Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of 3T3-L1 adipocytes with insulin resulted in activation of 2-deoxyglucose transport activity and translocation of glucose transporters (GLUT4 and GLUT1) from the cytoplasmic space to the plasma membrane. ML-9 (a myosin light chain kinase inhibitor) inhibited insulin stimulation of 2-deoxyglucose transport activity by 80% at 100 microM (IC50 = 27 microM) without affecting 2-deoxyglucose transport activity in the basal state. The inhibition was independent of extracellular Ca2+ concentration and almost fully reversible at 40 microM ML-9. ML-9 did not inhibit insulin-stimulated tyrosine phosphorylation of 95-kDa protein in the wheat germ agglutinin-purified preparation and of 95- and 160-kDa proteins in intact cells. However, ML-9 inhibited insulin-induced translocation of both GLUT4 and GLUT1 in a dose-dependent manner. The dose-response curves were similar to those observed for the inhibition of insulin stimulation of 2-deoxyglucose transport activity. Neither insulin nor ML-9 affected the phosphorylation state of both heavy and light chains of myosin. Therefore, it seems likely that ML-9 inhibits the insulin-induced translocation of glucose transporters at a step beyond the insulin receptor kinase activity by a mechanism different from that affecting phosphorylation of the myosin light chain. Phosphorylating activity of microtubule-associated protein 2 and myelin basic protein was stimulated by insulin, and this stimulation was not affected by ML-9. ML-9, however, inhibited the phosphorylating activity in vitro and insulin stimulation of the phosphorylating activity of ribosomal protein S6 in intact cells in a dose-dependent manner similar to that observed for the inhibition of insulin stimulation of glucose transport. These results suggest that mitogen-activated protein kinase may be one of the constituents in intracellular insulin signaling to the glucose transport system.
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PMID:Effects of ML-9 on insulin stimulation of glucose transport in 3T3-L1 adipocytes. 768 Mar 48

Ca(2+)-dependent myosin light chain (MLC) phosphorylation is an important step in the initiation of smooth muscle contraction. However, MLC phosphorylation alone cannot account for all aspects of contractile regulation, suggesting the involvement of other elements. In this article we present evidence obtained from Triton X-100 detergent skinned and intact tissue which demonstrates that vascular smooth muscle contraction can be initiated by a Ca(2+)-dependent mechanism that does not require prior MLC phosphorylation. We show that Ca2+ can initiate contractions supported by cytidine triphosphate (CTP) and that these contractions are inhibited by calmodulin antagonists, suggesting a Ca(2+)-calmodulin dependence of force distinct from that for MLC phosphorylation. Evidence is presented to demonstrate that carotid medial fibers contain a mitogen-activated protein (MAP) kinase which is activated by Ca2+ and may catalyze caldesmon phosphorylation. Based in part on our results and those of other investigators, we propose that direct Ca(2+)-calmodulin binding to caldesmon or phosphorylation of caldesmon by a Ca(2+)-dependent MAP kinase disinhibits caldesmon. Disinhibition of caldesmon allows an inherent basal level of actin-activated myosin ATPase activity to be expressed. The result is the slow development of force.
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PMID:Regulation of vascular smooth muscle contraction: myosin light chain phosphorylation dependent and independent pathways. 776 83

