By definition, GISTs are KIT(CD117)-positive. Positivity for nestin (90-100%) and CD34 (70%) are also characteristic but less specific features. Smooth muscle actins (20-30%) and heavy caldesmon (80%) are often expressed, whereas desmin is usually absent. Predictive of malignancy are mitotic rate over 5 per 50 HPF or size over 5 cm. However, mitotically inactive intestinal tumors can metastasize, and gastric tumors are in average less often malignant than the intestinal ones. True smooth muscle tumors, GI-schwannoma and undifferentiated sarcomas are the most important differential diagnoses. KIT activating mutations occur in 70-80% of cases. Their signaling consequences, clinical correlation and response to tyrosine kinase inhibitors, and specific genetic alterations are under intense investigation. Majority of these mutations are in-frame-deletions and missense mutations clustering in the 5'-end of juxtamembrane domain (exon 11). A rare mutation, an Ala502-Tyr503 duplication in exon 9, is specific for intestinal GISTs.
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PMID:Gastrointestinal stromal tumors (GISTs): definition, occurrence, pathology, differential diagnosis and molecular genetics. 1281 76

Inflammatory fibroid polyps (IFPs) are rare mesenchymal tumors of the gastrointestinal tract that consist of spindle-shaped stromal cells and an inflammatory infiltrate rich in eosinophils. Their etiology and histogenesis remain unknown. Based on previous reports of their immunoreactivity for CD34 and c-kit biomarkers, IFPs have been thought to be related to gastrointestinal stromal tumors (GISTs). After reviewing the current literature and examining IFPs at the light microscopic level, we evaluated a series of IFPs using an extensive panel of immunohistochemical and in situ hybridization markers in an effort to gain insight into their etiology and histogenesis and to determine their true relationship to GISTs. Sixteen routinely processed IFP specimens (14 gastric, 1 ileal, and 1 rectal) were immunohistochemically stained for antibodies to CD34, HMB-45, desmin, smooth muscle actin, calponin, h-caldesmon, anaplastic lymphoma kinase, S-100 protein, epithelial membrane antigen, c-kit (CD117), stem cell factor (SCF/N19 or kit ligand), p53, bcl-2, cyclin D1, and human herpesvirus-8 (HHV8). In situ hybridization for Epstein-Barr virus-encoded RNA (EBER) was also performed. Ten cases were further evaluated for the dendritic cell markers fascin, CD21, CD23, and CD35. Stromal cells were diffusely positive for CD34 and fascin in all (100%) cases, and these stromal cells were, in addition, immunoreactive for calponin and smooth muscle actin in 88% and 25% of cases, respectively. CD35 was also found to be focally reactive in the stromal cells. Cyclin-D1 was overexpressed in all (100%) IFPs. All other immunohistochemical markers and EBER were negative in the stromal cells. These findings suggest that the proliferating stromal cells in IFPs are of dendritic cell origin, with some cases also exhibiting myofibroblastic features. Absence of c-kit, SCF, and h-caldesmon immunoreactivity fails to support a relationship to GISTs. We also conclude that Epstein Barr virus and HHV8 are unlikely etiologic agents of IFPs. Overexpression of cyclin D1 in all cases suggests that a defect in cell-cycle regulation may be involved in the growth of IFPs.
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PMID:Inflammatory fibroid polyps of the gastrointestinal tract: evidence for a dendritic cell origin. 1470 72

