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)

1. Endothelin-1 (ET-1) caused a concentration-dependent contraction of helical strips from rat thoracic aorta in the absence of extracellular Ca2+. The Ca(2+)-depleted muscle strips, prepared by three repeated applications of 10(-2) M caffeine or 10(-6) M noradrenaline in Ca(2+)-free buffer, were contracted by 10(-8) M ET-1 in the same manner as non-treated strips. 2. In the absence of extracellular Ca2+, 10(-7) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, induced a small but sustained contraction of the rat thoracic aorta strips within 60 min. Preincubation of the strips with 10(-7) M PMA for 60 min in Ca(2+)-free buffer, did not affect the 10(-8) M ET-1-induced contraction, but decreased the 5 x 10(-8) M phorbol 12,13-dibutyrate (PDB)-, or the 10(-7) M PMA-induced contraction, and potentiated the contraction induced by 10(-8) M urotensin II. Preincubation with 10(-8) M ET-1 (which induced maximum contraction) for 25 min in Ca(2+)-free buffer did not change the subsequent contraction induced by PMA (10(-7) M) or urotensin II (10(-8) M) but gave a somewhat lower maximum tension than in non-treated strips. 3. Calyculin-A, a potent inhibitor of phosphatase, also induced a contraction of the Ca(2+)-depleted muscle strips in Ca(2+)-free buffer. Preincubation of the strips with ET-1 (10(-8) M) or PMA (10(-7) M) decreased the calyculin-A (3 x 10(-8) M)-induced contraction.4. These results suggest that ET-1 may induce phosphorylation of an unknown protein either without an increase in myoplasmic Ca2 + concentration or, alternatively, with mobilization of intracellular Ca2+ from noradrenaline- and caffeine-insensitive Ca2 + sources, through a mechanism different from that of phorbol ester.
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PMID:Contraction of rat thoracic aorta strips by endothelin-1 in the absence of extracellular Ca2+. 181 May 98

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, alpha-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120-270 to 500-700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca(2+)-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation-dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca(2+)-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C).
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PMID:The Ayerst Award Lecture 1990. Calcium-dependent mechanisms of regulation of smooth muscle contraction. 181 84

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.
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PMID:The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C. 183 77

Incubation of plasma membranes isolated from bovine aorta with either 0.5 mM CaCl2 or with a phorbol ester (1 microM phorbol 12,13-dibutyrate) and phosphatidylserine in an EGTA-containing buffer resulted in the phosphorylation of 10 proteins (Mr of 158, 105, 75, 62, 44, 39, 33, 22, 15 and 9 kDa), presumably due to activation of endogenous protein kinase C (PKC). After heat treatment of the aortic plasma membranes at 80 degrees C for 5 min in order to inactivate all endogenous protein kinase, phosphatase and ATPase activities, membrane phosphorylation was absolutely-dependent upon the addition of an exogenous, partially-purified PKC preparation from bovine aorta. Under these conditions, a total of 17 phosphoproteins could be detected (Mr of 158, 105, 75, 44, 39, 33, 30, 29, 27, 25, 22, 17.5, 16, 15, 11, 10 and 9 kDa). The most prominent phosphoprotein band in native membranes had a molecular weight of 75 kDa (p75); several characteristics suggest that p75 might be autophosphorylated PKC. The phosphorylation of aortic plasma membranes by exogenous PKC required phosphatidylserine and was calcium-dependent (10(-5) to 10(-7) M Ca2+); the addition of diolein resulted in little or no enhancement of phosphorylation. Replacement of phosphatidylserine with oleic acid resulted in the same number of phosphoproteins, but the extent of phosphorylation was diminished. The phosphorylation pattern was altered slightly if the aortic plasma membranes were isolated in the presence of 1 mM Ca2+ instead of EGTA buffers as in the standard procedure. Experiments were performed to determine if the p39 substrate of PKC in aortic plasma membranes was calpactin II (lipocortin I). Immunoblotting established that calpactin II was present in aortic plasma membranes, but there was no corresponding phosphoprotein on the autoradiographs.
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PMID:Phosphorylation of aortic plasma membranes by protein kinase C. 183 27

Protein phosphorylation and dephosphorylation are involved in regulation of cell growth. We tested the hypothesis that the growth inhibitory effect of transforming growth factor beta 1 (TGF-beta 1) involves activation of protein phosphatases. Exposure of human keratinocytes in culture to 400 pM TGF-beta 1 for 48 h led to 80% inhibition of DNA synthesis as measured by nuclear labeling. Incubation of cultured keratinocytes with 400 pM TGF-beta 1 rapidly activated (within 30 min) protein serine/threonine phosphatase, measured using phosphorylase as a substrate. Based on several criteria, including neutralization of activity with specific antibodies and inhibitor-2, TGF-beta 1-activated phosphorylase phosphatase was identified as protein phosphatase 1. TGF-beta 1 did not have rapid effects on protein serine/threonine phosphatase activity (type 2A) measured with histone phosphorylated by protein kinase C or on protein tyrosine phosphatase activity. However, protein tyrosine phosphatase was activated at 48 h, coincident with growth arrest. Differentiation, induced by the combination of TGF-beta 1 plus calcium or by serum, was not accompanied by further serine/threonine or tyrosine phosphatase activation. We conclude that induction of growth arrest in keratinocytes by TGF-beta 1 involves acute activation of protein phosphatase 1, while activation of protein tyrosine phosphatase may represent an additional mechanism for maintaining cells in a growth-arrested state.
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PMID:Growth arrest induced by transforming growth factor beta 1 is accompanied by protein phosphatase activation in human keratinocytes. 184 73

