EC:2.7.11.13 - FACTA Search

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)

To test directly whether protein kinase C activation is one of the required events leading to stimulation of prostaglandin production by bone cells, protein kinase C activity and prostaglandin E2 release were measured in monolayer cultures of the clonal human osteosarcoma cell lines G-292 and SaOS-2 after exposure to phorbol myristate acetate (PMA). Both cell lines have specific receptors for PMA but only G-292 cells respond with increased prostaglandin E2 production (M. A. Shupnik and A. H. Tashjian, Jr., J. Biol. Chem., 257: 12161-12164, 1982). The subcellular distribution of protein kinase C in both unstimulated osteosarcoma cell lines was similar; in an EDTA- and leupeptin-containing homogenization buffer, between 70 and 80% of the total enzyme activity was cytosolic. Short (less than 60 min) incubations with PMA induced marked decreases in cytosolic enzyme activity and parallel increases in particulate protein kinase C; thereafter, total measured cellular protein kinase C activity declined, mediated by decreases in both cytosolic and particulate protein kinase C specific activities. By 24 h cytosolic, particulate, and total protein kinase C activities were less than 10% of basal. Because the protein kinase C responses in both cell types were essentially the same, but only G-292 cells give a prostaglandin response to PMA, we conclude that protein kinase C activation by PMA is itself insufficient to stimulate prostaglandin E2 production and that the lack of a prostaglandin response in SaOS-2 cells cannot be explained by lack of protein kinase C activation.
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PMID:Time-dependent changes in protein kinase C distribution and disappearance in phorbol ester-treated human osteosarcoma cells. 347 24

Phorbol 12-myristate 13-acetate (PMA) induces time-dependent changes in protein kinase C subcellular distribution and enzymatic activity in the human osteosarcoma cell line SaOS-2. Short (less than 60 min) incubations with PMA caused decreased cytosolic enzyme activity and a concomitant increase in particulate protein kinase; after 3 h, particulate protein kinase C activity also declined to reach less than 10% of basal activity by 24 h (Krug, E., and Tashjian, Jr., A. H., (1987) Cancer Res. 47, 2243-2246). In order to determine whether the loss in enzyme activity was due to decreased enzyme protein, Western blot analyses were performed using a polyclonal antibody against protein kinase C raised in rabbits. This approach confirmed the previously reported time-related changes: 80-kDa immunoreactive protein kinase C initially translocated from the cytosol to the particulate cell fraction and later disappeared completely from the particulate fraction. Loss of protein kinase C enzymatic activity thus results from actual loss of the 80-kDa protein; we found no evidence for generation of a calcium/phospholipid-independent protein kinase C-like form of the enzyme. Membrane association was confirmed by immunoprecipitation experiments using [35S]methionine-labeled cells. Brief exposure to PMA caused a marked loss in the [35S]methionine-labeled cytosolic protein kinase C band and an increase in the labeled particulate band. Protein kinase C immunoprecipitated from cells treated with PMA for 14 h displayed an increase in [35S]methionine label despite a greater than 80% loss of enzyme activity. The high specific radioactivity of the remaining 80-kDa protein leads us to conclude that long term treatment with PMA causes an increase in the rate of protein kinase C synthesis accompanied by a still greater increase in the rate of enzyme degradation in SaOS-2 cells.
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PMID:Evidence for increased synthesis as well as increased degradation of protein kinase C after treatment of human osteosarcoma cells with phorbol ester. 347 87

We present evidence for the presence of specific, high-affinity binding sites for tritiated phorbol 12,13-dibutyrate on osteosarcoma-derived (HT-3) cells. Activation of protein kinase C by a phorbol ester resulted in an inhibition of alkaline phosphatase activity and the accumulation of prostaglandin E2. Indomethacin blocked prostaglandin E2 production and enhanced alkaline phosphatase activity. These data suggest that prostaglandin E2 is enhanced by activation of protein kinase C, and in turn, alkaline phosphatase activity is reduced.
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PMID:Activation of protein kinase C and the involvement of prostaglandin E2 in the inhibition of osteosarcoma-derived cell alkaline phosphatase activity. 766 74

