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)

We have been examining the role of protein kinase C (PKC) in synovial cell activation in response to interleukin-1 (IL-1). Attempts to measure PKC in soluble extracts of synovial fibroblasts by standard techniques failed. Western blotting with anti-PKC antibodies detected only a low level of PKC in synovial cells compared to rat basophilic leukemia cells and crude brain extracts. However, synovial PKC could be detected by measuring the Ca(2+)- and phospholipid-dependent phosphorylation of endogenous substrates. In this way, a 35 kDa protein was identified as the major endogenous cytosolic substrate for PKC. Treatment of synoviocytes with phorbol myristate acetate (PMA) strongly induced the synthesis of neutral metalloproteinases (NPs) and prostaglandin E2 (PGE2). Both Western blotting and assays based upon phosphorylation of the 35 kDa protein confirmed translocation of PKC from the cytosol in response to PMA. Although IL-1 induced the NPs and PGE2, it did so without detectable translocation of PKC. There thus appear to be PKC-dependent and PKC-independent routes of synovial cell activation. Our data suggest that IL-1 uses the latter.
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PMID:Interleukin-1 and synovial protein kinase C: identification of a novel, 35 kDa cytosolic substrate. 166

It has been suggested that the Ca2+ and phospholipid dependent protein kinase (protein kinase C; PKC) plays some intermediary roles in the regulation of the zona pellucida-induced acrosome reaction in mouse sperm. We here demonstrated that PKC activity is in the cytosol fraction of mouse sperm and that treatment of sperm with a PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), induces translocation of PKC to the membrane fraction. Treatment of epididymal sperm with 20 ng/ml TPA or 20 microM of the Ca2+ ionophore A23187 did not induce any specific protein phosphorylation. However, two specific proteins, with molecular weights of 215 kDa and 35 kDa, were significantly phosphorylated when sperm were incubated with A23187 prior to TPA treatment. A similar synergistic effect of TPA and A23187 was observed in Ca2+ accumulation in sperm. We also demonstrated that exogenous PKC purified from human pancreatic cells catalyzes the phosphorylation of these two proteins in vitro as well. The present data support the idea that the activation of PKC and subsequent protein phosphorylation are involved in the regulation of the zona pellucida-induced acrosome reaction.
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PMID:Protein kinase C activity and protein phosphorylation in mouse sperm. 199 9

Follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin, a novel gonadal glycoprotein hormone, has been shown to have chronic inhibitory effects on the secretion of both FSH and luteinizing hormone (LH) in response to gonadotropin-releasing hormone (GnRH) in vitro. The present study was designed to investigate the acute effects of bovine FSP on GnRH-stimulated gonadotropin secretion and to examine the potential subcellular sites of this action of FSP using cultured pituitary cells. Anterior pituitaries from adult male Sprague-Dawley rats were enzymatically dispersed and cultured for 48 h, after which the cells were treated with bovine FSP for 6 h, followed by a 4 h stimulation with secretagogues in the continued presence of FSP. Results showed that the 35 kDa form of bovine FSP (0.1-3 nM) dose-dependently suppressed GnRH-stimulated FSH and LH secretion, with inhibition of 38 and 25%, respectively, at 3 nM. In addition, FSP suppressed gonadotropin secretion in response to activators of protein kinase C (phorbol 12-myristate 13-acetate (PMA) and mezerein) and a calcium ionophore (A23187). However, FSP had no effect on gonadotropin secretion evoked by melittin, an activator of phospholipase A2. Furthermore, 35 kDa bovine FSP did not compete with GnRH for GnRH binding sites in a direct competition study and treatment of cultured pituitary cells with FSP (0.1-3 nM) for 10 h did not alter the number of GnRH binding sites on the cell membranes. Finally, similar inhibitory effects on gonadotropin secretion in response to GnRH, PMA and mezerein were obtained with the 31 and 39 kDa forms of bovine FSP, each at a concentration of 1 nM. We conclude from the present study that FSP acutely inhibits GnRH-stimulated gonadotropin secretion in cultured pituitary cells, and that FSP exerts its action beyond the GnRH receptor, possibly by affecting the protein kinase C and/or the calcium-calmodulin systems.
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PMID:Acute inhibitory effect of follicle-stimulating hormone-suppressing protein (FSP) on gonadotropin-releasing hormone-stimulated gonadotropin secretion in cultured rat anterior pituitary cells. 212 65

