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)

We expressed the C-terminal 99 amino acids of chicken gizzard caldesmon (658C) and two point mutants in which the preferred phosphorylation sites of MAP kinase and p34cdc2 kinase, Ser702 and Thr673 were altered to aspartic acid. The T673D mutant was indistinguishable from 658C but S702D was not phosphorylated by MAP kinase, was significantly less potent as an inhibitor of actin-tropomyosin activation of myosin MgATPase, and bound less actin-tropomyosin at low concentrations. Thus Ser702 is involved in the tropomyosin-dependent inhibitory mechanism of caldesmon, and its phosphorylation by MAP kinase or p34cdc2 kinase could modulate caldesmon function.
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PMID:The functional effects of mutations Thr673-->Asp and Ser702-->Asp at the Pro-directed kinase phosphorylation sites in the C-terminus of chicken gizzard caldesmon. 839 47

h-Caldesmon in vascular smooth muscle is phosphorylated in response to pharmacologic stimulation. Although many kinases phosphorylate h-caldesmon, in vitro, the responsible kinase in intact tissue is unknown. The sites of phosphorylation in caldesmon from intact canine aortas have recently been identified and are consensus sequences for a proline-directed protein kinase. In this study, we investigated the phosphorylation of h-caldesmon by mitogen-activated protein kinase (MAPK). Purified, recombinant MAPK phosphorylated porcine stomach h-caldesmon to a stoichiometry approaching 2 mol phosphate/mol protein. Phosphorylated h-caldesmon was subjected to proteolysis and the phosphopeptides were purified by high performance liquid chromatography. Two major phosphopeptides were identified and sequenced. These two peptides, VTS*PTKV and S*PAPK, were identical to the sequences of the sites phosphorylated in intact tissue. Antibodies to several enzymes implicated in the cascade of activation of MAPK were used to evaluate vascular smooth muscle by Western blotting. All components were found to be present. These data suggest that MAPK can function as a 'caldesmon kinase' in vascular smooth muscle.
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PMID:Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-Caldesmon. 848 68

1. Recombinant, activated mitogen-activated protein kinase (3.3 microM; p42mapk) phosphorylated caldesmon in phasic (rabbit portal vein) and tonic (rabbit femoral artery) smooth muscle strips permeabilized with Triton X-100. 2. Phosphorylation of caldesmon by p42mapk neither induced contraction of relaxed smooth muscle nor affected the Ca2+ sensitivity of submaximally contracted permeabilized phasic or tonic smooth muscle.
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PMID:Phosphorylation of caldesmon by mitogen-activated protein kinase with no effect on Ca2+ sensitivity in rabbit smooth muscle. 855 63

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

Arterial smooth muscle stretch is an important physiological modulator of vascular function. To identify intracellular processes altered during muscle stretch, we found previously that extracellular signal-regulated kinase-mitogen-activated protein kinase (MAPK) activity increased in response to the application of mechanical loads. In the present study, stretch-dependent activation of MAPK in porcine carotid arteries was investigated as was the phosphorylation of the thin filament-binding protein caldesmon, which is known to be a substrate for the kinase in fully differentiated smooth muscle. MAPK activity was 67 pmol.min-1.mg protein-1 in unloaded muscle strips immediately after attachment to force transducers and 139 pmol.min-1.mg protein-1 within 30 s of muscle stretch. When muscle strips were continually stretched, MAPK activity remained elevated for approximately 2 h and then decreased over 16 h to 16 pmol.min-1.mg protein-1. When muscle strips were stretched and then unloaded, MAPK activity decreased within 1 h to the level present in the muscle before the stretch. These effects of muscle stretch on MAPK activity were additive to the effects of KCl or phorbol ester stimulation and were partially inhibited by reducing extracellular Ca2+. Eliminating extracellular Ca2+ had no effect on phorbol 12,13-dibutyrate (PDBu)-dependent contractions or MAPK activity; however, KCl-dependent contractions and MAPK activity were completely abolished by this procedure. An antibody specific for detecting caldesmon phosphorylated by MAPK, vs. protein kinase C (PKC), was developed and used to assess relative caldesmon phosphorylation in unstimulated and PDBu-stimulated muscle strips. In all cases investigated, the level of MAPK activity correlated with phosphocaldesmon immunoreactivity. Because arterial MAPK activity is regulated by PKC- and stretch-dependent mechanisms, these data are consistent with a role for MAPK and the subsequent phosphorylation of caldesmon as mediators in the stretch activation of vascular smooth muscle.
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PMID:Stretch-dependent activation and desensitization of mitogen-activated protein kinase in carotid arteries. 943 85

