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)

Human monoblastoid leukemia U937 cells differentiate to monocyte/macrophage upon treatment with phorbol ester, 12-o-tetradecanoylphorbol-13-acetate (TPA). Previous studies, including our own, have demonstrated that drug-induced differentiation of leukemia cells is associated with genetic and enzymatic activations of protein tyrosine phosphatases (PTPases). In this study, to further investigate a relationship between PTPase activation and leukemic differentiation, we established TPA-resistant U937 variant UT16 cells. Unlike known TPA-resistant cells whose resistance is mainly due to lack or down modulation of protein kinase C (PKC), UT16 cells showed TPA-induced activation of PKC, Raf-1, and ERK/MAP kinases similar to the parental U937 cells. Interestingly, however, UT16 cells exhibited altered binding activity of AP-1 complexes, decreased ability to induce c-jun and c-fos gene expressions, and failure to differentiate to a monocytic lineage. Based on these observations, UT16 cells could be considered a novel type of TPA-resistant cell. Among UT16 cells, most of TPA-inducible PTPase genes, PTP-1C, PTP-MEG2, P19-PTP, HPTP epsilon, and PTP-U1, did not respond to TPA. Consistently, TPA increased PTPase enzymatic activity in U937 but not in UT16 cells. Taken together, activation of PTPases is well correlated with TPA-induced differentiation of U937 cells. These findings indicate that gene expression and enzymatic activity of some PTPase isozymes described here are regulated by a TPA-mediated signaling event and are likely to be used as biomarkers for the monocytic differentiation of myeloid leukemia cells.
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PMID:Phorbol ester-resistant monoblastoid leukemia cells with a functional mitogen-activated protein kinase cascade but without responsive protein tyrosine phosphatases. 747 24

The protein tyrosine phosphatase PTP-PEST is an 88 kDa cytosolic enzyme which is ubiquitously expressed in mammalian tissues. We have expressed PTP-PEST using recombinant baculovirus, and purified the protein essentially to homogeneity in order to investigate phosphorylation as a potential mechanism of regulation of the enzyme. PTP-PEST is phosphorylated in vitro by both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) at two major sites, which we have identified as Ser39 and Ser435. PTP-PEST is also phosphorylated on both Ser39 and Ser435 following treatment of intact HeLa cells with TPA, forskolin or isobutyl methyl xanthine (IBMX). Phosphorylation of Ser39 in vitro decreases the activity of PTP-PEST by reducing its affinity for substrate. In addition, PTP-PEST immunoprecipitated from TPA-treated cells displayed significantly lower PTP activity than enzyme obtained from untreated cells. Our results suggest that both PKC and PKA are capable of phosphorylating, and therefore inhibiting, PTP-PEST in vivo, offering a mechanism whereby signal transduction pathways acting through either PKA or PKC may directly influence cellular processes involving reversible tyrosine phosphorylation.
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PMID:PTP-PEST: a protein tyrosine phosphatase regulated by serine phosphorylation. 752 Aug 67

Ninety minutes after i.v. injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) into rats, phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide anion (O2-) secretion was enhanced in suspensions of in vivo LPS-treated alveolar macrophages (AM phi) when compared with saline (SAL)-treated AM phi. The purpose of this investigation was to dissect the in vitro mechanism of PMA-stimulated O2- generation in both LPS and SAL-treated rat AM phi, with a panel of inhibitors of protein kinase C (PKC), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), phospholipase A2 (PLA2), cyclooxygenase (CO) and 5-lipoxygenase (5-LO). The following agents blocked PMA-stimulated O2- generation in both LPS- and SAL-treated AM phi (expressed as percentage of control): 1) PKC inhibitors: staurosporine: 100 nM, 7.0% (LPS) and 5.6% (SAL); sphingosine: 10 microM, 21% (LPS) and 10.5% (SAL); 2) PTK inhibitor: genistein: 100 microM, 44% (LPS) and 31% (SAL); 3) PTP inhibitors: phenylarsine oxide, 10 microM, 12.1% (LPS) and 18% (SAL); diamide, 1000 microM, 10.1% (LPS) and 10.5% (SAL); and 4) PLA2 inhibitors: manoalide: 1 microM, 29.3% (LPS) and 5.2% (SAL); scalaradial: 1 microM, 7.7% (LPS) and 7.1% (SAL); and WAY 125,984: 10 microM, 17.1% (LPS) and 14.5% (SAL). In addition, it was observed that exogenously added arachidonic acid (AA)-stimulated O2- generation in a time- and dose-dependent manner in both LPS and SAL-treated AM phi. The following inhibitors enhanced or did not affect PMA-stimulated O2- generation in LPS- and SAL-treated AM phi (expressed as percentage of of control): 1) PSP inhibitors: okadaic acid: 0.5 microM, 117% (LPS) and 153% (SAL); calyculin A: 1 microM, 112% (LPS) and 101% (SAL); 2) CO and 5-LO inhibitors: indomethacin: 10 microM, 107% (LPS) and 90% (SAL); WY 50, 295: 1 microM, 99% (LPS) and 103% (SAL); and 3) the PTP inhibitor orthovanadate upon prolonged preincubation. In both in vivo LPS- or SAL-primed AM phi, PMA-stimulated O2- generation appears to be modulated by PKC, PLA2, AA, PTK, PTP and PSP. No modulatory role was evident for either CO or 5-LO metabolites. These findings might bear on the design of therapeutic approaches for the modulation of O2- release by AM phi in the early stages of sepsis and adult respiratory distress syndrome.
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PMID:Modulation of superoxide generation in in vivo lipopolysaccharide-primed rat alveolar macrophages by arachidonic acid and inhibitors of protein kinase C, phospholipase A2, protein serine-threonine phosphatase(s), protein tyrosine kinase(s) and phosphatase(s). 761 27