Cell interaction with extracellular matrix (ECM) modulates cell growth and differentiation. By using in vitro culture systems, we tested the effect of type I collagen (Coll-I) on signal transduction mechanisms in the osteosarcoma cell line UMR-106 and in primary cultures from neonatal rat calvariae. Cells were cultured for 72 h on Coll-I gel matrix and compared with control cells plated on plastic surfaces. Agonist-dependent and voltage-dependent rises in cytosolic Ca2+ concentration ([Ca2+]i; measured by fura 2 fluorometry) were significantly blunted in cells cultured on Coll-I compared with cells grown on plastic. In UMR-106 cells, the collagen matrix effect was mimicked by 24-h incubation with soluble Coll-I or short peptides containing the arginine-glycine-aspartate motif. Accumulation of cellular adenosine 3',5'-cyclic monophosphate (cAMP) stimulated by parathyroid hormone, cholera toxin, and forskolin was augmented (50-150%) in cells plated on Coll-I vs. control. The collagen effect on both [Ca2+]i- and adenylate cyclase-signaling pathways in UMR-106 cells was abrogated in the presence of protein kinase C (PKC) depletion or inhibition. Also, Coll-I induced a twofold increase in membrane-bound PKC without changing cytosolic PKC activity. Thus, by altering PKC activity, Coll-I modulates the [Ca2+]i- and cAMP-signaling pathways in osteoblasts. This, in turn, may influence bone remodeling processes.
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PMID:Cell-matrix interaction in bone: type I collagen modulates signal transduction in osteoblast-like cells. 776 1

The rat osteosarcoma cell line UMR-106 has an osteoblast-like phenotype and possesses parathyroid hormone (PTH)-responsive dual signal transduction systems [adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) and calcium-protein kinase C (Ca-PKC)]. These cells transport inorganic phosphate (Pi) by a Na(+)-dependent carrier under stimulation by PTH. The present study aimed to clarify PTH-responsive signal transduction mechanisms in the regulation of Na(+)-dependent Pi transport by PTH in UMR-106 cells. Exposure of these cells to 10(-7) mol/l PTH induced a significant increase in Pi uptake within 30 min of incubation and it became maximal after 2 h. Parathyroid hormone (10(-9)-10(-7) mol/l) stimulated Pi uptake dose dependently. Activation of PKC by 12-O-tetradecanoyl phorbol-13-acetate (TPA) also increased Pi uptake in time- and dose-dependent manners similar to PTH. In contrast, neither PKA activation by 10(-4) mol/l forskolin or by 10(-4) mol/l dibutyryladenosine 3',5'-cyclic monophosphate nor calcium ionophore treatment with 10(-7) mol/l A23187 or with 10(-7) mol/l ionomycin during 3-h incubations affect Pi uptake, except its increase by 10(-4) mol/l forskolin at a 3-h incubation. These agents had no influence on Pi uptake even in combined treatments with TPA. The PTH-induced increase in Pi uptake was abolished almost completely by pretreating cells with PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) (50 mumol/l) or staurosporin (10 and 50 nmol/l), and by down-regulating PKC with a prolonged TPA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Involvement of protein kinase C in the stimulation of sodium-dependent phosphate transport by parathyroid hormone in osteoblast-like cells. 780 49

