Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tyrosine phosphorylation has been linked to plasmalemmal targeting of src homology-2-containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation-independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation-dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes.
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PMID:Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells. 753 16

Vascular smooth muscle contraction is thought to occur by a mechanism similar to that described for striated muscles, i.e., via a cross-bridge cycling--sliding filament mechanism. This symposium focused on Ca2+ signalling and the role of intracellular free Ca2+ concentration, [Ca2+]i, in regulating vascular tone: how contractile stimuli leading to an increase in [Ca2+]i trigger vasoconstriction and how relaxant signals reduce [Ca2+]i causing vasodilation. M.P. Walsh opened the symposium with an overview emphasizing the central role of myosin phosphorylation-dephosphorylation in the regulation of vascular tone and identifying recent developments concerning regulation of [Ca2+]i, Ca2+ sensitization and desensitization of the contractile response, Ca(2+)-independent protein kinase C induced contraction, and direct regulation of cross-bridge cycling by the thin filament associated proteins caldesmon and calponin. The remainder of the symposium focused on three specific areas related to the regulation of vascular tone: Ca2+ signalling in relation to smooth muscle structure, structure-function relations of myosin, and the role of cyclic GMP (cGMP) dependent protein kinase. G.J. Kargacin described how smooth muscle cells are structured and how second messenger signals such as Ca2+ might be modified or influenced by this structure. J. Kendrick-Jones then discussed the results of mutagenesis studies aimed at understanding how the myosin light chains, particularly the phosphorylatable (Ca(2+)-calmodulin dependent) regulatory light chains, control myosin. The vasorelaxant effects of signalling molecules such as beta-adrenergic agents and nitrovasodilators are mediated by cyclic nucleotide dependent protein kinases, leading principally to a reduction in [Ca2+]i. T.M. Lincoln described the roles of cyclic nucleotide dependent protein kinases, in particular cyclic GMP dependent protein kinase, in vasodilation.
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PMID:Intracellular mechanisms involved in the regulation of vascular smooth muscle tone. 758 22

The fact that smooth muscle exists in almost every hollow organ and is involved in a large number of disease states has led to a vast increase in smooth muscle research, covering areas from testing response to antagonists and agonists to measuring the molecular force generated by a single actin filament. Yet, the exact mechanisms regulating contractile response of smooth muscle remain unsolved. Calcium has been a central player in mediating smooth muscle contraction through binding with calmodulin, although there is evidence showing that under special circumstances smooth muscle can contract without change in intracellular Ca2+. In addition to the major regulatory pathway of Ca(2+)-calmodulin-myosin light chain kinase, there are other thin filament linked regulatory mechanisms in which Ca(2+)-calmodulin dependent phosphorylation of calponin and caldesmon may be involved. Ca2+ sensitivity of smooth muscle contraction may vary under different situations and this has recently been recognized as an important regulatory mechanism. Examples are protein kinase C (PKC) dependent phosphorylation of myosin light chain kinase which results in partial inhibition of contraction, and activation of myosin light chain phosphatase. There is new evidence showing that not only does Ca2+ regulate contraction by regulating the interaction of contractile proteins in smooth muscle, but also that shortening of smooth muscle itself reduces intracellular Ca2+ concentration, via a negative feedback.
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PMID:Calcium and smooth muscle contraction. 781 50

The interaction of phosphatidylserine with intact smooth muscle caldesmon and caldesmon fragments obtained by bacterial expression was investigated by means of light scattering. Among these fragments only those derived from the C-terminal part of caldesmon (so-called domain 4) were able to interact with phospholipids. Fragments 606C (residues 606-756), H7 (566-710) and H2 (626-710) form tight complexes with phosphatidylserine, whereas fragments H8 (658-737), H9 (669-737) and fragment H4 (566-624) interact with phospholipids less effectively. It is concluded that the phospholipid-binding site is located in the sequence 626-710 of caldesmon. This sequence contains calmodulin-binding sites and serine residues phosphorylated by protein kinase C and pro-directed protein kinases. This could explain the effects of calmodulin and phosphorylation on the caldesmon-phospholipid interaction described earlier.
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PMID:Localization of phospholipid-binding sites of caldesmon. 814 73

Endogenous caldesmon kinase activity in sheep aorta smooth muscle was purified and characterized. The enzyme was identified as a proteolytic fragment of protein kinase C by cross-reactivity with anti-protein kinase C antibodies, autophosphorylation, substrate specificity and the primary structure of the sites of phosphorylation on caldesmon. The enzyme phosphorylated aorta caldesmon both in native thin filaments and in the isolated state. Up to 2.9 mols of phosphate per mol of caldesmon were transferred. Prolonged incubation of caldesmon with the kinase resulted in phosphorylation of Ser-127, Ser-587, Ser-600, Ser-657, Ser-686, and Ser-726 (numbering corresponds to chicken gizzard caldesmon sequence). Ser-600 and Ser-587 were the major sites of phosphorylation containing more than 30% of phosphate transferred. Phosphorylation did not significantly affect the interaction of caldesmon with Ca(2+)-calmodulin. However, phosphorylation of both intact caldesmon and of its C-terminal fragment (658C), containing residues 658-756, significantly decreased their ability to inhibit acto-heavy meromyosin ATPase. This seems to be partially due to a decrease in the binding of caldesmon and 658C to actin-tropomyosin and partly due to an uncoupling of the binding-inhibition relationship.
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PMID:Phosphorylation of aorta caldesmon by endogenous proteolytic fragments of protein kinase C. 818 8