When cultured in the absence of thyreostimulin (TSH), thyroid cells lose some of their differentiated functions such as iodide transport and its incorporation into thyroglobulin. In the presence of TSH (0.1 mU/ml), these differentiated functions are preserved ("TSH cells"). The addition of tetradecanoyl phorbol 13 acetate (TPA) inhibits some differentiated functions of the cells and provokes important modifications of bio-signalling pathways. The protein kinase C (pKC) activity, unchanged in "control" and "TSH cells", was dramatically modified in TPA treated cells. After translocation, the pKC activity was down-regulated and the phosphorylation of its endogenous substrates (35-38 kDa) disappeared. Among these substrates, we identified the lipocortin I (LC I) (35 kDa), a phospholipase A2 inhibitory protein related to the Ca2+ binding protein family. By monodimensional electrophoresis (PAGE-SDS) and western-blot, we evidenced the presence of LCI in cytosols and particulate extracts. By 2 dimensional electrophoresis (PAGE-SDS and IEF) and western-blot we identified a phosphorylated and unphosphorylated LCI protein. The phosphorylation of LCI by pKC decreased its isoelectric point from 6.9-6.6. The modifications of pKC activity and LCI phosphorylation and the changes in the bio-signalling pathways can partly account for the loss of differentiation observed in control or TPA treated cells.
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PMID:[Modifications of protein kinase C activity and phosphorylation of lipocortin I in cultures of pig thyroid cells]. 214 21

PKC (80 kDa) can be cleaved by limited proteolysis into distinct catalytic (50 kDa) and regulatory (32-35 kDa) fragments. After cleavage, the catalytic fragment is active in the absence of Ca2+, phospholipid, or DAG while the regulatory fragment is found associated with phospholipid and continues to bind phorbol esters in a Ca2(+)- and PS-dependent manner (28, 29). In the holoenzyme, the association of the regulatory domain with the membrane may be important to release the catalytic domain from inhibition by the regulatory domain. We have presented evidence indicating that effective membrane binding occurs through interaction with the hydrophobic and/or interfacial regions of the bilayer, and does not result from binding to individual phospholipids. In vivo and in vitro studies suggest that the binding event is carefully regulated. An important function of Ca2+ may be to modify the local structure of the membrane, and thus affect the ability of PKC to associate with it. For at least one of the isozymes, however, Ca2+ may also play an additional role at a site distant from the membrane, suggesting the possibility that the isozymes may be differentially regulated.
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PMID:Activation of protein kinase C by short chain phospholipid micelles. 261 67

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.
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PMID:Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity. 297 Oct 38

Lipocortin I, a 35-kDa protein, has been detected in terminally differentiated monocytes and neutrophils. This calcium-phospholipid binding protein appears to be identical to a 35-kDa protein that can serve as a substrate for the EGF-receptor/tyrosine kinase. We have used the human myelocytic cell line HL-60 to explore whether differentiation of hematopoietic cells is associated with changes in the level of lipocortin I. We find that differentiation of HL-60 cells toward the macrophage lineage by the addition of phorbol esters or vitamin D3 or toward neutrophils with dibutyryl cyclic AMP or dimethyl sulfoxide is accompanied by an increase in the cellular content of lipocortin I. In comparison, treatment of HL-60 cells with bryostatin 1, a compound that activates protein kinase C but does not differentiate HL-60 cells, did not effect the level of 35 kDa protein. We have developed a radioimmunoassay to quantitate this protein by using a polyclonal antibody to a synthetic amino terminal peptide of the 35-kDa protein. This antibody recognizes purified pig lung 35-kDa protein as well as a single 35-kDa protein in HL-60 and A-431 cells as determined by Western blotting and immune precipitation. Differentiated HL-60 cells contain 2.6-fold the amount of 35-kDa protein found in undifferentiated HL-60 cells. Our findings that the addition of phorbol esters to HL-60 cells results in an increase in the mRNA for the 35-kDa protein and in an increase in the incorporation of 35S-methionine into the protein suggest that transcriptional activation or increased stability of the mRNA is responsible for the increased rate of synthesis and accumulation of lipocortin I during differentiation of these cells. In the absence of added divalent cations, we have determined that in differentiated HL-60 cells 79% of lipocortin I protein is located in the cytosol while 21% of the total cellular protein is bound to the particulate fraction. The 35-kDa protein can be removed from the particulate fraction by incubation with chelators or treatment with phospholipase A2 or phospholipase C. Addition of the calcium ionophore A23187 to intact differentiated HL-60 cells causes the 35-kDa protein to associate with the particulate fraction of the cell, suggesting that modulation of intracellular calcium levels may play a role in changing the intracellular location of this protein.
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PMID:Differentiation of HL-60 cells is associated with an increase in the 35-kDa protein lipocortin I. 297 67

