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)

Phorbol esters bind with high affinity to protein kinase C (PKC) isozymes as well as to two novel receptors, n-chimaerin and Unc-13. The cysteine-rich regions present in these proteins were identified as the binding sites for the phorbol ester tumor promoters and the lipophilic second messenger sn-diacylglycerol. A 50-amino-acid peptide comprising the second cysteine-rich region of PKC delta, expressed in Escherichia coli as a glutathione S-transferase (GST)-fusion protein, bound [3H]phorbol 12,13-dibutyrate (PDBu) with high affinity (Kd = 0.8 nM). Using the cDNA of that cysteine-rich region as a template, a series of 37 point mutations was generated by site-directed mutagenesis, and the mutated proteins were analyzed quantitatively for binding of [3H]PDBu and, as appropriate, for binding of the ultrapotent analog [3H]bryostatin 1. Mutants displayed one of three patterns of behavior: phorbol ester binding was completely abolished, binding affinity was reduced, or binding was not significantly modified. As expected, five of the six cysteines as well as the two histidines involved in Zn2+ coordination are critical for the interaction of the protein with the phorbol esters. In addition, mutations in several positions, including phenylalanine 3, tyrosine 8, proline 11, leucines 20, 21 and 24, tryptophan 21, glutamine 27, and valine 38 drastically reduced the interaction with the ligands. The effect of these mutations can be rationalized from the three-dimensional (NMR) structure of the cysteine-rich region. In particular, the C-terminal portion of the protein does not appear to be essential, and the loop comprising amino acids 20 to 28 is implicated in the binding activity.
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PMID:Residues in the second cysteine-rich region of protein kinase C delta relevant to phorbol ester binding as revealed by site-directed mutagenesis. 766 8

Antigenic cross-linking of the high affinity IgE receptor (Fc epsilon R1) on mast cells results in protein tyrosine kinase activation. The object of the present study was to explore the regulation of the SH2 and SH3 domain containing adapter molecule Grb2 by Fc epsilon R1-stimulated PTK signal transduction pathways. Affinity purification of in vivo Grb2 complexes together with in vitro experiments with Grb2 glutathione S-transferase fusion proteins were used to analyze Grb2 complexes in the mast cell line RBL2H3. The data show that in RBL2H3 cells several different proteins are complexed to the SH3 domains of Grb2. These include the p21ras guanine nucleotide exchange factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for Fc epsilon R1-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins which suggests that Grb2 may be a pleiotropic adapter. Two Grb2 SH2-binding proteins were also characterized in RBL2H3 cells; the adapter Shc and a 33-kDa molecule. Shc is constitutively tyrosine phosphorylated in unstimulated cells and Fc epsilon R1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for Fc epsilon R1-activated PTKs and binds to Grb2 SH2 domains in Fc epsilon R1 activated but not quiescent cells. The beta subunit of the Fc epsilon R1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2-binding protein described in the present report is not the Fc epsilon R1 beta chain and its identity is unknown. The present report thus demonstrates that there are multiple Grb2 containing protein complexes in mast cells of which a subset are Fc epsilon R1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to Fc epsilon R1 ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain. We propose that protein complex formation by Grb2 is an important consequence of Fc epsilon R1 cross-linking and that this may be a signal transduction pathway which acts synergistically with calcium/PKC signals to bring about optimal mast cell end function.
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PMID:Regulation of the adapter molecule Grb2 by the Fc epsilon R1 in the mast cell line RBL2H3. 772 78

The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), known to induce murine glutathione S-transferase (GST) Ya, was examined for its effect on the expression of human GST alpha. Unexpectedly, 24-h treatment of the human hepatoma cell line HepG2 with 100 nmol/l TPA caused a decrease of the GST alpha mRNA level to below 5% of controls, i.e. opposite to the known response in the mouse. The level of mRNA for GST Mu was also decreased, but the mRNAs of c-jun and jun-B were elevated after 2 h. The decrease of GST alpha mRNAs was inhibited by staurosporine, suggesting an involvement of protein kinase C. Inhibition of transcription and translation by actinomycin D and cycloheximide also partially inhibited the effect of TPA on the expression of GST alpha. In the presence of actinomycin D, GST alpha mRNA halflife was 14.5 h, compared to 3.5 h in the presence of TPA. The calcium ionophore A23187 caused a loss of GST alpha mRNAs to levels almost as low as those obtained with TPA. The effects of TPA and the calcium ionophore were also observed in CaCo2 colon carcinoma cells. As a consequence of the decrease of mRNA levels, GST alpha protein levels and total GST enzyme activity were also diminished. Also, the morphology of the cells was changed after 3 h exposure to TPA. These data suggest that human GST alpha expression can be regulated at the level of mRNA stability by a pathway involving protein kinase C.
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PMID:Turnover of glutathione S-transferase alpha mRNAs is accelerated by 12-O-tetradecanoyl phorbol-13-acetate in human hepatoma and colon carcinoma cell lines. 774 32

