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)

Human leukotriene C4 (LTC4) synthase was purified > 25,000-fold to homogeneity from the monocytic leukemia cell line THP-1. Beginning with taurocholate-solubilized microsomal membranes, LTC4 synthase was chromatographically resolved by (i) anion exchange, (ii) affinity chromatography (through a resin of biotinylated LTC2 immobilized on streptavidin-agarose), and then (iii) gel filtration. The final preparation contained only an 18-kDa polypeptide. The molecular mass of the pure polypeptide was consistent with an 18-kDa polypeptide from THP-1 cell membranes that was specifically photolabeled by an LTC4 photoaffinity probe, 125I-labeled azido-LTC4. On calibrated gel-filtration columns, purified LTC4 synthase activity eluted at a volume corresponding to 39.2 +/- 3.3 kDa (n = 12). The sequence of the N-terminal 35 amino acids was determined and found to be a unique sequence composed predominantly of hydrophobic amino acids and containing a consensus sequence for protein kinase C phosphorylation. We therefore conclude that human LTC4 synthase is a glutathione S-transferase composed of an 18-kDa polypeptide that is enzymatically active as a homodimer and may be phosphoregulated in vivo.
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PMID:Purification to homogeneity and the N-terminal sequence of human leukotriene C4 synthase: a homodimeric glutathione S-transferase composed of 18-kDa subunits. 844 23

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14-3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14-3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.
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PMID:Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity. 858 68

Coronary heart disease is a major complication of diabetic subjects, and platelet-derived growth factor (PDGF) has been implicated in the development of atherosclerosis. We investigated the effects of high glucose on expression of PDGF-beta receptor. In a binding assay with 125I-labeled PDGF-BB homodimer, high concentrations of glucose increased high-affinity binding of PDGF-BB on human monocyte-derived macrophages and rabbit aortic medial smooth muscle cells. Northern blot analysis confirmed the enhanced effect of glucose on expression of PDGF-beta receptor mRNA in human monocyte-derived macrophages. The protein kinase C inhibitor, staurosporin, completely suppressed an increase in PDGF-BB binding by high glucose, and high glucose significantly activated protein kinase C. These results indicated that PDGF-beta receptor expression was enhanced by high glucose through the activation of protein kinase C. Furthermore, we observed similar effects of high glucose on both PDGF-beta receptor expression and protein kinase C activation in rat mesangial cells and human capillary endothelial cells. Our results suggest that stimulation of the PDGF system is significantly involved in the development not only of diabetic atherosclerosis but also of microangiopathy.
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PMID:Enhanced expression of platelet-derived growth factor-beta receptor by high glucose. Involvement of platelet-derived growth factor in diabetic angiopathy. 860 74

The sphingomyelin pathway is an important signal transduction system regulating various cellular functions. However, little is known about the effect of sphingomyelin metabolites on vasomotor function. We examined the vascular effects of sphingomyelin, sphingosine, and sphingomyelinase (SPMase) in vitro. In pig coronary rings precontracted with prostaglandin F2 alpha, sphingosine and SPMase evoked initial contraction and subsequent gradual relaxation; however, sphingomyelin did not influence the tone. The initial contractions in response to either SPMase (40 microU/ml to 0.4 U/ml) or sphingosine (0.5-10 microM) treatment were abolished in rings denuded of endothelium. This initial contraction in response to sphingosine treatment was significantly attenuated by a cyclo-oxygenase inhibitor indomethacin, but not altered by either a nitric oxide synthase inhibitor, N omega-monomethyl-L-arginine (L-NMMA), a protein kinase C (PKC) inhibitor staurosporine, or superoxide dismutase (SOD, 100 U/ml). Incubation of coronary rings with sphingosine (10 microM) or SPMase (0.4 U/ml) for 120 min significantly attenuated subsequent endothelium-dependent relaxation in response to thrombin and A23187, but did not affect endothelium-independent relaxation in response to sodium nitroprusside. In contrast, sphingomyelin (10 microM) did not alter the endothelium-dependent relaxation. In conclusion, in the sphingomyelin pathway, sphingosine induces vasoconstriction in coronary arteries that seems to be mediated by the release of cyclooxygenase-sensitive vasoconstrictor prostanoids from the endothelium. Sphingosine also induced endothelial dysfunction characterized by impaired endothelium-dependent relaxation. Thus, the sphingomyelin pathway may be an important regulator of vascular function.
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PMID:Effects of sphingomyelinase and sphingosine on arterial vasomotor regulation. 882 30