Protein kinase C (PKC) was first implicated in the regulation of smooth muscle contraction with the observation that phorbol esters induce slowly developing, sustained contractions. In some vascular smooth muscles, e.g., ferret aorta, phorbol ester induced contractions occur without an increase in sarcoplasmic free-Ca2+ concentration ([Ca]i) or myosin light chain phosphorylation. This response appears to be mediated by a Ca(2+)-independent isoenzyme of PKC (probably PKC epsilon), since saponin-permeabilized single ferret aortic smooth muscle cells, which retain receptor coupling, developed force in response to phenylephrine at low free [Ca2+] (pCa 7.0-8.6) and the constitutively active proteolytic fragment of PKC (PKM) elicited a contraction at pCa 7 comparable with the phenylephrine-induced contraction. Both contractions were reversed by a pseudo-substrate peptide inhibitor of PKC. These observations suggest a mechanism whereby alpha-adrenergic agonists may elicit a contractile response without a Ca2+ signal: alpha-adrenergic stimulation of phosphatidylcholine-specific phospholipase C or D (the latter in conjunction with phosphatidate phosphohydrolase) generates diacylglycerol. In the absence of an increase in [Ca2+]i, diacylglycerol specifically activates so-called novel PKCs, of which epsilon is the only isoenzyme known to be expressed in vascular smooth muscle. Recent evidence suggests that PKC may trigger a cascade of phosphorylation reactions, resulting in activation of mitogen-activated protein kinase and phosphorylation of the thin filament associated protein caldesmon. Alternatively, or additionally, PKC may directly phosphorylate calponin, another thin filament associated protein. These phosphorylations are predicted to alleviate inhibition of the cross-bridge cycling rate by these thin-filament proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Smooth muscle protein kinase C. 776 84

We have expressed two truncated isoforms of chicken nonmuscle myosin II-B using the baculovirus expression system. One of the expressed heavy meromyosins (HMMexp) consists of two 150-kDa myosin heavy chains (MHCs), comprising amino acids 1-1231 as well as two pairs of 20-kDa and 17-kDa myosin light chains (MLCs) in a 1:1:1 molar ratio. The second HMMexp was identical except that it contained an insert of 10 amino acids (PESPKPVKHQ) at the 25-50-kDa domain boundary in the subfragment-1 region of the MHC. These 10 amino acids include a consensus sequence (SPK) for proline-directed kinases. Expressed HMMs were soluble at low ionic strength and bound to rabbit skeletal muscle actin in an ATP-dependent manner. These properties afforded a rapid purification of milligram quantities of expressed protein. Both isoforms were capable of moving actin filaments in an in vitro motility assay and manifested a greater than 20-fold activation of actin-activated MgATPase activity following phosphorylation of the 20-kDa MLC. HMMexp with the 10-amino acid insert was phosphorylated by Cdc2, Cdk5, and mitogen-activated protein kinase in vitro to 0.3-0.4 mol of PO4/mol of MHC. The site phosphorylated in the MHC was identified as the serine residue present in the 10-amino acid insert and its presence was confirmed in bovine brain MHCs. Characterization of the baculovirus expressed noninserted and inserted MHC isoforms with respect to actin-activated MgATPase activity and ability to translocate actin filaments in an in vitro motility assay produced the following average values following MLC phosphorylation: noninserted HMMexp, Vmax = 0.28 s-1, Km = 12.7 microM; translocation rate = 0.077 micron/s; inserted HMMexp, Vmax = 0.37 s-1, Km = 15.1 microM; translocation rate = 0.092 micron/s.
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PMID:Baculovirus expression of chicken nonmuscle heavy meromyosin II-B. Characterization of alternatively spliced isoforms. 857 42

To investigate the mechanisms for intracellular signaling and increased vascular tone by 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha), we measured mitogen-activated protein kinase (MAPK) activity and myosin regulatory light chain (LC20) phosphorylation in porcine carotid arteries incubated with 8-epi-PGF2 alpha or PGF2 alpha. With stimulation by either 8-epi-PGF2 alpha or PGF2 alpha. MAPK activity and the force of contraction rose in parallel and were maintained during the time of exposure to agonist (2 hours). LC20 phosphorylation levels rose and then partially declined during stimulation with either agonist. The effects of 8-epi-PGF2 alpha on contraction, MAPK activity, and myosin light chain phosphorylation were completely inhibited by the receptor antagonists, SQ-29548 and BMS-180291; the effects of PGF2 alpha were only partially inhibited by these compounds. Thus, intracellular signaling by 8-epi-PGF2 alpha in fully differentiated vascular smooth muscle, resulting in MAPK activation and increased myosin phosphorylation, is specifically mediated by an activation of thromboxane A2/prostaglandin endoperoxide receptors. Lipid peroxidation and 8-epi-PGF2 alpha production, resulting from such vascular pathological processes as atherosclerosis, lead to an activation of two intracellular signaling pathways in smooth muscle: one pathway results in the activation of MAPK, while the other results in myosin light chain phosphorylation.
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PMID:Intracellular signaling by 8-epi-prostaglandin F2 alpha is mediated by thromboxane A2/prostaglandin endoperoxide receptors in porcine carotid arteries. 878 Jul 11

