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)

In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP-binding protein or Ca2+.
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PMID:Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+. 803 89

To help elucidate the physiological role of calponin (a thin-filament-linked regulatory protein) in smooth muscle contraction, the effects of its exogenous application were investigated on actin-activated MgAT-Pase activity in crude actomyosin from chicken gizzard, and on contraction induced by Ca(2+)-dependent and -independent means in arterial smooth muscle strips skinned by saponin or beta-escin. Calponin concentration dependently inhibited actin-activated MgATPase activity with a proportional increase in its binding to actomyosin and also attenuated Ca(2+)-induced contractions, in the presence or absence of calmodulin, in skinned arterial strips. Calponin, when phosphorylated by protein kinase C, reduced both its ability to bind to actomyosin and its inhibitory action on actomyosin MgATPase. The phosphorylated calponin also had no effect on the maximum Ca(2+)-induced contraction in skinned smooth muscle, suggesting that these actions of calponin are not nonspecific. Calponin attenuated the Ca(2+)-independent contraction observed in myosin light chain thio-phosphorylated strips, or on application of trypsin-treated myosin light chain kinase. However, calponin had no effect on maintained rigor contraction. These results suggest that in vascular smooth muscle, calponin may play a physiological role in the inhibition of Ca(2+)-regulated force, possibly through a direct action on active actin-myosin interactions.
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PMID:Effects of exogenously applied calponin on Ca(2+)-regulated force in skinned smooth muscle of the rabbit mesenteric artery. 807 50

Calponin is a basic, approximately 34,000 M(r), smooth muscle-specific protein which is developmentally expressed in up to four isoforms. Calponin binds very strongly to actin in a Ca(2+)-independent manner and is localized to the thin filaments in smooth muscle, where it is present at a stoichiometry of 1 mol calponin/7 mol actin. The interaction of calponin with actin inhibits the actomyosin MgATPase (cross-bridge cycling rate) without affecting myosin phosphorylation. The calponin-actin interaction is blocked and calponin-mediated inhibition of the actomyosin MgATPase is reversed upon phosphorylation of calponin by either PKC or CaM kinase II; these properties are restored upon dephosphorylation of calponin by a type 2A protein phosphatase. Consistent with these in vitro findings, calponin is phosphorylated in intact smooth muscle in response to contractile stimuli. The increasing body of evidence, both in vitro and in vivo, strongly supports calponin phosphorylation-dephosphorylation as a thin filament-linked regulatory system in smooth muscle.
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PMID:Calponin: thin filament-linked regulation of smooth muscle contraction. 813 72

The mechanisms of smooth muscle tissue contractile system functioning are in many respects unexplained. According to the existing hypothesis, the mechanism of smooth muscle contraction is based on the interaction between myosin and actin. The change of muscle tissue stiffness during the contractive process is the important and obligatory feature of this actomyosin interaction. Earlier we have shown that, together with the smooth muscle cells, the connective tissue matrix can also produce the active mechanical strength. This process is not accompanied by changes of stiffness. We suggested that in some cases the induced contraction of smooth muscles is fulfilled, entirely or in part, by the connective tissue matrix. We report here that contractive reaction induced by the phorbol ester--activator of protein kinase C, which is one of the most important enzymes involved in the regulation of the smooth muscle contraction--occurs without any stiffness changes. The results obtained can not be explained in terms of the generally accepted hypothesis of actomyosin interaction. The conclusion is made that phorbol ester during the action on the smooth muscle tissue activates the mechanical strength generation by the connective tissue matrix.
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PMID:Activation of the non-actomyosin component of aortic wall contraction by phorbol ester. 813 44

This article describes a model of reversible disassembly of a cultured human intestinal epithelial monolayer by prolonged exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Prolonged phorbol ester exposure reduces protein kinase C (PKC) levels in numerous cell types including T84, as shown here. Under PKC-downregulated conditions, T84 monolayers, which simulate the highly organized structure of native intestinal crypt cells, become disassembled into 2 or 3 layers of rounded cells. Proliferation does not account for these morphological changes as assessed by thymidine incorporation studies. The effects of structural disorganization on epithelial barrier function was also examined. The permeability of disassembled monolayers was significantly greater than that of controls. Flux studies localized the permeability defect to the tight junction. PKC-associated alterations in the perijunctional ring of actin and myosin, one of the putative regulators of flow across the tight junction, were found to correlate with the observed functional changes. Most interesting was the fact that monolayer reassembly to the original columnar epithelial phenotype and reestablishment of barrier function occurred upon normalization of PKC levels. This model of reversible monolayer disassembly will allow investigation into the relationship between epithelial structure and function and examination of factors that govern monolayer formation.
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PMID:Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester. 814 Dec 94

It has been believed that cytosolic Ca2+ level ([Ca2+]i) is the only regulator of smooth muscle contraction. Measuring [Ca2+]i and muscle force simultaneously, it is evident that changes in Ca2+ sensitivity of contractile elements is also an important regulator. Although the mechanisms for Ca2+ sensitization is not fully understood yet, Ca2+ sensitization of myosin phosphorylation and activation of a Ca(2+)-sensitive mechanism which is not dependent on myosin phosphorylation have been proposed. Receptor-agonists seem to activate both of these mechanisms although protein kinase C does not necessary be involved in these mechanisms.
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PMID:[Calcium sensitivity of vascular smooth muscle and effects of drugs]. 816 17