In voltage-dependent Ca2+ channels, the beta subunit interacts with the alpha 1 subunit via a cytoplasmic site. A biochemical assay has been developed to quantitatively describe the interaction between both subunits. In vitro synthesized 35S-labeled beta subunits specifically bind to a glutathione S-transferase (GST) fusion protein containing the alpha 1A interaction domain (AIDA, located between the amino-acids 383 and 400 of the cytoplasmic loop between the hydrophobic domains I and II). Kinetic analysis demonstrates that the association of 35S-labeled beta 1b subunit to the AIDA GST fusion protein occurs with a fast rate constant at 4 degrees C. The binding is almost irreversible as demonstrated by the absence of dissociation observed after an 8-h incubation with an 18-amino acid synthetic AIDA peptide. The alpha 1-beta binding site does not seem to be a target for cytoplasmic regulation. The interaction is mostly unaffected by changes in ionic strength, pH, and Ca2+ concentration or by protein kinase C phosphorylation. The specificity of subunit interaction in voltage-dependent Ca2+ channels was also followed by saturation analyses. The data obtained show that the AIDA GST fusion protein binds to a single site on the beta 1b with an apparent Kd of 5 nM. The affinities of AIDA GST fusion protein for various beta subunits was measured and demonstrate that beta subunits associate with different affinities to each alpha 1 interaction domain. The rank order of AIDA affinity for each beta subunit is as follows: beta 4 > beta 2a > beta 1b >> beta 3. The binding of the beta subunit to alpha 1 subunit can be inhibited in vitro by the AIDA synthetic peptide with an apparent Ki of 285 nM. This interaction can also be prevented in heterologous Ca2+ channels by the injection of the AIDA GST fusion protein into Xenopus oocytes. Our results demonstrate that the site of interaction between AID and beta subunit is responsible for anchoring the beta subunit to the alpha 1 subunit and thus allowing the beta subunit to modify Ca2+ channel activity.
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PMID:Properties of the alpha 1-beta anchoring site in voltage-dependent Ca2+ channels. 774 54

We reported previously that recombinant myristoylated, alanine-rich protein kinase C substrate (MARCKS) expressed in Escherichia coli as well as MARCKS purified from rat brain specifically bound to phosphatidylserine (PS) in a calcium-independent manner and that the binding was regulated through phosphorylation of MARCKS (Nakaoka, T., Kojima, N., Hamamoto, T., Kurosawa, N., Lee, Y. C., Kawasaki, H., Suzuki, K., and Tsuji, S. (1993) J. Biochem. (Tokyo) 114, 449-452). In this study, to identify the minimum PS-binding region of MARCKS and the regulatory phosphorylation site, the binding of MARCKS to PS was examined in deletion mutants producing glutathione S-transferase (GST) fusion proteins. The mutant proteins GST-6-180 and GST-127-160 had almost the same ability to bind to immobilized PS as MARCKS purified from rat brain, whereas GST-127-152 did not bind to it. In addition, the binding of GST-6-156 to immobilized PS was 62% of that of GST-6-180, but that of GST-6-152 was only 8% and that of GST-6-135 was not detected. The effect of phosphorylation by protein kinase C was examined in several mutants of GST-6-180 whose serine residues were substituted with alanine. After phosphorylation, the mutants GST-6-180[S156A and S163A], GST-6-180]S156A], and GST-6-180[S163A] did not bind to immobilized PS like native MARCKS and GST-6-180. However, even after phosphorylation, GST-6-180-[S152A] and GST-6-180[S152A and S156A] could bind to immobilized PS. These results strongly suggest that MARCKS binds to PS molecules in the inner leaflet of the plasma membrane through residues 127-156, with residues 153-156 (FKKS) being particularly important in the binding of MARCKS to PS, and that the binding is regulated through the protein kinase C-catalyzed phosphorylation of the serine at residue 152.
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PMID:Characterization of the phosphatidylserine-binding region of rat MARCKS (myristoylated, alanine-rich protein kinase C substrate). Its regulation through phosphorylation of serine 152. 774 64

