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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Superprecipitation of reconstituted actomyosin composed of smooth muscle myosin, skeletal muscle actin and smooth muscle native tropomyosin was studied. When the actomyosin solution was preincubated in the presence of ATP and the absence of Ca2+, or in the relaxed state, superprecipitation was markedly suppressed. The extent of suppression was correlated with the inhibition of the phosphorylation of the 20,000-dalton light chain of smooth muscle myosin. This is consistent with the theory that the interaction of smooth muscle actomyosin is regulated by the phosphorylation of myosin light chain through a system of myosin light chain kinase and phosphatase. However, further studies showed that the myosin light chain kinase and phosphatase system could not explain the present suppression of superprecipitation, even if a cyclic AMP-dependent protein kinase system was also involved. A new regulatory factor should be taken into account in the regulation of smooth muscle actomyosin interaction.
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PMID:Suppression of superprecipitation of contractile proteins from chicken gizzard muscle by preincubation in the presence of adenosine triphosphate without Ca2+. 627 37

1. Porcine cardiac native tropomyosin was phosphorylated by bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Most of the phosphate incorporation was observed in troponin I, the maximum of which was 0.7 mol of Pi per mol of troponin I. 2. In the presence of phosphorylated native tropomyosin, actomyosin ATPase activity was 15-40% lower than that in the presence of the unphosphorylated preparation at all calcium ion concentrations (1.5 x 10(-8) M-2.4 x 10(-5) M). Half-maximum activation of ATPase was obtained with a concentration of 7 x 10(-7) M Ca2+ (unphosphorylated) and 1.3 x 10(-6) M Ca2+ (phosphorylated), respectively. Maximum ATPase activity was reached with 3 x 10(-6) M Ca2+ (unphosphorylated) and 1.0 x 10(-5) M Ca2+ (phosphorylated). 3. Porcine cardiac troponin I isolated by affinity chromatography inhibited ATPase activity of desensitized actomyosin in the presence of tropomyosin. There was little difference between phosphorylated troponin I and a control preparation with regard to the inhibitory effect of ATPase activity. 4. Troponin C from rabbit skeletal muscle neutralized the inhibitory effect of troponin I. The minimum amount of troponin C required for complete neutralization was approximately equimolar to troponin I. The inhibitory effect of phosphorylated troponin I was neutralized by troponin C less effectively than that of unphosphorylated preparation.
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PMID:Effect of phosphorylation of porcine cardiac troponin I by 3':5'-cyclic AMP-dependent protein kinase on the actomyosin ATPase activity. 628 30

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.
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PMID:Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells. 629 4

Cardiac phospholipid-sensitive Ca2+-dependent protein kinase phosphorylated cardiac troponin inhibitory subunit (troponin I) and tropomyosin-binding subunit (troponin T), present either as the free form or as the troponin-tropomyosin complex. Exhaustive phosphorylation of troponin I and of troponin T revealed that 1.7 and 2 mol of phosphate was incorporated/mol of the subunits respectively. Cyclic AMP-dependent protein kinase, though incorporating 0.8 mol of phosphate/mol of troponin I, was unable to phosphorylate troponin T. Phosphorylation of troponin I (apparent Km = 3.4 microM; Vmax. = 2.6 mumol/min per mg of enzyme) or troponin T (apparent Km = 0.3 microM; Vmax. = 0.5 mumol/min per mg of enzyme) by the Ca2+-dependent enzyme was inhibited by various agents, such as adriamycin, palmitoylcarnitine, trifluoperazine, melittin and N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide (compound W-7). Ca2+ antagonists (such as verapamil), forskolin and ouabain were ineffective. These findings indicate that troponin I and troponin T were effective substrates for this species of Ca2+-dependent protein kinase, suggesting its potential regulatory role in the contractile activity of myofibrils modulated by troponin.
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PMID:Phosphorylation of cardiac troponin inhibitory subunit (troponin I) and tropomyosin-binding subunit (troponin T) by cardiac phospholipid-sensitive Ca2+-dependent protein kinase. 630

The src gene product of Rous sarcoma virus (pp60(src)) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.
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PMID:Highly purified pp60src induces the actin transformation in microinjected cells and phosphorylates selected cytoskeletal proteins in vitro. 640 62

It has previously been shown that the regulatory light chains of myosin from Limulus, the horseshoe crab, can be phosphorylated either by purified turkey gizzard smooth muscle myosin light chain (MLC) kinase or by a crude kinase fraction prepared from Limulus muscle [Sellers, J. R. (1981) J. Biol. Chem. 256, 9274-9278]. This phosphorylation was shown to be associated with a 20-fold increase in the actin-activated MgATPase activity of the myosin. We have now purified the Ca2+-calmodulin-dependent MLC kinase from Limulus muscle to near homogeneity by using a combination of low ionic strength extraction, ammonium sulfate fractionation, and chromatography on Sephacryl S-300 and DEAE-Sephacel. The final purification was achieved by affinity chromatography on a calmodulin-Sepharose 4B column. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed 95% of the protein to be comprised of a doublet with Mr = 39000 and 37000. Electrophoresis of the kinase fraction under nondenaturing conditions resulted in a partial separation of the two major bands and demonstrated that each had catalytic activity. An SDS-polyacrylamide gel overlayed with 125I-calmodulin demonstrated that both the Mr 39K and the Mr 37K proteins bind calmodulin. Neither of the bands could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. With Limulus myosin light chains as a substrate, the Vmax was 15.4 mumol min-1 mg-1, and the Km was 15.6 microM. The KD for calmodulin was determined to be 6 nM. The enzyme did not phosphorylate histones, casein, actin, or tropomyosin.
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PMID:Purification of myosin light chain kinase from Limulus muscle. 654 61