Two separate signal transduction pathways exist in vascular smooth muscle: one for cell growth, proliferation, and differentiation and the other for contraction. Although activation of protein tyrosine kinases is intimately involved in the signaling pathway that induces cell growth, proliferation, and differentiation, activation of myosin light chain kinase (MLCK) is an important step in the pathway leading to smooth muscle contraction. Indirect evidence suggests that "cross talk" exists between these two signaling pathways, but the common intermediates are not well defined. The purpose of this study was to determine whether a vasoconstrictor and a mitogen initiate crossover signaling between the tyrosine kinase pathway and the MLCK pathway in vascular smooth muscle. Rat aorta and pulmonary arteries were isolated and stimulated with either fetal calf serum (FCS) or phenylephrine in the presence or absence of a tyrosine kinase inhibitor (genistein) or tyrosine phosphatase inhibitor [sodium o-vanadate (Na3 VO4)]. Isometric force was recorded as a function of time; myosin light chain phosphorylation, protein tyrosine phosphorylation, and mitogen-activated protein kinase (MAPK) mobility were determined by immunoblotting. The results demonstrate that FCS, which contains a variety of growth factors known to activate tyrosine kinases, induced myosin light chain phosphorylation and contraction in vascular smooth muscle. Phenylephrine, a vasoconstrictor known to activate MLCK, induced tyrosine phosphorylation of a 42-kDa protein identified as MAPK. Tyrosine phosphorylation of this protein was inhibited by genistein and enhanced by vanadate. Genistein significantly inhibited both serum- and phenylephrine-induced myosin light chain phosphorylation as well as the serum- and phenylephrine-induced force generation, whereas vanadate enhanced these responses. These data demonstrate interrelationship between activation of the tyrosine kinase pathway and the MLCK pathway in vascular smooth muscle. These interactions may influence smooth muscle contraction and be important in the regulation of smooth muscle cell proliferation.
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PMID:Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle. 889 27

Thrombin treatment causes a dose-dependent rounding of 1321N1 astrocytoma cells. This cytoskeletal response is rapid, peaking 2 h after thrombin stimulation, and reverses by 50% after 24 h. The thrombin receptor peptide SFLLRNP also induces cell rounding, whereas other G protein-linked receptor agonists such as carbachol, lysophosphatidic acid, or bradykinin fail to do so. Results of studies using pharmacological inhibitors do not support a requirement for phosphatidylinositol 3-kinase, mitogen-activated protein kinase, or Ca2+ mobilization in this response. Inhibition of protein kinase C or tyrosine kinase produces minimal blockade. Pertussis toxin treatment is also without effect. However, thrombin-induced rounding is fully blocked by the C3 toxin from Clostridium botulinum, which specifically ADP-ribosylates and inactivates the small G protein Rho. Thrombin also leads to a rapid, 2.4-fold increase in 32P incorporation into myosin light chain while carbachol does not. Myosin phosphorylation, like cell rounding is inhibited by inactivation of Rho with C3 exoenzyme, suggesting that myosin phosphorylation is necessary for this cytoskeletal response. This is supported by the observation that thrombin-induced rounding is also blocked by the myosin light chain kinase inhibitor KT5926. However, treatment with KT5926 fails to inhibit mitogenesis. Thus, cell rounding is not prerequisite to thrombin-induced DNA synthesis. We conclude that stimulation of the heterotrimeric G protein-coupled thrombin receptor in 1321N1 cells activates Rho-dependent pathways for both DNA synthesis and cell rounding, the cytoskeletal response being mediated in part through increases in myosin phosphorylation.
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PMID:Requirement for Rho-mediated myosin light chain phosphorylation in thrombin-stimulated cell rounding and its dissociation from mitogenesis. 955 56