Granulosa cell-derived inhibin A (a dimer of alpha- and beta A-subunits), activin A (a homodimer of beta A-subunits) and the activin-binding protein follistatin are important regulators of human ovarian steroidogenesis. We here studied how 8-bromo-cAMP (8br-cAMP), a protein kinase A activator, and 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, affect the steady-state levels of alpha- and beta A-subunit and follistatin mRNAs in cultured human granulosa-luteal cells. 8br-cAMP induced alpha- and beta A-subunit and follistatin steady-state mRNA levels in a time- and concentration-dependent manner. The levels of alpha-subunit mRNAs were stimulated by 8br-cAMP in a sustained manner with a maximal induction seen at the time points 24 and 48 h. By contrast, beta A-subunit and follistatin mRNA levels were rapidly and transiently induced by 8br-cAMP with maximal effects observed at 3 h and 8 h, respectively. TPA did not affect basal alpha-subunit mRNA levels but it rapidly induced beta A-subunit mRNAs at 3 h and the stimulation was still evident at 48 h. TPA induced follistatin mRNA levels with kinetics similar to 8br-cAMP but to a lesser extent. Moreover, 8br-cAMP and TPA stimulated beta A-subunit and follistatin mRNA levels synergistically at 3 h. By contrast, TPA had a potent inhibitory effect on 8br-cAMP- and hCG-induced alpha-subunit levels. Neither 8br-cAMP nor TPA regulated inhibin/activin beta B-subunit mRNA levels. Taken together the activation of protein kinase-A and -C by 8br-cAMP and TPA, respectively, lead to clearly differential responses in the steady-state levels of inhibin activin alpha- and beta A-subunit and follistatin mRNAs. These results suggest that the inhibin A vs. activin A ratio as well as follistatin levels are regulated by multiple second-messenger pathways in the human ovary.
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PMID:Differential regulation of inhibin/activin alpha- and beta A-subunit and follistin mRNAs by cyclic AMP and phorbol ester in cultured human granulosa-luteal cells. 886 60

Experiments were designed to clarify the role of c-Jun/c-Fos and of putative phorbol 12-myristate-13-acetate-(PMA)-responsive elements (TREs) in the induction of plasminogen-activator inhibitor 1 (PAI-1) gene transcription in the human hepatoma cell line HepG2 by activators of protein kinase C (PKC). Treatment of HepG2 cells with the phorbol ester PMA or serum rapidly and transiently increased c-Jun and c-Fos mRNA and protein levels prior to PAI-1 induction. This induction of PAI-1 gene transcription was found to be dependent on ongoing protein synthesis. An essential role of c-Jun and c-Fos in basal and PMA-stimulated transcription of the PAI-1 gene is demonstrated by our finding that antisense c-jun and c-fos oligodeoxynucleotides both strongly reduced basal and PMA-stimulated PAI-1 synthesis. Since it has already been shown that two TREs between positions -58 and -50 and between -79 and -72 of the PAI-1 promoter are essential for basal and PMA-induced PAI-1 promoter activity ([16]), we examined binding of nuclear proteins to these elements. The protein-binding activity to the TRE between positions -79 and -72 shows very strong PMA induction of an unknown factor, which is not related to c-Jun or c-Fos. The TRE binding between positions -58 and -50 forms two complexes, both containing c-Jun protein. The faster migrating complex primarily contains c-Jun homodimers. The amount of the faster migrating complex is enhanced more than 30-fold in PMA-treated cells, due to a strongly increased binding of c-Jun homodimers and, to a minor extent, to binding of c-Jun/c-Fos heterodimers. Dissociation experiments suggest that the c-Jun/c-Fos heterodimers bind with much lower affinity compared to binding of c-Jun homodimers. Together with the finding that both antisense c-jun and antisense c-fos oligodeoxynucleotides reduced the amount of c-Jun homodimer, we conclude that binding of c-Jun homodimer to the TRE at positions -58 to -50 is important in the basal activity and PMA activation of the PAI-1 promoter in HepG2 cells.
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PMID:Role of c-Jun and proximal phorbol 12-myristate-13-acetate-(PMA)-responsive elements in the regulation of basal and PMA-stimulated plasminogen-activator inhibitor-1 gene expression in HepG2. 891 35