Pleckstrin homology (PH) domains are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. We have previously reported that the PH domains of several proteins, including beta ARK1, PLC gamma, IRS-1, Ras-GRF, and Ras-GAP, expressed as glutathione S-transferase fusion proteins, can reversibly bind purified bovine brain G beta gamma subunits in vitro with varying affinity. To determine whether PH domain peptides would behave as antagonists of G beta gamma subunit-mediated signal transduction in intact cells, plasmid minigene constructs encoding these PH domains were prepared, which permit transient cellular expression of the peptides. Pertussis toxin-sensitive, G beta gamma subunit-mediated inositol phosphate (IP) production was significantly inhibited in COS-7 cells transiently coexpressing the alpha 2-C10 adrenergic receptor (AR) and each of the PH domain peptides. Pertussis toxin-insensitive, Gq alpha subunit-mediated IP production via coexpressed M1 muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the PLC gamma PH domain peptide, suggesting that the inhibitory effect of most of the PH domain peptides was G beta gamma subunit-specific. Stimulation of the mitogen-activated protein (MAP) kinase pathway by Gi-coupled receptors in COS-7 cells has been reported to require activation of p21ras and to be independent of protein kinase C. Since several proteins involved in activation contain PH domains, the effect of PH domain peptide expression on alpha 2-C10 AR-mediated p21ras-GTP exchange and MAP kinase activation as well as direct G beta gamma subunit-mediated activation of MAP kinase was determined. In each assay, coexpression of the PH domain peptides resulted in significant inhibition. Increasing G beta gamma subunit expression surmounted PH domain peptide-mediated inhibition of MAP kinase activation. These data suggest that the PH domain peptides behave as specific antagonists of G beta gamma-mediated signaling in intact cells and that interactions between PH domains and G beta gamma subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.
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PMID:Effect of cellular expression of pleckstrin homology domains on Gi-coupled receptor signaling. 776 89

Extracellular signal-regulated kinase (Erk) (mitogen-activated protein (MAP) kinase) is rapidly activated when neutrophils are stimulated. Several isoforms of MAP/Erk kinase (MEK), a kinase capable of phosphorylating and activating Erk, have been identified, but their distribution and differential roles in leukocytes are unknown. We studied the effect of chemotactic stimulation on MEK-1, using isoform-specific antibodies. MEK-1 was found to be phosphorylated on serine and threonine residues in unstimulated human neutrophils. Stimulation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced serine/threonine phosphorylation of MEK-1, while reducing its electrophoretic mobility. MEK-1 activity, measured as autophosphorylation or as phosphorylation of a glutathione S-transferase-Erk fusion protein, was undetectable in unstimulated cells but became evident after treatment with chemoattractant. Phosphorylation and activation of MEK-1 were rapid and transient, peaking after 1-2 min and returning to base line by 10 min. Experiments using electropermeabilized cells indicated that elevation of cytosolic Ca2+ is not required for activation of MEK-1 by fMLP. Moreover, MEK-1 was not stimulated by either platelet-activating factor or thapsigargin, which increase Ca2+ to levels comparable with those attained in chemoattractant-activated cells. In contrast, activation of MEK-1 was induced by phorbol esters, and the stimulatory effect of fMLP was blocked by an antagonist of protein kinase C. Stimulation of MEK-1 was also blocked by concentrations of erbstatin that prevent the fMLP-induced accumulation of tyrosine-phosphorylated proteins. The data suggest that MEK-1 is largely responsible for the activation of Erk in chemoattractant-stimulated neutrophils and that protein kinase C and/or tyrosine kinases mediate this effect, whereas elevated cytosolic Ca2+ is not essential.
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PMID:Chemotactic peptides induce phosphorylation and activation of MEK-1 in human neutrophils. 803 95