The presence of endogenous substrates of the protein kinase C (PKc) in rat thyroid glands has been demonstrated in in vitro phosphorylated cytosolic proteins by polyacrylamide gel electrophoresis (PAGE). Rat thyroid PKc specifically catalyzes the phosphorylation of the 35 kDa and 18 kDa proteins. These proteins were not labelled in the presence of Ca2+ alone, but they were phosphorylated when phospholipids alone were added. In hyperplastic glands the total phosphorylation of endogenous proteins is stimulated, due to the increased labelling of the 35 kDa and 18 kDa proteins. No extra phosphorylated bands were revealed by PAGE analysis. After suppression of growth activity the labelling of the two PKc-specific substrates was strongly inhibited.
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PMID:Endogenous substrates of protein kinase C in experimentally induced and regressed rat thyroid goitres. 380 4

A novel stimulatory monoclonal antibody (Mab) termed Mab.F11 induces granular secretion and subsequent aggregation of human platelets. Mab.F11 recognizes a unique 32 and 35 kDa protein duplex on the platelet membrane surface, called the F11 receptor; binding of Mab.F11 to its receptor results in increased intracellular phosphorylation of P47, the known protein kinase C (PKC) substrate pleckstrin. In order to determine whether the mechanism of action of Mab.F11 involves direct activation of PKC, two types of functional assays for measuring PKC activity were performed. Measurement of PKC activity in digitonin-permeabilized platelets revealed that Mab.F11 produced a rapid, 2-3 fold increase in the control value in the phosphorylation of the PKC peptide substrate, PKC(19-31) Ser25. The increase in PKC activity induced by Mab.F11 was found to be associated with the platelet membrane; a 1.6-fold control value increase in membrane PKC activity occurred rapidly, within 10 s of the addition of Mab.F11. The translocation from the cytoplasm to the membrane induced by Mab.F11 in PKC isoenzymes alpha and zeta was reversible, whereas translocation of the PKC isoenzymes delta, beta, eta' and theta was irreversible, with PKC levels remaining elevated in the membrane for at least 15 min. Taken together, our results demonstrate that in the initial stages of platelet activation by this stimulatory antibody, the enhanced membrane PKC activity reflects the presence of all six isoenzymes. At later stages, PKC activity is reflective of four isoenzymes. These results demonstrate that separate groups of PKC isoenzymes must be involved in different aspects of platelet activation. The long lag period and prolonged activation time of platelets by Mab.F11 renders this agonist most suitable for identifying the isoenzymes and their specific endogenous protein substrates involved in platelet secretion and aggregation induced by platelet membrane protein antibodies.
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PMID:Stimulatory antibody-induced activation and selective translocation of protein kinase C isoenzymes in human platelets. 748 74

The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of 125I-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single approximately 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.
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PMID:Mechanisms of platelet activation by a stimulatory antibody: cross-linking of a novel platelet receptor for monoclonal antibody F11 with the Fc gamma RII receptor. 764 39


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