The microtubule-associated protein tau of normal brains is attached to tubulin through its 18-amino-acid repeat units. In the paired helical filaments (PHF) of Alzheimer's disease, however, tau is oligomerized in an abnormally hyperphosphorylated from (PHF-tau). tau contains two cysteine residues in repeat units 2 and 3, but only the R3-R3 homodimer is present in PHF-tau. A serine residue two amino acids downstream of the R3 cysteine is a major phosphate acceptor site for protein kinase C. In the work repeated here, we used synthetic peptides corresponding to R2, R3 and phosphorylated R3 to determine the binding of the tau repeat peptides to a peptide fragment corresponding to the C-terminal domain of beta-tubulin and to study the kinetics of homo- and heterodimer formation. Additionally, we studied two major biochemical properties of the peptides that distinguish between normal tau and PHF-tau: conformation and metabolic stability. All R2 and R3 peptides bound specifically to the tubulin peptide regardless of the state of phosphorylation or dimerization. The reverse-turn conformation of the tau repeat peptides in the presence of the tubulin peptide remained unaffected. Phosphorylation slightly loosened the turn structure of the monomeric and dimeric peptides, and did not univocally affect the serum stability of the peptides or the ability of the peptides to form dimers. The isolated R2 and R3 units formed homodimers approximately in the same rate. When the two peptides were mixed, however, the R2-R3 heterodimer was formed preferentially over the homodimers. The dimers were generally more stable in human serum than the monomers. Our results with the synthetic peptide fragments of tau indicate that neither oxidation nor phosphorylation of the repeat units is able to generate extended structure such as that found in PHF-tau. Additionally, phosphorylation of Ser324 does not appear to modulate the kinetics of oligomerization of tau, and in general biochemistry terms, does not affect disulfide bridge formation nearby. In agreement with studies at the full-protein level, the formation of homodimers of the peptides, a model of the self-association of tau, is not preferred. If the dimers are formed, however, their clearance is considerably slower than that of the monomers, explaining the remarkable protease resistance of PHF-tau in the affected brains.
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PMID:Oxidized and phosphorylated synthetic peptides corresponding to the second and third tubulin-binding repeats of the tau protein reveal structural features of paired helical filament assembly. 927 97

Recent evidence demonstrates that the proto-oncogene product, Bcl-2 can protect cells from a variety of cell death-inducing stimuli. Because previous studies have demonstrated that protein kinase (PK) pathways may be involved in the regulation of cell death, we tested various PK inhibitors for their effects on cell death in a dopaminergic neuronal cell line, MN9D, as well as the potential of Bcl-2 family members and structural mutants to block this process. Cells expressing either human Bcl-2 (MN9D/Bcl-2), or neomycin (MN9D/Neo; control cells) were treated with either staurosporine (0.25-2 microM) or trifluoperazine (10-100 microM). In control MN9D/Neo cells, both reagents led to a dose-dependent cell death with morphological features of apoptosis. Overexpression of Bcl-2 rescued cells from staurosporine-induced but not trifluoperazine-induced apoptotic cell death. Cell death induced by the specific PKC inhibitor, calphostin C was also significantly attenuated in MN9D/Bcl-2 cells indicating that a PKC pathway represents one mechanism by which Bcl-2 prevents staurosporine-induced cell death. Similarly, the Bcl-2 family member, Bcl-X(L) also blocked staurosporine-induced cell death in MN9D cells whereas overexpression of Bcl-X(S) or Bax did not. Finally, staurosporine-induced cell death was still blocked by the expression of clones encoding mutations in the Bcl-2 homology domains, BH1 and BH2, as well as C-terminally truncated Bcl-2. These data suggest that in the staurosporine-mediated cell death model Bcl-2 is not heterodimerizing to related proteins through these highly conserved structural domains nor does it need to be membrane-anchored. Thus, in this paradigm, either Bcl-2 functions as a homodimer or essential sequences lie outside of the BH1 or BH2 domains.
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PMID:Regions outside of the Bcl-2 homology domains, BH1 and BH2 protect a dopaminergic neuronal cell line from staurosporine-induced cell death. 942 15