The regulatory domain of protein kinase C gamma (PKC gamma) contains the following functional elements which can interact with lipids: the pseudosubstrate motif within the first variable region (V1), cysteine-rich domains, Cys1 and Cys2 which contain zinc and bind phorbol dibutyrate (PDBu)/diacylglycerol, and the calcium-dependent lipid binding domain (CaLB). The function of individual or combined segments of the regulatory domain was investigated, using glutathione S-transferase (GST) fusion proteins and mixed micellar or liposomal assays. GST-Cys1 and GST-Cys2 bound PDBu with comparable affinity (Kd = 14-17 nM). GST-Cys1Cys2 yielded a protein with a PDBu binding affinity of 3.4 nM, in the presence of calcium, similar to that of intact PKC gamma (Kd = 2.6 nM). The phosphatidylserine (PS) dependence of PDBu binding was highly cooperative for all fusion proteins tested with Hill numbers (n) lying in the range of 3.5-4.8, similar to values obtained for intact PKC gamma. While Hill numbers were similar under all conditions, the PS concentration necessary for half-maximal PDBu binding was dependent upon the nature and presence of divalent cations. The PS requirement was lowest in the presence of calcium for GST-Cys1, GST-Cys2, and GST-Cys1Cys2 (Km for PS = 11, 14, and 12 mol %, respectively) but still significantly above the value for intact PKC gamma (5.4 mol %). The data establish Cys1 and Cys2 as independent PDBu binding domains that are modulated by divalent cations. While PDBu binding affinity to a GST-V1Cys1 fusion protein (Kd = 36 nM) was comparable to that of GST-Cys1, the CaLB domain dramatically reduced PDBu binding affinity of GST-Cys2CaLB (Kd = 912 nM). This effect of the CaLB domain on PDBu binding to Cys2 suggests that PDBu/diacylglycerol binding to native PKC gamma may occur at Cys1 and that the Cys2 domain may serve another regulatory function.
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PMID:The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations. 805 Oct 84

The activity of several proteins involved in the development of antitumor drug resistance is regulated by protein phosphorylation. These proteins include the mdr-1-encoded P-glycoprotein (Pgp) and topoisomerase II (topo II). The corresponding evidence is reviewed and attempts to modulate multidrug resistance (MDR) by protein kinase C inhibitors are described. The expression of several proteins which are essential in drug resistance is regulated at the transcriptional level, involving protein phosphorylation by members of the protein kinase C (PKC) family, casein kinase II (CKII), and others. These proteins include mdr-1-encoded P-glycoprotein, metallothionein, glutathione S-transferase (GST), dTMP synthase, and the proteins Fos and Jun. The corresponding genes are under positive regulation of ras, which in turn requires the activation of a protein kinase cascade for its function. Protein kinases are therefore potentially useful targets in reducing the expression of proteins involved in the development of multifactorial drug resistance caused by the expression of transforming ras-genes. Attempts to inhibit the ras-induced fos expression by an inhibitor of protein kinase C (ilmofosine) are described. Protein kinase inhibitors are also able to synergistically enhance the cytotoxicity of cis-platinum, which is discussed as resulting from a reduction of PKC-dependent fos expression.
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PMID:Role of protein kinases in antitumor drug resistance. 806 Nov 7

Resistance of tumor cells to doxorubicin is a multifactorial phenomenon. In the present investigation, the ability of resistance modifiers against different resistance mechanisms was analysed. Substances which block P-glycoprotein (P-170) function circumvented resistance of doxorubicin-resistant sarcoma 180 (S180) cells completely (verapamil, thioridazine) or partially (hycanthone), whereas inhibitors of glutathione S-transferase (ethacrynic acid, N-ethylmaleimide, buthionine sulfoximine), and protein kinase C (staurosporine, acridine orange) caused only a partial reversion of resistance. In contrast, an inhibitor of alkaline phosphatase (levamisole) did not overcome doxorubicin-resistance. These results indicate that P-glycoprotein blockers might be more effective to modulate doxorubicin-resistance of S180 cells as compared to other modifiers. Further investigations using other MDR cell lines are required to clarify the generality of these findings.
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PMID:Reversal of doxorubicin-resistance in sarcoma 180 tumor cells by inhibition of different resistance mechanisms. 810 93


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