Incorporation of 32P into telokin, a smooth muscle-specific, 17-18-kDa, acidic (pI 4.2-4.4) protein, was increased by forskolin (20 microM) in intact rabbit ileum smooth muscle (ileum) and by 8-bromo-cyclic GMP (100 microM) in alpha-toxin-permeabilized ileum. Native telokin (5-20 microM), purified from turkey gizzard, and recombinant rabbit telokin, expressed in Escherichia coli and purified to >90% purity, induced dose-dependent relaxation, associated with a significant decrease in regulatory myosin light chain phosphorylation, without affecting the rate of thiophosphorylation of regulatory myosin light chain of ileum permeabilized with 0.1% Triton X-100. Endogenous telokin was lost from ileum during prolonged permeabilization (>20 min) with 0.1% Triton X-100, and the time course of loss was correlated with the loss of 8-bromo-cyclic GMP-induced calcium desensitization. Recombinant and native gizzard telokins were phosphorylated, in vitro, by the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and p42/44 mitogen-activated protein kinase; the recombinant protein was also phosphorylated by calmodulin-dependent protein kinase II. Exogenous cGMP-dependent protein kinase (0.5 microM) activated by 8-bromo-cyclic GMP (50 microM) phosphorylated recombinant telokin (10 microM) when added concurrently to ileum depleted of its endogenous telokin, and their relaxant effects were mutually potentiated. Forskolin (20 microM) also increased phosphorylation of telokin in intact ileum. We conclude that telokin induces calcium desensitization in smooth muscle by enhancing myosin light chain phosphatase activity, and cGMP- and/or cAMP-dependent phosphorylation of telokin up-regulates its relaxant effect.
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PMID:Acceleration of myosin light chain dephosphorylation and relaxation of smooth muscle by telokin. Synergism with cyclic nucleotide-activated kinase. 955 31

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in smooth muscle contraction by monitoring MAP kinase activation, caldesmon phosphorylation, and contractile force during agonist stimulation. Isometric tension in response to KCl and phenylephrine (PE) was measured from strips of ferret aorta. MAP kinase activation was monitored by Western blot using a phosphospecific p44/p42 MAP kinase antibody. Caldesmon phosphorylation was assessed using specific phosphocaldesmon antibodies. We report here that treatment of smooth muscle strips with PD-098059, a specific inhibitor of MAP kinase kinase, did not detectably modify the KCl-evoked contraction but significantly inhibited the contraction to PE in the absence of extracellular Ca2+. In this experimental condition, where the contraction occurs in the absence of increases in 20-kDa myosin light chain phosphorylation, PD-098059 also inhibited significantly MAP kinase and caldesmon phosphorylation. Collectively, these results demonstrate a direct cause-and-effect relationship between MAP kinase activation and Ca2+-independent smooth muscle contraction and support the concept of caldesmon phosphorylation as the missing link between both events.
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PMID:A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha-adrenoceptor stimulation. 975 61

The role of myosin light chain phosphorylation in regulating the mechanical properties of the cytoskeleton was studied in NIH/3T3 fibroblasts expressing a truncated, constitutively active form of smooth muscle myosin light chain kinase (tMK). Cytoskeletal stiffness determined by quantifying the force required to indent the apical surface of adherent cells showed that stiffness was increased twofold in tMK cells compared with control cells expressing the empty plasmid (Neo cells). Cytoskeletal stiffness quantified using magnetic twisting cytometry showed an approximately 1.5-fold increase in stiffness in tMK cells compared with Neo cells. Electronic volume measurements on cells in suspension revealed that tMK cells had a smaller volume and are more resistant to osmotic swelling than Neo cells. tMK cells also have smaller nuclei, and activation of mitogen-activated protein kinase (MAP kinase) and translocation of MAP kinase to the nucleus are slower in tMK cells than in control cells. In tMK cells, there is also less bromodeoxyuridine incorporation, and the doubling time is increased. These data demonstrate that increased myosin light chain phosphorylation correlates with increased cytoskeletal stiffness and suggest that changing the mechanical characteristics of the cytoskeleton alters the intracellular signaling pathways that regulate cell growth and division.
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PMID:Regulation of cytoskeletal mechanics and cell growth by myosin light chain phosphorylation. 981 84


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