This report describes the cloning and characterization of rat leukocyte common antigen-related protein (rLAR), a receptor-like protein tyrosine phosphatase (PTPase). The recombinant cytoplasmic PTPase domain was expressed at high levels in bacteria and purified to homogeneity. Kinetic properties of the PTPase were examined along with potential modulators of PTPase activity. Several sulfhydryl-directed reagents were effective inhibitors, and a surprising distinction between iodoacetate and iodoacetamide was observed. The latter compound was an extremely poor inhibitor when compared to iodoacetate, suggesting that iodoacetate may interact selectively with a positive charge at or near the active site of the enzyme. Site-directed mutants were made at 4 highly conserved cysteine residues found at positions 1434, 1522, 1723, and 1813 within the protein. The Cys-1522/Ser mutation resulted in a 99% loss of enzymatic activity of the pure protein. This observation is consistent with greater than 99% of the PTPase activity being found in the first domain of the PTPase and demonstrates the critical importance of this cysteine residue in catalysis. The recombinant C1522S mutant phosphatase could also be phosphorylated in vitro by protein kinase C and p43v-abl tyrosine kinase. When pure recombinant PTPase was mixed with 32P-labeled tyrosine substrate and then rapidly denatured, a 32P-labeled enzyme intermediate could be trapped and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The catalytically inactive C1522S mutant did not form the phosphoenzyme intermediate.
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PMID:Cloning, bacterial expression, purification, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase. 191 76

The RL cell line is an EBV-negative, surface IgM, IgD-positive B lymphoma line, which is significantly growth arrested in the presence of acrylamide-linked antibodies to the surface IgM receptor. We demonstrate here that activation of protein kinase C (PKC) with PMA abrogates anti-IgM-induced phosphoinositide turnover and Ca2+ mobilization; however, growth inhibition is not affected. In addition, inhibitors of PKC are unable to reverse the anti-IgM-mediated growth inhibition. Two-dimensional gel electrophoresis reveals a different pattern of protein phosphorylation after treatment of RL with PMA or anti-IgM. These data strongly suggest that anti-IgM-induced growth inhibition does not rely on phospholipase C-mediated phosphoinositide turnover, Ca2+ mobilization, or PKC activation. On the other hand, the phosphatase inhibitor orthovanadate results in an augmentation of proteins phosphorylated on tyrosine and the growth inhibition which follows anti-IgM treatment. Furthermore, protein tyrosine kinase inhibitors, genistein and herbimycin A, are able to reverse the anti-IgM-induced inhibition of growth. These data demonstrate that multiple signaling pathways are activated by the interaction of anti-IgM with its ligand, and suggest that tyrosine kinase activation is a critical component of the inhibitory response.
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PMID:Anti-IgM-mediated growth inhibition of a human B lymphoma cell line is independent of phosphatidylinositol turnover and protein kinase C activation and involves tyrosine phosphorylation. 191 71

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.
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PMID:In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon. 193 6

MCH (melanin concentrating hormone) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which stimulates melanosome (melanin granule) aggregation to a perinuclear position within teleost fish integumental melanocytes, resulting in lightening of the skin. The mechanisms of action of MCH are unknown. Drugs that affect the diacylglycerol/inositol triphosphate pathway were used to investigate the possible roles of this pathway in the mechanisms of action of MCH on Synbranchus marmoratus (teleost) melanocytes. The shift of the dose-response curve to MCH in the presence of various concentrations of 4-bromophenacyl bromide and neomycin sulphate, phospholipase C inhibitors, suggests that phospholipase C is stimulated after MCH receptor activation. Low concentrations (10(-9) to 10(-8) M) of the phorbol ester TPA exhibited MCH-like activity, eliciting a dose-dependent melanosome aggregation. Higher doses, however, displaced to the right the dose-response curve to MCH, as did the protein kinase C inhibitors, dibucaine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). These results support the assumption that protein kinase C mediates the pigment aggregating activity of MCH. Both MCH and norepinephrine lightening actions were abolished by beta-glycerophosphate, a phosphatase inhibitor, suggesting that a protein dephosphorylation occurs during melanosome aggregation, and is, therefore, a common event triggered by MCH and norepinephrine, although both agonists act through separate receptors and exhibit different transduction mechanisms.
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PMID:Protein-kinase C mediates MCH signal transduction in teleost, synbranchus marmoratus, melanocytes. 194 11

Laminin mediates neural adhesion and process formation. A possible signal transduction pathway for laminin was investigated in both NG108-15 and PC12 neuronal cells using radiolabeling studies as well as various stimulators and inhibitors of phosphatases and kinases. Using [32P]-ortho-phosphate, laminin caused a decrease in the TCA-precipitable counts. Further, laminin stimulated dephosphorylation of laminin binding proteins of 110 kDa, 67 kDa, and 45 kDa and this dephosphorylation was blocked by the phosphatase inhibitor, okadaic acid, and the protein kinase C stimulator, TPA. The phosphatase inhibitors okadaic acid and vanadate, as well as the protein kinase C stimulators, TPA and DAG, blocked laminin-mediated process formation. Inhibitors of kinase activity such as H-7, H-8, and H-9 increased laminin-mediated neural process formation. Since phosphate incorporation into laminin-binding proteins is decreased by laminin and because both phosphatase inhibitors and kinase stimulators inhibit laminin-mediated process formation, we conclude that dephosphorylation events promote the neural cell response to laminin.
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PMID:Laminin-mediated process formation in neuronal cells involves protein dephosphorylation. 196 40


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