Recent cloning and sequencing studies suggest that heavy chains of all non-muscle myosins II have a protein kinase C (PKC) phosphorylation site within their tail regions. A fragment of human macrophage myosin heavy chain, encompassing its COOH-terminal 396 amino acids (MIIAF46), was expressed in Escherichia coli to provide a model system for study of PKC-mediated phosphorylation. PKC phosphorylated this fragment when phosphatidylserine (PS) liposomes were present, but not when liposomes made from PS/phosphatidylcholine (PC) were used. The reaction required Ca2+, but not other activators such as diacylglycerol (DG) or phosphatidylinositol 4,5-bisphosphate. Phosphorylation of MIIAF46 was not observed in the presence of micelles of PS or PS/DG. Similar results were obtained using native myosin II purified from bovine brain and chicken intestine brush border. Phosphorylation of light chains, in contrast, occurred even with PS/PC liposomes if DG was present. Addition of the PS and PS/DG liposomes significantly increased the turbidities at 340 nm of MIIAF46 and native myosin II, and the extent of increase depended upon the type of myosin used. Also, PS and PS/DG liposomes shifted the gel filtration elution positions of MIIAF46 and myosin II. In contrast, liposomes of PS/PC and PS/PC/DG gave only a slight increase in turbidity with all myosins and fragments and did not noticeably shift their gel filtration elution positions. These results suggest that myosins II bind to PS liposomes via the COOH-terminal regions of their heavy chains with affinities specific to each myosin isoform, that the binding is dependent upon the PS composition, and that PKC phosphorylates the PS-bound heavy chains.
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PMID:Direct binding of myosin II to phospholipid vesicles via tail regions and phosphorylation of the heavy chains by protein kinase C. 820 8

The fatty acid 12(S)-HETE may be a new second messenger capable of activating PKC. In tumor cells 12(S)-HETE stimulates cytoskeleton-dependent cellular responses such as adhesion and spreading. Analysis of 12(S)-HETE effects on B16a melanoma cell cytoskeleton revealed reversible rearrangement of microtubules, microfilaments, the actin-binding proteins, vinculin, myosin heavy (MHC) and light chains (MLC), as well as bundling of vimentin intermediate filaments. The alterations in microfilaments and intermediate filaments occurred very rapidly, i.e., 5 min after exposure of tumor cells to 12(S)-HETE. The 12(S)-HETE-induced cytoskeletal alterations were accompanied by centrifugal organelle-translocation. Interestingly, MLC exhibited clear association with the cytoplasmic organelles. Biochemical analysis of the 12(S)-HETE effect indicated a PKC-mediated reversible hyperphosphorylation of MLC, vimentin, and a 130 kD cytoskeletal-associated protein. Optimal effects were obtained after 5 min treatment with 12(S)-HETE at 0.1 microM concentration. 12(S)-HETE pretreatment induced tumor cell spreading on a fibronectin matrix which required the intactness of all three major cytoskeletal components. The spreading process was dependent upon the activity of PKC. Our data suggest that 12(S)-HETE is a physiological stimulant of PKC. Further, it induces rearrangement of the cytoskeleton of tumor cells in interphase resulting in the stimulation of cytoskeleton-dependent cell activity such as spreading.
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PMID:PKC mediates 12(S)-HETE-induced cytoskeletal rearrangement in B16a melanoma cells. 822 7

Actin polymerization accompanies receptor-mediated responses and is correlated with motility-related events. In T lymphocytes, there is a lateral redistribution of surface receptors into caps and aggregation of actin-myosin in cytoplasmic subcaps, and these are impaired in T cells from aged individuals. This study documents marked changes in age-related cytoskeletal actin filament function which may account for the reduced motility. Basal levels of filamentous actin (F-actin) are significantly higher in purified G(o) T cells from aged C57BL/6 mice, due to a preferential increase in the CD8+ subpopulation. Following activation of the resting T cells with Concanavalin A (Con A), F-actin depolymerized in cells from young mice for 2 min, followed by rapid polymerization, reaching a plateau 200% above resting levels. In cells from 15-17-month-old mice, an attenuated depolymerization phase was seen for 45 sec, followed by little polymerization. No depolymerization or polymerization phases occurred in cells from aged mice. Phorbol 12 myristate 13-acetate (PMA), which activates protein kinase C (PKC), bypassing receptor mediated signals, induced actin polymerization to 57% of the levels of that after Con A stimulation in cells from both young and old animals and partially eliminated the differences in actin filament assembly due to age. Perturbation of the cytoskeleton with cytochalasin E (CE) potentiated proliferation of Con A-stimulated T cells from aged mice but did not completely restore the deficit attributed to immunosenescence. The results show an age-related impairment of cytoskeletal functions and suggest that differences in early signal transduction events contribute to the decrements in surface receptor motility and subsequent proliferation of T lymphocytes from older individuals.
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PMID:Differential regulation of actin polymerization following activation of resting T lymphocytes from young and aged mice. 822 68

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.
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PMID:Characterization and confocal imaging of calponin in gastrointestinal smooth muscle. 823 86


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