Phosphorylation of docking proteins is essential for signal transduction. In this report we provide evidence that activation of human thymocytes in culture induces the phosphorylation of the protein tyrosine phosphatase 1C (PTP 1C). Thymocytes were activated with Con A, PMA or Con A+PMA. The enzyme is phosphorylated on its serine and threonine residues. Phosphorylation occurs within 5 min and lasts for 24 h. PTP 1C is phosphorylated by PKC in vivo and in vitro; however, phosphopeptide mapping suggests that in addition to PKC other kinases phosphorylate the enzyme. On the maps of tryptic digests of cultured thymocytes more radiolabeled phosphopeptides are visualized than on the maps of digests of PTP 1C phosphorylated with partially purified PKC alpha or beta. Phosphorylation of PTP 1C decreases its activity, whereas dephosphorylation increases its activity, suggesting that phosphorylation of PTP 1C takes part in the regulation of signal transduction.
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PMID:Activation of human thymocytes induces the phosphorylation of protein tyrosine phosphatase 1C. 778 5

The protein-tyrosine phosphatase epsilon (PTP epsilon) is a transmembranal, receptor-type protein that possesses two phosphatase catalytic domains characteristic of transmembranal phosphatases. Here we demonstrate the existence of a nontransmembranal isoform of PTP epsilon, PTP epsilon-cytoplasmic. PTP epsilon-cytoplasmic and the transmembranal isoform of PTP epsilon have separate, nonoverlapping expression patterns. Further, the data clearly indicate that control of which of the two isoforms is to be expressed is initiated at the transcriptional level, suggesting that they have distinct physiological roles. PTP epsilon-cytoplasmic mRNA is the product of a delayed early response gene in NIH 3T3 fibroblasts, and its transcription is regulated through a pathway that requires protein kinase C. The human homologue of PTP epsilon-cytoplasmic has also been cloned and is strongly up-regulated in the early stages of phorbol 12-tetradecanoate 13-acetate-induced differentiation of HL-60 cells. Sequence analysis indicates and cellular fractionation experiments confirm that this isoform is a cytoplasmic molecule. PTP epsilon-cytoplasmic is therefore the initial example to our knowledge of a nontransmembranal protein-tyrosine phosphatase that contains two tandem of catalytic domains.
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PMID:Identification of a cytoplasmic, phorbol ester-inducible isoform of protein tyrosine phosphatase epsilon. 861 76

The purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O2-) generation in all-trans retinoic acid-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), secretory phospholipase A2, cyclooxygenase (CO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O2- generation significantly in the all-trans retinoic acid-treated HL-60 cells (expressed as percentage of control, P < .05): 1) PKC inhibitors: staurosporine (100 nM, 3 +/- 1%); Ro 31-8220 (1 microM, 3 +/- 2%); sphingosine (100 microM, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 microM, 35 +/- 1%); calyculin A (10 microM, 73 +/- 1%); 3) MAPK inhibitor: SB-203580 (100 microM, 62 +/- 1%); 4) PTP inhibitors: phenylarsine oxide (1 microM, 12 +/- 9%); diamide (1 mM, 21 +/- 11%); and 5) secretory phospholipase A2 inhibitors: manoalide (1 microM, 24 +/- 10%); scalaradial (1 microM, 11 +/- 4%). Exogenously added arachidonic acid-stimulated O2- generation in a time- and dose-dependent manner. The following inhibitors enhanced or did not significantly affect phorbol 12-myristate 13-acetate-stimulated O2- generation (expressed as percentage of control): 1) PTK inhibitors: genistein (100 microM, 69 +/- 12%); CGP 53716 (100 microM, 67 +/- 10%); herbimycin A (10 microM, 67.4 +/- 1%); 2) PSP 2b inhibitors: cyclosporin A (30 microM, 71 +/- 5%); FK506 (30 microM, 88 +/- 7%); 3) CO inhibitor: indomethacin (100 microM, 111 +/- 12%); 4) 5-lipoxygenase inhibitor: WY 50,295 (100 microM, 140 +/- 23%); 5) MEK inhibitor: PD98059 (100 microM, 94 +/- 6.7%); and 6) the PTP inhibitor: orthovanadate (100 microM, 131 +/- 25%). Our pharmacological study suggests that, in neutrophil-like HL-60 cells, the signaling pathways leading to PMA-stimulated O2- generation appear to involve PKC, MAPK, phospholipase A2, arachidonic acid, PSP 1 and 2a and PTP. Furthermore, PTK, MEK, CO, 5-lipoxygenase and PSP 2b do not appear to participate in the modulation of PKC-stimulated O2- generation.
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PMID:Pharmacological targeting of signaling pathways in protein kinase C-stimulated superoxide generation in neutrophil-like HL-60 cells: effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s) and phospholipase A2. 893 Jan 66