Smooth muscle contraction is initiated by myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+) dependent MLC kinase. However, many aspects of smooth muscle contraction cannot be accounted for by MLC phosphorylation. One hypothesis that has received experimental support involves the thin filament protein caldesmon. Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon relieves this inhibitory effect. The primary candidates for catalysis of caldesmon phosphorylation are the p42/p44 ERK MAP kinases. However, we and others have shown that inhibition of the ERK MAP kinases has no effect on many smooth muscles. The goal of this study was to determine if evidence for a second endogenous caldesmon kinase may be obtained. We used Triton X-100 skinned and intact tissues of the swine carotid artery to address this goal. Caldesmon phosphorylation was evident in resting and Ca(2+) stimulated Triton X-100 skinned fibers. Ca(2+)-dependent caldesmon phosphorylation was partially sensitive to the ERK MAP kinase inhibitor PD98059, whereas all caldesmon phosphorylation was sensitive to the general kinase inhibitor, staurosporine. Histamine increased caldesmon phosphorylation levels in intact swine carotid artery, which was sensitive to both PD98059 and staurosporine. Histamine increased ERK MAP kinase activity, which was reversed by PD98059, staurosporine, and EGTA. Histamine-induced contractions were inhibited by staurosporine but not by PD98059. We interpret these results to suggest that although ERK MAP kinases catalyze caldesmon phosphorylation, a second staurosporine sensitive kinase is also important in caldesmon phosphorylation and it is this pathway that may be more important in contractile regulation.
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PMID:Caldesmon phosphorylation is catalyzed by two kinases in permeabilized and intact vascular smooth muscle. 1475 51

The present study tested the hypothesis that ERK activation is an essential step in the onset of labor in a rat model of preterm labor. The administration of RU-486, an antiprogesterone agent, to rats induced preterm delivery 22.2 +/- 0.24 h after treatment. Changes in basal signaling events were studied in myometrial tissue from CO(2)-euthanized rats. Rats treated with RU-486 displayed a dramatically increased in vitro uterine contractility compared with gestational stage-matched, sham-treated rats. In vitro contractility was not significantly different from that during spontaneous labor. During RU-486-induced preterm labor, as previously described for spontaneous labor, ERK phosphorylation levels increased, as did phosphorylation of caldesmon at Ser(789), an ERK phosphorylation site. Also, a small but significant increase in 20-kDa myosin light chain phosphorylation was seen at a constant intracellular pCa of 7. When rats were chronically treated with an agent that prevents ERK activation, U-0126, the onset of RU-486-induced preterm labor was delayed in a statistically significant manner. Chronic in vivo treatment with U-0126 also significantly inhibited the RU-486-induced increase in in vitro contractility and ERK and caldesmon phosphorylation but did not alter the RU-486-induced increase in 20-kDa myosin light chain phosphorylation. These data indicate that ERK activation is a component of the multiple events leading to the development of labor in this rat model. We suggest that the ERK pathway could possibly be used to identify targets for the development of a novel class of tocolytic agents.
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PMID:Role of ERK1/2 in uterine contractility and preterm labor in rats. 1507 63

To confirm the usefulness of an immunohistochemical panel of antibodies for KIT (c-kit/CD117), CD34, desmin, smooth-muscle actin (SMA), h-caldesmon (HCD), S-100 protein, neuron-specific enolase (NSE), and beta-catenin, 297 mesenchymal and peripheral nerve-sheath tumors of the gastrointestinal tract and intra-abdominal locations including 211 gastrointestinal stromal tumors (GISTs), 12 leiomyomas, 18 leiomyosarcomas, 17 solitary fibrous tumors (SFTs), 14 schwannomas, and 25 desmoid-type fibromatoses (DTFs) were analyzed immunohistochemically. Consistent (100%) immunoreactivity for KIT, CD34, desmin and S-100, and nuclear accumulation of beta-catenin were detected in GISTs, SFTs, smooth-muscle tumors, schwannomas, and DTFs, respectively. Immunoreactivity for SMA, HCD, and NSE was observed in a wide range of these tumors. In addition, 418 bone and soft tissue tumors were enrolled in this study for KIT immunostaining. As a result, a limited number of these tumors were KIT positive, including synovial sarcoma that showed morphological similarity to GISTs. These findings suggest that KIT, CD34, desmin, S-100, and beta-catenin are key markers for clinical diagnosis of GISTs and other spindle cell tumors that may involve the gastrointestinal tract, whereas SMA, HCD, and NSE have only limited value.
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PMID:Differential diagnosis of gastrointestinal stromal tumor and other spindle cell tumors in the gastrointestinal tract based on immunohistochemical analysis. 1523 41


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