A myofibrillar protein extract has been isolated from the muscle of Ascaris suum. Two-dimensional electrophoresis of this extract revealed that the myosin light chain 1 (ALC1) migrates as 3 components with approximate isoelectric points in the range of 5.3-5.6. The most acidic component of ALC1 appeared to be phosphorylated when the myofibrillar extract was incubated for 10 s with catalytic subunit of cAMP dependent protein kinase and [gamma-32P] ATP. The myosin light chain 2 (ALC2) migrated as a single component in isoelectric focusing with an approximate isoelectric point of 5.5 Actin was resolved into 2 components with identical molecular weight but isoelectric points differing by approximatley 0.2 pH units. A protein was tentatively identified in the myofibrillar extract as tropomyosin. It migrated as a single band with an approximate isoelectric point of 5.0 and a molecular weight of 39 000. None of the troponin components could be identified in the myofibrillar extract. It is postulated that muscle contraction in A. suum muscle could be controlled by phosphorylation of myosin.
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PMID:Electrophoretic analyses of myofibrillar proteins from the body wall muscles of Ascaris suum. 689 80

A protein has been purified from human brain that appears to be the human equivalent of bovine 14-3-3 protein. On polyacrylamide gel electrophoresis the protein migrates as a faster major component, termed 14-3-3-2 protein, and a slower minor component, termed 14-3-3-1 protein, which consists of approximately 12% of the total protein. Both 14-3-3-1 and 14-3-3-2 have a native molecular weight of approximately 67,000. 14-3-3-2 appears to have the subunit composition alpha beta; 14-3-3-1 has the composition beta'beta'. Peptide mapping with Staphylococcus aureus V8 proteinase shows that alpha and beta subunits are unrelated but the beta and beta' subunits show some common peptides. Immunoperoxidase labelling shows that 14-3-3 is localised in neurones in the human cerebral cortex. 14-3-3 shows no enolase, creatine kinase, triose phosphate isomerase, ATPase, cyclic nucleotide-dependent protein kinase, or purine nucleoside phosphorylase activity. 14-3-3 does not bind calcium and does not appear to be related to calmodulin, calcineurin, tubulin, neurofilament proteins, clathrin-associated proteins, or tropomyosin. The functional significance of this neuronal protein remains obscure.
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PMID:Purification, properties, and immunohistochemical localisation of human brain 14-3-3 protein. 703 50

We have previously shown that Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein dramatically increases during the early period of myoblast fusion and treatment of calmodulin antagonists, such as trifluoperazine, blocks the fusion. Here, we show that cAMP treatment of primary cultures of chick embryonic myoblasts blocks 100-kDa protein phosphorylation. This effect is dose-dependent and can be reversed upon removal of the nucleotide from the culture media. However, cAMP shows little or no effect on accumulation of the 100-kDa protein. Furthermore, phosphorylation of the 100-kDa protein by the partially purified Ca2+/calmodulin-dependent protein kinase (CaM kinase III) from cAMP-treated cells occurs to a much lower extent than that from untreated cells. Nevertheless, cAMP-sensitive protein kinase does not seem to be directly involved in phosphorylation and inactivation of CaM kinase III, because preincubation of cAMP with the myoblast extracts lacking the endogenous 100-kDa protein does not show any effect on activity of CaM kinase III. Similar to its effect on 100-kDa protein phosphorylation, cAMP reversibly inhibits the fusion of cultured myoblasts. Moreover, treatment with forskolin or theophylline, which is known to elevate the intracellular cAMP level, also reversibly blocks both protein phosphorylation and myoblast fusion. On the other hand, cAMP shows little or no effect on accumulation of muscle-specific proteins, such as creatine kinase and tropomyosin. These results suggest that cAMP is involved in down-regulation of both 100-kDa protein phosphorylation and membrane fusion of cultured myoblasts. These results also suggest that the cAMP-mediated inhibition of 100-kDa protein phosphorylation may be associated with its inhibitory effect on myoblast fusion.
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PMID:Cyclic AMP negatively modulates both Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein and membrane fusion of chick embryonic myoblasts. 808 35

Smooth muscle caldesmon was phosphorylated by casein kinase II, and the effects of phosphorylation on the interaction of caldesmon and its chymotryptic peptides with myosin and tropomyosin were investigated. The N-terminal chymotryptic peptide of caldesmon of molecular mass 27 kDa interacted with myosin. Phosphorylation of Ser-73 catalysed by casein kinase II resulted in a 2-fold decrease in the affinity of the native caldesmon (or its 27 kDa N-terminal peptide) for smooth muscle myosin. At low ionic strength, caldesmon and its N-terminal peptides of molecular masses 25 and 27 kDa were retarded on a column of immobilized tropomyosin. Phosphorylation of Ser-73 led to a 2-4-fold decrease in the affinity of caldesmon (or its N-terminal peptides) for tropomyosin. Thus phosphorylation of Ser-73 catalysed by casein kinase II affects the interaction of caldesmon with both smooth muscle myosin and tropomyosin.
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PMID:Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin. 845 32


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