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

Prostacyclin (1 ng to 2 micrograms per ml), which effectively inhibits platelet secretion and aggregation, does not affect adhesion of a proportion of platelets (10-38%) to collagen (50-100 micrograms/ml). Adhesion is not detectable by changes of light transmission (as measured in the optical aggregometer) and is not affected by inhibitors of cyclooxygenase and lipoxygenase enzymes such as indomethacin and compound BW 755C. This adhesion is independent of the collagen concentration (50-400 micrograms/ml) and the incubation time (5-20 min). This suggests that adhesion to collagen is related to a specific platelet population. Adhesion in the presence of prostacyclin, indomethacin and BW 755C occurs in parallel with the formation of a limited amount of phosphatidic acid. Under those conditions it is also possible to observe some phosphorylation of a 40,000 dalton protein which is a substrate for protein kinase C activity. Phosphorylation of the 20,000 dalton protein, or myosin light chain, is less evident. Chlorpromazine (25-100 micrograms/ml) inhibited the adhesion of platelets to collagen, but propanolol (0.5-4 microM) was inactive. The adhesion of platelets to collagen in these experiments parallels the formation of a fraction of phosphatidic acid and 40,000 dalton protein phosphorylation, which are independent of the increased levels of platelet cyclic-AMP induced by high concentrations of prostacyclin. It is also independent of the formation of cyclooxygenase or lipoxygenase products.
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PMID:Adhesion of human platelets to collagen in the presence of prostacyclin, indomethacin and compound BW 755C. 308 71

Tetradecanoylphorbol acetate (TPA) activates primarily only the protein kinase C pathway not the calcium ion-dependent pathway in platelets. The net effect of this split activation is that only the pseudopodal cytoskeleton assembles, not the contractile cytoskeleton needed for rapid secretion. In this study, platelets were first activated with TPA, then activated secondarily with either thrombin or arachidonate and the subsequent dense body secretion, calcium-ion mobilization, protein phosphorylation and cytoskeletal assembly compared to these same processes in control platelets activated solely with either thrombin or arachidonate. Secretion was reduced as the length of time between the primary and secondary activation was increased; but at a 2-3 min interval, where the activation by TPA was essentially complete, the reduction in the total radiolabeled serotonin secreted was small. Furthermore, nearly normal cytosolic calcium-ion increases, phosphorylation of myosin light chain and contractile cytoskeletal development were induced by thrombin or arachidonate after this interval. Prior treatment of the platelets with 100 microM acetylsalicylate to block the cyclooxygenase-dependent pathway caused minor reduction in dense-body secretion induced by TPA or thrombin or the combination of both, but otherwise the relative results were comparable to the untreated platelets. Therefore, short-term prior activation of gel-filtered platelets with TPA, even at concentrations in excess of 100-times that required to saturate protein kinase C, does not prevent normal activation of the calcium ion dependent processes through either the cyclooxygenase-dependent or -independent pathway. Longer-term preincubations with TPA differentially inhibit the secretion response induced by thrombin and arachidonate.
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PMID:The effect of tetradecanoylphorbol acetate on calcium-ion mobilization, protein phosphorylation and cytoskeletal assembly induced by thrombin or arachidonate. 308 29

The Ca dependence of contraction and myosin phosphorylation was investigated in canine tracheal smooth muscle stimulated with carbachol, K or serotonin. Previous studies of tracheal muscle showed carbachol concentration-response curves for contraction and myosin phosphorylation were superposable. In contrast, there was a striking difference in the Ca++ sensitivities of tension and myosin phosphorylation when Ca++ concentration-response curves were constructed in the presence of 10(-7) M carbachol. Significant phosphorylation (greater than 0.3 moles phosphate/mole 20,000 dalton myosin light chain) was observed in the absence of active tension. In the present study, carbachol (10(-7) and 10(-6) M) and serotonin (10(-5) M) also induced significant myosin phosphorylation in low Ca++ solutions (0-0.025 mM CaCl2) without proportional increases in tension. K+ depolarization in Ca++-free physiological salt solution (60 mM KCl, 10(-6) M atropine) yielded phosphorylation not significantly different from basal levels. All stimulants induced active stress after readmission of Ca. The Ca++ dependence curve for myosin phosphorylation in muscles stimulated with carbachol was shifted up and to the left of the force curve. Atropine (10(-6) M) significantly reduced phosphorylation induced by carbachol in Ca++-free solutions, as did 3 X 10(-6) M nifedipine and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Phorbol 12-myristate, 13-acetate or phorbol 12,13-dibutyrate did not increase basal phosphorylation or phosphorylation in low Ca++ solutions, suggesting that protein kinase C did not phosphorylate myosin in this case. Myosin phosphorylation under these conditions is not sufficient to support contraction, and is reduced by treatments that decrease Ca++ entry.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle. 310 Jul 73