There is relatively little known about expression and activation of p38 mitogen-activated protein kinases (MAPKs) through G protein-linked, seven-transmembrane-spanning (STM) receptors in mammalian smooth muscle. To investigate the role of p38 MAPK in smooth muscle, we cloned and sequenced the p38 MAPK expressed in canine smooth muscles. A full-length clone of the canine p38 MAPK expressed in colonic smooth muscle was obtained by RT-PCR. The deduced amino acid sequence revealed 99% identity to the human p38 MAPK and differed from the human enzyme in only two conservative substitutions. The deduced molecular mass of the canine p38 MAPK is 41.2 kDa, with a calculated isoelectric point of 5.41. Canine p38 MAPK was found to be expressed in colonic, tracheal, and vascular smooth muscles and underwent increased tyrosine phosphorylation in response to motor neurotransmitters, acetylcholine (ACh) and neurokinin A (NKA), in colonic smooth muscle. There was an eightfold increase in p38 MAPK phosphorylation after a 10-min incubation with ACh and a threefold increase with NKA. We also identified a p38 immunoreactive kinase activity isolated from colonic smooth muscle homogenate by Mono Q chromatography. Partially purified p38 MAPK and activated recombinant p38 MAPK (Mpk2) phosphorylated both the known p38 MAPK substrate ATF2, as well as porcine stomach h-caldesmon in vitro. The results suggest that elements of the "stress-response" pathway may be coupled to transcriptional control as well as to cytoskeletal and possibly contractile protein phosphorylation in mammalian smooth muscle.
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PMID:p38 mitogen-activated protein kinase expression and activation in smooth muscle. 968 7

Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon reverses the inhibition. The caldesmon kinase is believed to be mitogen-activated protein (MAP) kinase. MAP kinases are activated during vascular stimulation, but a cause-and-effect relationship between kinase activity and contraction has not been established. We examined the role of MAP kinase in contraction using PD-098059, an inhibitor of MAP kinase kinase (MEK). MAP kinase activity was assessed using an anti-active MAP kinase antibody and direct measurement of MAP kinase catalyzed phosphorylation of myelin basic protein, MBP-(95-98). MAP kinase phosphorylation, stimulated by histamine (50 microM) or phorbol 12,13-dibutyrate (PDBu, 0.1 microM), was inhibited by PD-098059 (100 microM). PD-098059 did not alter the sensitivity or the maximal level of force in smooth muscle stimulated by histamine or PDBu, nor did PD-098059 affect contraction of beta-escin-permeabilized tissue. Our data suggest that p44 and p42 MAP kinases are not involved in regulation of vascular smooth muscle contraction. These results do not, however, preclude a role for other isoforms of the MAP kinase family.
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PMID:Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery. 968 5

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 effect of direct phosphorylation by recombinant p44erk1 mitogen-activated protein kinase on the inhibitory activity of caldesmon and its C-terminal fragment H1 was studied in vitro. Neither inhibition of actin-tropomyosin activated ATPase of heavy meromyosin by caldesmon or H1, nor inhibition of the actin-tropomyosin motility over heavy meromyosin by H1 was significantly affected by the phosphorylation while only a moderate effect on the actin-activated component of heavy meromyosin ATPase inhibition was observed. Phosphopeptide mapping of caldesmon immunoprecipitated from [32P]PO4-labelled intact gizzard strips revealed that it is predominantly phosphorylated at mitogen-activated protein kinase sites in unstimulated tissue and that it is stimulated for 1 h with phorbol 12,13-dibutyrate. We find that phorbol 12,13-dibutyrate also induces a transitory phosphorylation of caldesmon peaking at 15 min after addition and this phosphorylation is not attributed to mitogen-activated protein kinase, protein kinase C, Ca2+/calmodulin-dependent kinase II or casein kinase II. We suggest that a yet unidentified kinase, rather than mitogen-activated protein kinase, may be involved in regulation of the caldesmon function in vivo.
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PMID:Evidence against the regulation of caldesmon inhibitory activity by p42/p44erk mitogen-activated protein kinase in vitro and demonstration of another caldesmon kinase in intact gizzard smooth muscle. 1038 1


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