The regulation of vitamin D receptor (VDR) abundance in MC3T3-E1 mouse osteoblasts and UMR 106-01 rat osteosarcoma cells by rat PTH 1-34, human PTH-related protein 1-34, and agents that activate specific signal transduction pathways was studied. Treatment of these cells with forskolin (FSK) caused up-regulation of VDR, whereas treatment with phorbol esters suppressed VDR levels. PTH or PTH-related protein treatment induced a 2- to 3-fold increase in VDR, which was equivalent to that elicited by FSK in UMR 106-01 cells but less than the FSK-induced increase (approximately 8-fold) in MC3T3-E1 cells. PTH treatment of MC3T3-E1 cells resulted in an approximately 3-fold increase in VDR levels with maximum stimulation occurring at 10(-9) M PTH after 4 h of treatment. In UMR 4-7 cells, a subclone of UMR 106-01 cells that express cAMP resistance due to regulated expression of a mutant form of the type 1 regulatory subunit of the cAMP-dependent protein kinase A (PKA), the up-regulation of VDR abundance due to FSK and PTH treatment was mostly prevented. Pretreatment of MC3T3-E1 cells with staurosporine, an inhibitor of PKC, resulted in an approximately 3-fold increase in basal VDR levels but did not enhance the PTH-mediated up-regulation of VDR. Collectively, these data suggest that the increase in VDR abundance observed in these target cells is mainly due to the activation of the PKA signal transduction pathway. Treatment of UMR 106-01 cells with PTH for 4 h before exposure of the cells to 1,25-dihydroxyvitamin D3 resulted in a 2-fold increase in the induction of 25-hydroxyvitamin D3-24 hydroxylase messenger RNA. Thus, exposure of target cells to PTH augments their response to 1,25-dihydroxyvitamin D3 due to up-regulation of VDR abundance.
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PMID:Regulation of 1,25-dihydroxyvitamin D3 receptors by parathyroid hormone in osteoblastic cells: role of second messenger pathways. 783 3

Epidermal growth factor (EGF) induces a rapid increase in the phosphorylation of extracellular signal-regulated kinases (ERKs) in the human osteosarcoma osteoblastic cell line G292 and in primary cultures of rat osteoblastic cells. This phosphorylation is transient and time-dependent. Maximal stimulation is attained within 1 min in G292 and within 5 min in rat osteoblastic cells. Enzymatic activity in G292 cells is also induced rapidly after EGF stimulation. Western blot analysis revealed that enhancement of the phosphorylation of ERKs in the EGF-stimulated cells is not due to an increase in ERK protein, since EGF-treatment does not lead to an increase in the absolute amount of ERKs present even after 2 days of stimulation. The pattern of expression of the ERKs observed in the two cell types differs in the apparent molecular weights observed. The most slowly migrating immunoreactive protein (approximately 45 kDa) in normal rat osteoblastic cells is ERK1, identified by an ERK1-selective antiserum. The same antiserum reacts only weakly with one of the ERK proteins (44 kDa) blotted from the human osteosarcoma cell line G292. Phorbol 12-myristate 13-acetate (PMA) is also capable of inducing ERK phosphorylation, albeit to a lasser degree. The combination of PMA and EGF does not produce a greater response than EGF alone. The role of protein kinase C (PKC) in the EGF-stimulated ERK signaling pathway was further examined by inhibition of PKC with the staurosporine analog, CGP41251, and by down-regulation of PKC via chronic treatment with PMA. Chronic PMA treatment results in a partial inhibition of the EGF-mediated phosphorylation. CGP41251 completely abolishes the increased ERK activity produced by PMA, but the effect of EGF in this regard is potentiated. We conclude that PKC and EGF act through parallel pathways to stimulate ERK phosphorylation and activity. The inhibitor studies, in addition, indicate that activation of PKC may moderate the actions of the EGF pathway via a tonic inhibitory feedback.
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PMID:EGF-mediated phosphorylation of extracellular signal-regulated kinases in osteoblastic cells. 786 Jun 43

Five analogues of human parathyroid hormone (hPTH-(20-34)-NH2, I; cyclo[Lys26-Asp30]-hPTH-(20-34)-NH2, II; cyclo[Glu22-Lys26]-hPTH-(20-34)-NH2, III; cyclo[Lys27-Asp30]- hPTH-(20-34)-NH2, IV; and [Leu27]-hPTH-(20-34)-NH2 V) were tested for their ability to promote membrane-bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in alpha-helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix-promoting solvent. The remaining analogues showed a three- to four-fold enhancement of alpha-helix in this solvent. Analogues II and III had increased retention times in reversed-phase chromatography, as compared to I and IV. This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV. The data suggest that in the region bounded approximately by residues 24-32, an amphiphilic alpha-helix is important for correct functional binding to the PTH receptor.
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PMID:Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment. 792 86