Smooth muscle calponin bound to the biologically active fluorescent calmodulin [2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin] (MIANS.CaM) with a Kd of 80 nM and produced a 3.4-fold fluorescence enhancement. PKC-phosphorylated calponin (1.3 mol of Pi/mol) bound to CaM with approximately 15-fold lower affinity. Calponin inhibited CaM (10 nM) activation of the Ca(2+)-/CaM-activated cyclic nucleotide phosphodiesterase (PDE) with an IC50 of 138 nM. The calponin-CaM interaction was Ca(2+)-dependent: half-maximal binding of calponin to MIANS.CaM occurred at pCa 6.6 with a Hill coefficient of 2.4. Stopped-flow fluorescence kinetic analysis demonstrated that EGTA chelation of Ca2+ from CaM disrupted the MIANS.CaM-calponin complex at a rate of 1 s-1. Calponin bound MIANS.CaM at a rate of (6.0 +/- 1.8) x 10(6) M-1s-1, and melittin and unlabeled brain CaM both disrupted the MIANS.CaM-calponin complex at a rate of 0.3 +/- 0.1 s-1. These studies suggest that calponin binds CaM with 80-fold lower affinity than myosin light-chain kinase and that calponin associates with CaM much slower than it associates with caldesmon or myosin light-chain kinase. The physiological relevance of the CaM-calponin interaction was evaluated by analysis of the effects of Ca(2+)-CaM on (i) the interaction of calponin with actin and (ii) calponin-mediated inhibition of actin-activated myosin MgATPase activity. Ca(2+)-CaM half-maximally inhibited calponin (2 microM) binding to smooth and skeletal muscle actins (9 microM) at 5.4 and 11 microM CaM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calponin-calmodulin interaction: properties and effects on smooth and skeletal muscle actin binding and actomyosin ATPases. 824 Nov 89

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.
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PMID:Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase. 839 39

Airway smooth muscle plays a principal role in the pathogenesis of asthma. Primary cultures are being used to investigate airway myocyte proliferation and cellular pathways regulating contraction. Airway smooth muscle cells (SMC) modulate from a contractile to a noncontractile phenotype in culture, but no systematic study of the concomitant changes in expression of cytocontractile and cytoskeletal proteins has been reported. We measured temporal changes in protein marker expression of canine tracheal SMC in primary culture, using specific antibodies and cDNA probes. Immunoblot analysis revealed that when cells became proliferative after 5 days of culture, the content of smooth muscle myosin heavy chain (sm-MHC), calponin, sm-alpha-actin, and desmin diminished by > 75%; myosin light chain kinase, h-caldesmon, and beta-tropomyosin had also decreased significantly (P < 0.05). Northern blots revealed that mRNA levels for sm-MHC and sm-alpha-actin were also significantly reduced in proliferative SMC. Conversely, immunoblotting demonstrated the content of non-muscle myosin heavy chain, l-caldesmon, vimentin, alpha/beta-protein kinase C (PKC), and CD44 homing cellular adhesion molecule (HCAM) increased one- to sixfold as cells became proliferative. The content of sm-MHC and sm-alpha-actin protein increased after confluence, suggesting that cultured airway SMC are capable of phenotypic plasticity. Marker protein contents were also compared, by immunoblot assay, between SMC dissociated from trachealis or pulmonary arterial media. Cytocontractile protein content was higher in the trachea, which shortens faster than the pulmonary artery. The identification of these markers provides tools for assessing the phenotype of airway SMC in culture and the airways of asthmatic patients.
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PMID:Markers of airway smooth muscle cell phenotype. 876 31

We provide here the first direct evidence for in situ functional specificity of protein kinase C (PKC)-epsilon as a regulator of smooth muscle contractility. PKC is known to cause a Ca(2+)-independent contraction of ferret aortic smooth muscle, and the expression of two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, has been demonstrated in this tissue. To test directly the hypothesis that one of these isoenzymes regulates contractility, constitutively active forms of PKC-epsilon and PKC-zeta were applied to saponin-permeabilized single ferret aortic smooth muscle cells. PKC-zeta caused no significant force response, but PKC-epsilon induced contraction of a magnitude (105 +/- 8 micrograms) similar to that produced by phenylephrine (110 +/- 10 micrograms), a relatively selective alpha 1-adrenergic agonist that triggers a PKC-dependent contraction. The PKC-epsilon-induced contraction was reversed by the PKC pseudosubstrate inhibitory peptide, PKC19-31. The myosin light chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) did not affect the force response of PKC-epsilon-activated cells, suggesting that PKC-epsilon may induce this contraction solely via thin filament disinhibition. In support of this conclusion, calponin and caldesmon were shown to be good in vitro substrates of PKC-epsilon but not of PKC-zeta.
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PMID:Epsilon-isoenzyme of protein kinase C induces a Ca(2+)-independent contraction in vascular smooth muscle. 876 99

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


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