Phosphorylation of myosin light chain (LC) isoforms in arterial actomyosin can be induced by endogenous kinases upon addition of Mg2+ and ATP. The extent of phosphorylation in the presence of 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 0.2 mM Ca2+, or 0.2 mM Ca2+ plus calmodulin is 1.6, 2.1, and 2.3 mol phosphate/mol LC, respectively. Two-dimensional gel electrophoresis of actomyosin shows that the LC isoforms may be mono-, di-, and triphosphorylated. Tryptic phosphopeptide mapping of LC indicates that the radioactive phosphate is distributed to six peptides referred to as A through F. Phosphoamino acid analyses and the phosphopeptide maps of isolated LC, phosphorylated by either purified myosin light chain kinase or protein kinase C, reveal that myosin light chain kinase phosphorylates a serine residue in peptides A and B, and threonine plus serine residues in peptides C and D. Peptides E and F are phosphorylated by protein kinase C in serine and threonine residues, respectively. In actomyosin, with EGTA, phosphorylation of peptides E and F proceeds while phosphorylation of peptides A, B, C, and D is inhibited. Ca2+ and calmodulin enhance the phosphorylation of peptides A, B, C, and D, while phosphorylation of peptides E and F is decreased. In isolated LC, myosin light chain kinase preferentially phosphorylates the peptides A and B over C and D. Phosphorylation of peptides E and F in LC by protein kinase C promotes additional phosphorylation of peptides C and D by myosin light chain kinase, whereas phosphorylation of peptides A and B is diminished. The present data suggest that the phosphorylation of distinct sites in arterial myosin light chain by myosin light chain kinase and protein kinase C is interrelated.
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PMID:Phosphorylation of the 20,000-Da myosin light chain isoforms of arterial smooth muscle by myosin light chain kinase and protein kinase C. 314 63

The phosphorylation of synthetic peptides derived from the NH2-terminal sequence of smooth-muscle myosin was studied with purified protein kinase C. The protein kinase C phosphorylation domain included both serine residues and threonine residues in the sequence SSKRAKAKTTKKR(G), denoted myosin light chain (1-13) (MLC(1-13)). Kinetic analysis of MLC(1-13) and truncated peptides derived from the parent peptide established that removal of the serine residues had little effect on protein kinase C reactivity. MLC(1-13) had a V/K of 2.4 min-1.mg-1, whereas the V/K of MLC(3-13) was 3.0 min-1.mg-1. Removal of Lys-3 resulted in a 50% decrease in V/K which was attributable to a 50% decrease in apparent Vmax.Arg-4 was established as a significant protein kinase C specificity determinant, since the apparent Km increased 7-fold and the Vmax decreased 3-fold when the parent peptide was truncated at that residue. All peptides studied required calcium and lipid effectors for full activity with protein kinase C, indicating that they are Class C substrates as defined by Bazzi and Nelsestuen (Biochemistry 26 (1987) 5002) for protein kinase C. Other protein kinases, including cyclic AMP- and cyclic GMP-dependent protein kinase, S6/H4 kinase, myosin light-chain kinase and calcium/calmodulin-dependent kinase II, had little or no activity with these peptides. In studies on the purification of lymphosarcoma protein kinase C by several chromatographic procedures, the results showed that the myosin light-chain peptides can provide convenient and well-characterized substrates for purification and mechanistic studies of protein kinase C biochemistry.
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PMID:Synthetic peptides derived from the nonmuscle myosin light chains are highly specific substrates for protein kinase C. 317 14

The effects of 12-o-tetradecanoylphorbol-13-acetate (TPA) on isometric tension, unloaded shortening velocity determined using the slack test (Vmax), and phosphorylation of myosin light chain (MLC20) were investigated in intact and skinned muscle tissues of the rabbit mesenteric artery. In intact muscles exposed to 128 mM K+, the Vmax reached a maximum before the peak of the phasic component of contraction and reduced proportionally with changes in the amplitude of the subsequent tonic contraction. TPA (0.1 microM) enhanced to a greater extent the amplitude of the tonic than the phasic contraction. During the tonic phase, TPA enhanced the Vmax and amplitude of the contraction more than the phosphorylation of MLC20. In skinned muscles, Ca2+ (0.3-1.0 microM) increased the force, Vmax and MLC20 phosphorylation, in a dose dependent manner. The relation between any two of the above three parameters was always linear. TPA (0.1 microM) with phosphatidylserine (50 micrograms/ml) time-dependently increased the force and Vmax observed with 0.3-0.5 microM Ca2+. The relation between the force and Vmax shifted in the direction of that observed in the presence of high Ca2+, but in 0.5 microM Ca2+, TPA with phosphatidylserine showed little change in the phosphorylation of MLC20. As estimated from the actions of TPA, protein kinase C may contribute to activation of contractile proteins through MLC20 phosphorylation dependent and other regulatory processes.
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PMID:Actions of a phorbol ester on factors regulating contraction in rabbit mesenteric artery. 318 Mar 54

Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation.
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PMID:Sites phosphorylated in myosin light chain in contracting smooth muscle. 319 18

Phorbol diesters, potent activators of protein kinase C, can produce a slow contraction in arterial smooth muscle. Such observations have prompted proposals that protein kinase C may have direct regulatory functions in contraction. In this paper, we present evidence that [Ca2+]-dependent myosin light chain phosphorylation is responsible for the contraction induced by low-dose phorbol diester and during force development in response to high-dose phorbol diester stimulation. The relationships between myoplasmic [Ca2+], myosin phosphorylation, and steady-state stress induced by low-dose phorbol dibutyrate were similar to those observed with contractile agonists. However, prolonged exposure to high-dose phorbol dibutyrate induced high stress with elevated phosphorylation that was not associated with elevations in aequorin-estimated [Ca2+]. Our results suggest that phorbol diesters can increase myoplasmic [Ca2+], and the resulting increase in myosin phosphorylation quantitatively explains the contraction.
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PMID:[Ca2+]-dependent myosin phosphorylation in phorbol diester stimulated smooth muscle contraction. 320 46

The protein phosphorylation changes associated with the contraction and relaxation of bovine carotid artery smooth muscle were studied using two-dimensional gel electrophoresis of labeled phosphoproteins. Muscle was stimulated with histamine, angiotensin II, 12-deoxyphorbol 13-isobutyrate (DPB) or high extracellular K+. Histamine induced a rapid and sustained contraction which was associated with an early (2 min) phosphorylation of 20 kDa myosin light chain (MLC) and two cytosolic proteins, Nos. 1 and 2, and with the late (60 min) phosphorylation of MLC, two isoelectric variants of desmin and ten other cytosolic proteins. Additionally, there was a decrease in the extent of phosphorylation of two cytosolic proteins, Nos. 9 and 10. Angiotensin II induced a rapid but transient contraction which was associated with the same early (2 min) phosphorylation changes, but with none of the late (60 min) changes. Elevation of the extracellular K+ concentration to 110 mM led to a sustained contraction which was associated with the phosphorylation of MLC and proteins Nos. 1 and 2 at both 2 and 60 min, but none of the other late phase phosphoproteins were seen. Addition of DPB, an activator of protein kinase C, induced a slowly developing but sustained contractile response which was associated with none of the early (5 min) phosphorylation changes. However, nearly all of late (60 min) protein phosphorylation changes were the same as those seen after histamine action. Addition of forskolin to either control or histamine-treated muscle led to an increase in the phosphorylation of three cytosolic proteins (Nos. 3, 8 and 13), and in the histamine-contracted muscle the dephosphorylation of MLC and proteins Nos. 4, 9, 10, 15 and 16. Similarly, forskolin induced a relaxation of DPB-treated muscle and the dephosphorylation of proteins Nos. 4, 9, 10, 15 and 16. These results suggest that there are two pathways by which histamine activates contraction: a Ca2+-calmodulin pathway which initiates the response, and a protein kinase C pathway which, along with the Ca2+-calmodulin pathway, sustains contraction.
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PMID:Protein phosphorylation changes in bovine carotid artery smooth muscle during contraction and relaxation. 321 89

Three forms of 20-kDa myosin light chain (MLC), unphosphorylated, monophosphorylated, and diphosphorylated MLC (designated 20K, 20K-P, and 20K-PP) were demonstrated in thrombin-stimulated human platelets by two different gel electrophoretic methods: in the presence of glycerol urea or in two dimensions (isoelectric and sodium dodecyl sulfate). The diphosphorylation of platelet 20-kDa MLC increased, dose dependently, up to 0.4 U/ml thrombin and reached 25% of platelet 20-kDa MLC. After mono- or diphosphorylated 20-kDa MLC from thrombin-stimulated platelets was digested with trypsin, the analysis using two-dimensional peptide mapping demonstrated that two different sites were phosphorylated by MLC kinase and protein kinase C, as noted in the case of 12-O-tetradecanoylphorbol-13-acetate-stimulated platelets (M. Naka, et al. (1983) Nature (London) 306, 490-492). The more rapid monophosphorylation was catalyzed preferentially by MLC kinase while the slower and additional phosphorylation was catalyzed mainly by protein kinase C. These results suggest the importance of distinguishing multiple site phosphorylation of 20-kDa MLC in thrombin-activated human platelets.
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PMID:Two phosphorylated forms of myosin in thrombin-stimulated platelets. 335 50


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