PTH is regarded conventionally as a catabolic hormone that stimulates osteoclastic resorption of bone. However, it has been known since 1932 that intermittent pulses of PTH stimulate bone formation in animals and humans. PTH independently activates two signal mechanisms: one that stimulates adenylyl cyclase and one that stimulates protein kinase C (PKC). The goal of this study was to use the 3- to 5-month-old ovariectomized (OVX) rat model to determine which of the two signal mechanisms is responsible for the anabolic action of PTH on bone. OVX triggered a large loss of trabecular bone without significantly affecting the normal slow growth of cortical bone in the distal halves of the femora. Daily injections of human hPTH(1-34) fragment (1 nmol/100 g body weight), which stimulated both adenylyl cyclase and membrane-associated PKC activity in osteoblast-like ROS 17/2 rat osteosarcoma cells, stimulated the growth of both cortical and trabecular bone in the OVX rats. Daily injections of the same dose of hPTH(1-31), which stimulated adenylyl cyclase but not PKC in ROS 17/2 cells, stimulated trabecular bone growth in the OVX rats less effectively than hPTH(1-34), but it stimulated cortical bone growth as rapidly and as dramatically as hPTH(1-34). Injections of equimolar amounts of desamino-hPTH(1-34) [N-propionyl(2-3)hPTH-amide], which stimulated PKC as strongly as hPTH(1-34) in ROS 17/2 cells but had a drastically reduced ability to stimulate adenylyl cyclase, or injections of recombinant hPTH(8-84) which stimulated PKC only in the ROS 17/2 cells, did not stimulate cortical or trabecular bone growth in the OVX animals. Thus, cyclic AMP and cyclic AMP-dependent protein kinases may be the primary mediators of the anabolic action of intermittent pulses of PTH on bone in OVX rats.
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PMID:Parathyroid hormone fragments may stimulate bone growth in ovariectomized rats by activating adenylyl cyclase. 797

The roles of three protein kinases, cyclic AMP-dependent protein kinase (protein kinase A), protein kinase C, and beta-adrenergic receptor kinase (beta ARK), implicated in agonist-induced desensitization of guanine nucleotide-binding protein (G-protein)-coupled receptors were explored in four different cell lines after 48 hr of incubation with oligodeoxynucleotides antisense to the mRNA encoding each kinase. Desensitization of beta 2-adrenergic receptors was analyzed in cell types in which the activities of the endogenous complement of protein kinases A and C and beta ARK were distinctly different. Protein kinase A was necessary for desensitization of rat osteosarcoma cells (ROS 17/2.8), whereas the contribution of beta ARK to desensitization was insignificant. In Chinese hamster ovary cells that stably express beta 2-adrenergic receptors and in smooth muscle cells (DDT1MF-2), oligodeoxynucleotides antisense to beta ARK mRNA nearly abolished desensitization, whereas oligodeoxynucleotides antisense to protein kinase A mRNA attenuated desensitization to a lesser extent. In human epidermoid carcinoma cells (A-431), oligodeoxynucleotides antisense to either protein kinase A mRNA or beta ARK mRNA attenuated agonist-induced desensitization, providing a third scenario in which two kinases constitute the basis for agonist-induced desensitization. In sharp contrast, oligodeoxynucleotides antisense to protein kinase C mRNA were found to enhance rather than attenuate desensitization in DDT1MF-2 and A-431 cell lines, demonstrating counterregulation between prominent protein kinases in desensitization. Using antisense oligodeoxynucleotides to "knock out" target protein kinases in vivo, we reveal distinctive cell-type-specific roles of protein kinase A, protein kinase C, and beta ARK in agonist-induced desensitization.
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PMID:Oligodeoxynucleotides antisense to mRNA encoding protein kinase A, protein kinase C, and beta-adrenergic receptor kinase reveal distinctive cell-type-specific roles in agonist-induced desensitization. 799 5

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