Fluorescence polarization (FP) has been used to develop high throughput screening (HTS) assays for nuclear receptor-ligand displacement and kinase inhibition. FP is a solution-based, homogeneous technique requiring no immobilization or separation of reaction components. The FP-based estrogen receptor (ER) assay is based on the competition of fluorescein-labeled estradiol and estrogen-like compounds for binding to ER. These studies determined the Kd for this interaction to be 3 nM for ERalpha and 2 nM for ERbeta; IC50 values for 17beta-estradiol, tamoxifen, 4-OH-tamoxifen, and diethylstibestrol were determined to be 5.6, 189, 26, and 3.5 nM, respectively. In a screen of 50 lead compounds from a transcriptional activation screen, 21 compounds had IC50 values below 10 microM, with one having an almost 100-fold higher affinity for ERbeta over ERalpha. These data show that an FP-based competitive binding assay can be used to screen diverse compounds with a broad range of binding affinities for ERs. The FP-based protein-tyrosine kinase (PTK) assay uses fluorescein-labeled phosphopeptides bound to anti-phosphotyrosine antibodies. Phosphopeptides generated by a kinase compete for this binding. In c-Src kinase reactions, polarization decreased with time as reaction products displaced the fluorescein-labeled phosphopeptide from the anti-phosphotyrosine antibodies. The experimentally determined IC50 of AG 1478 was 400 pM, while Genistein did not inhibit the epidermal growth factor receptor at similar concentrations. Like the FP-based PTK assay, the protein kinase C (PKC) assay utilizes competition. PKC isoforms had different turnover rates for the peptide substrate. The IC50 for staurosporine was less than 10 nM for all PKC isoforms. Tyrosine phosphatase assays use direct binding rather than competition. Increasing concentrations of T-cell protein-tyrosine phosphatase (TC PTP) increased the rate of dephosphorylation. This change in polarization was dependent on TC PTP and was inhibited by 50 microM Na3VO4. The IC50 of Na3VO4 was 4 nM for TC PTP. These data demonstrate that a FP-based assay can detect kinase and phosphatase activity. Homogeneous, fluorescent techniques such as FP are now methods of choice for screening many types of drug targets. New HTS instrumentation and assay methods like these make FP a technology easily incorporated into HTS.
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PMID:Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays. 1080 7

Prolactin (PRL) involvement in the regulation of luteal steroidogenesis in pigs during the early luteal phase and pregnancy is well documented. The intracellular mechanism of PRL action in steroidogenic cells, however, is not fully recognized yet. In the current study, we have tested the hypothesis that protein kinase C (PKC) and tyrosine kinases (PTK) as well as serine-threonine (PP) and tyrosine phosphatases (PTP) are involved in PRL signaling in luteal cells originated from the early corpora lutea (CL) of cyclic sows. Luteal cells (50 000 cells/ml M199) were incubated for 8 h (37 degrees C) with PRL (200 ng) and low density lipoproteins (LDL) to stimulate P(4) production. In addition, treatments included: PKC inhibitors--staurosporine and chelerythrine chloride; tyrosine kinase inhibitors--genistein and tyrphostin; serine-threonine phosphatase inhibitors--okadaic acid, cantharidin (inhibitors of PP1/2A) and cypermethrin (inhibitor of PP2B); and tyrosine phosphatase inhibitor--sodium orthovanadate. Moreover, after incubation (37 degrees C) with PRL (200 ng) for 2, 5, 10 or 20 min, luteal cells were homogenized and cytosolic as well as membrane fractions have been obtained. This was followed by partial purification of the subcellular fractions by DEAE-cellulose chromatography and determination of PKC activity by measuring the transfer of (32)P from [gamma-(32)P]ATP to histone III-S. In unstimulated porcine luteal cells the major proportion of PKC activity was present in the cytosol. Incubation of luteal cells with PRL resulted in a rapid, time dependent increase in the amount of PKC activity in the membrane fraction and a decrease in the amount of PKC activity in the cytosol fraction. PKC activity in the membrane fraction was maximal after 5 min of exposure the cells to PRL. Inhibitors of PKC and PTK suppressed PRL and LDL-induced P(4) production by porcine luteal cells. It is of interest that stimulated P(4) production was also reduced by inhibitors of PTP and PP1/2A (okadaic acid, cantharidin). In contrast, cypermethrin did not affect P(4) production stimulated by PRL and LDL. The results of the current study support the hypothesis that PKC and tyrosine kinases are intracellular mediators of PRL action in porcine luteal cells during the first days of the estrous cycle. The involvement of protein phosphatases in transmission of the PRL signal in early luteal cells in pigs is also suggested.
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PMID:Luteotrophic action of prolactin during the early luteal phase in pigs: the involvement of protein kinases and phosphatases. 1466 68