PRL is an anterior pituitary hormone that, along with GH and PLs, forms a family of hormones that probably resulted from the duplication of an ancestral gene. The PRLR is also a member of a larger family, known as the cytokine class-1 receptor superfamily, which currently has more than 20 different members. PRLRs or binding sites are widely distributed throughout the body. In fact, it is difficult to find a tissue that does not express any PRLR mRNA or protein. In agreement with this wide distribution of receptors is the fact that now more than 300 separate actions of PRL have been reported in various vertebrates, including effects on water and salt balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, and immune regulation and protection. Clearly, a large proportion of these actions are directly or indirectly associated with the process of reproduction, including many behavioral effects. PRL is also becoming well known as an important regulator of immune function. A number of disease states, including the growth of different forms of cancer as well as various autoimmune diseases, appear to be related to an overproduction of PRL, which may act in an endocrine, autocrine, or paracrine manner, or via an increased sensitivity to the hormone. The first step in the mechanism of action of PRL is the binding to a cell surface receptor. The ligand binds in a two-step process in which site 1 on PRL binds to one receptor molecule, after which a second receptor molecule binds to site 2 on the hormone, forming a homodimer consisting of one molecule of PRL and two molecules of receptor. The PRLR contains no intrinsic tyrosine kinase cytoplasmic domain but associates with a cytoplasmic tyrosine kinase, JAK2. Dimerization of the receptor induces tyrosine phosphorylation and activation of the JAK kinase followed by phosphorylation of the receptor. Other receptor-associated kinases of the Src family have also been shown to be activated by PRL. One major pathway of signaling involves phosphorylation of cytoplasmic State proteins, which themselves dimerize and translocate to nucleus and bind to specific promoter elements on PRL-responsive genes. In addition, the Ras/Raf/MAP kinase pathway is also activated by PRL and may be involved in the proliferative effects of the hormone. Finally, a number of other potential mediators have been identified, including IRS-1, PI-3 kinase, SHP-2, PLC gamma, PKC, and intracellular Ca2+. The technique of gene targeting in mice has been used to develop the first experimental model in which the effect of the complete absence of any lactogen or PRL-mediated effects can be studied. Heterozygous (+/-) females show almost complete failure to lactate after the first, but not subsequent, pregnancies. Homozygous (-/-) females are infertile due to multiple reproductive abnormalities, including ovulation of premeiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Twenty per cent of the homozygous males showed delayed fertility. Other phenotypes, including effects on the immune system and bone, are currently being examined. It is clear that there are multiple actions associated with PRL. It will be important to correlate known effects with local production of PRL to differentiate classic endocrine from autocrine/paracrine effects. The fact that extrapituitary PRL can, under some circumstances, compensate for pituitary PRL raises the interesting possibility that there may be effects of PRL other than those originally observed in hypophysectomized rats. The PRLR knockout mouse model should be an interesting system by which to look for effects activated only by PRL or other lactogenic hormones. On the other hand, many of the effects reported in this review may be shared with other hormones, cytokines, or growth factors and thus will be more difficult to study. (ABSTRACT TRUNCATED)
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PMID:Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. 962 54

PKN is a fatty acid-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. PKN has been recently reported to interact with a small GTP-binding protein Rho and cytoskeletal proteins such as neurofilament and alpha-actinin. To identify the new components of the PKN-signaling pathway, the yeast two-hybrid system was employed. Using the amino-terminal regulatory domain of PKN as a bait, cDNA encoding a neural antigen PCD17, which is recognized by characteristic antibodies of patients with paraneoplastic cerebellar degeneration, was isolated from a human brain cDNA library. The interaction between PKN and PCD17 was also determined by the in vitro binding analysis. PCD17 was coimmunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and the amino-terminal region of PCD17. PCD17 was phosphorylated by PKN, and the extent of this phosphorylation was enhanced by addition of 40 microM arachidonic acid. The amino-terminal region of PCD17 could form a homodimer in vitro, and PCD17 fused to the Gal4 DNA binding domain showed the transcriptional transactivation of the chloramphenicol acetyltransferase reporter gene linked to 5 Gal4 binding sites and minimal promoter in rat C6 glioma cells. These results suggest the participation of PCD17 in gene expression and lead to a clue for elucidating the PKN signaling pathway from the cytosol to the nucleus.
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PMID:PKN interacts with a paraneoplastic cerebellar degeneration-associated antigen, which is a potential transcription factor. 963 78


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