Previous experiments have shown that dietary n-6 and n-3 polyenoic fatty acids (PFA) have different effects on collagen production, a process that may be related to the formation of prostaglandins (PG). This study tested the hypothesis that fibroblast collagen production could be regulated by different n- 6:n-3 PFA ratios and that the effects were mediated by PGE(2) and altered signaling via the different PGE receptor subtypes. Compared to a bovine serum albumin control, eicosapentaenoic acid (EPA; 20:5 n-3) treated cells significantly (P < 0.05) increased both collagen production and collagen as a percentage of total cellular protein (C-PTP), but arachidonic acid (AA; 20:4 n-6) reduced collagen production and C-PTP. As the amount of AA decreased and that of EPA increased, collagen production and C-PTP increased, especially when ratio of n-6:n-3 PFA was less than 1:1. C-PTP was significantly correlated with the amount of PGE(2) in the medium. AA- or EPA-treated cells produced similar C-PTP when incubated with 10(-6) M indomethacin, a cyclooxygenase inhibitor. Addition of exogenous PGE(2) to cell cultures treated with 10(-6) M indomethacin for 48 hrs decreased C-PTP in both AA and EPA groups. Decreased C-PTP was observed in AA-treated cells exposed to EP1, EP2, and EP4 PGE receptor agonists and in EPA-treated cells exposed to EP2 and EP4 agonists. AA-treated cell responded to activators of cyclic adenosine monophosphate and protein kinase C by decreasing C-PTP, but EPA-treated cells were unresponsive. In conclusion, collagen production in 3T3-Swiss fibroblasts induced by different n-6:n-3 PFA ratios was correlated with PGE(2) production and altered responsiveness and signaling via the different PGE receptor subtypes.
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PMID:Polyenoic fatty acid ratios alter fibroblast collagen production via PGE2 and PGE receptor subtype response. 1522 62

Proteolytic processing and ectodomain shedding have been described for a broad spectrum of transmembrane proteins under both normal and pathophysiological conditions and has been suggested as one mechanism to regulate a protein's function. It has also been documented for the receptor-like protein tyrosine phosphatase PTP-LAR, induced by treating cells with the tumor promoter TPA or the calcium ionophor A23187. Here we identified the epidermal growth factor receptor (EGFR) as both an association partner of PTP-LAR, that mediates phosphorylation of the latter, as well as an inducer of LAR-cleavage. Both overexpression of this kinase and stimulation of endogenous EGFR in various tumor cell lines were shown to induce proteolytic processing of the catalytic LAR-P-subunit. In contrast to TPA-induced shedding of PTP-LAR, EGFR-mediated cleavage did not require PKC-activity. For both stimuli, however, processing of the P-subunit turned out to be dependent on the activation of the MAP kinases ERK1 and ERK2, and was completely abrogated upon pre-treating cells with Batimastat, indicating the involvement of a metalloproteinase in this pathway. Being strongly impaired in fibroblasts derived from ADAM-17/TACE-knockout-mice or tumor cells that express a dominant negative mutant of ADAM-17/TACE, cleavage of PTP-LAR is suggested to be mediated by this metalloproteinase. Paralleled by rapid reduction of cell surface-localized LAR-E-subunit, EGFR-induced cleavage could be shown to lead to degradation of the catalytic LAR-P-subunit, thereby resulting in a significantly reduced overall cellular phosphatase activity of PTP-LAR. These results for the first time identify a protein tyrosine phosphatase as a potential substrate of TACE and describe proteolytic processing of PTP-LAR as a means of regulating phosphatase activity downstream and thus under the control of EGFR-mediated signaling pathways.
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PMID:EGFR signaling leads to downregulation of PTP-LAR via TACE-mediated proteolytic processing. 1647 62

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