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)

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

TNF is a potent activator of neutrophil granulocytes which acts via two cell surface receptors: the p55-TNF receptor (TNF-R55) and the p75-TNF receptor (TNF-R75). The extracellular region of the receptors can be released by proteolytic cleavage and form soluble TNF-binding proteins, TNF-R55-BP and TNF-R75-BP, respectively. The phorbol ester PMA, the chemotactic peptide FMLP, and TNF were all found to induce release of TNF-R55-BP and TNF-R75-BP from neutrophils in suspension in a time- and dose-dependent manner as measured by ELISA. Exposure of neutrophils to 10 ng/ml of PMA for 60 min resulted in release of 900 pg of TNF-R55-BP and 350 pg of TNF-R75-BP per 5 million cells, corresponding to approximately 4800 receptors per cell. In addition, adherence by itself of neutrophils to fibrinogen-coated culture plates and other surfaces resulted in a release of TNF-R55-BP of the same magnitude as seen in response of neutrophils in suspension to 1 nM TNF, whereas the release of TNF-R75-BP was less pronounced. The protein kinase C inhibitors staurosporin and calphostin C inhibited both the TNF-, PMA-, and adherence-induced release of soluble forms of TNFRs. Ab to the common beta-chain of the leukocyte integrins (CD18) did not affect adherence-induced TNF-R55-BP release, indicating that non-integrin-dependent mechanisms are involved in receptor cleavage. However, cross-linking of anti-CD18 Ab (IB4) with a Fab2 fragment resulted in a decrease of specific binding of 125I-TNF to neutrophils indicating that the leukocyte integrins can modulate TNFR expression on neutrophils. Thus, adherence to a biological surface, without additional stimuli, induces release of soluble TNFR form from neutrophils. TNFR expression can be modulated by protein kinase C as well as both leukocyte integrins and non-integrin-dependent adherence mechanisms.
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PMID:Adherence of neutrophils induces release of soluble tumor necrosis factor receptor forms. 790 2

Rat lymphoblasts are arrested in the G1 phase of the cell cycle and can be promoted to proceed up to the S phase, when they are stimulated by phorbol ester. In this work, we have studied some details of the phorbol 12,13-dibutyrate (PBu2)-stimulated proliferation. We show that in response to PBu2 at least four different protein kinase C (PKC) isoforms translocate to the membrane. A specific PKC zeta antibody recognizes two bands of 75 and 82 kDa. These two activities are separated using a Mono Q chromatography and we show that p75 is the classical PKC zeta isoform, while p82 might be a related isoform which is PBu2 sensitive. Our data show that there is a correlation between the ability of PBu2 to promote mitogenesis and to activate ERK2 kinase, suggesting that ERK2 kinase might be the limiting step of the process. We also show that ERK kinase activation precedes Raf-1 kinase hyperphosphorylation, suggesting that Raf-1 kinase activation is not required for ERK kinase activation. This idea was checked using a Raf-1 kinase antisense (AS) oligonucleotide. The results obtained with the Raf-1 AS oligonucleotide indicate that this serine/threonine kinase is dispensable for ERK kinase activation, but needed for the PBu2 mitogenic signaling even as late as 7 h after the delivery of the signal.
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PMID:Raf-1 and ERK2 kinases are required for phorbol 12,13-dibutyrate-stimulated proliferation of rat lymphoblasts. ERK2 activation precedes Raf-1 hyperphosphorylation. 795 67

The expression and cytokine-mediated regulation of the two different receptors for tumor necrosis factor, TNF-R-75 and TNF-R-55, was investigated in human malignant epithelial cell lines. Here we show that cells treated with TNF-alpha up-regulate the TNF-R-75 mRNA and protein levels. No changes were seen regarding the level of TNF-R-55 transcripts. Phospholipase and protein kinase C inhibitors abrogated the signal transduction pathway of TNF-mediated TNF-R-75 mRNA up-regulation which proceeded in the absence of transcriptional activation. This process was also elicited by an agonistic antibody binding specifically to TNF-R-55. Ligand binding assays using specific inhibitory antibodies showed a marked shift in active binding sites from p55 to p75 without significant changes in the total binding for TNF-alpha after up-regulation of p75 TNF-R. This ligand-induced regulation of one of the corresponding receptors has so far only been detected in malignant epithelial cells and not in hematopoietic cell lines. In our search for a specific function we were able to show that p75 is the specific receptor for TNF-mediated up-regulation of transforming growth factor alpha mRNA, whereas p55 is the signal transducer for TNF-induced up-regulation of epidermal growth factor receptor mRNA. This is the first demonstration of an exclusive function of TNF-R-75 in cells of epithelial origin.
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PMID:Tumor necrosis factor (TNF) up-regulates the expression of p75 but not p55 TNF receptors, and both receptors mediate, independently of each other, up-regulation of transforming growth factor alpha and epidermal growth factor receptor mRNA. 838 14

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-1) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (alpha)-chain of the IL-2R in PER-117 cells. The synergistic increases in IL-2R alpha expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-117 cells IL-1 appears to induce expression of the alpha-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2R beta (p75) differed significantly from that seen for IL-2R alpha. Moreover, the requirements for IL-2R alpha induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-117 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation.
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PMID:Requirements for the induction of the interleukin-2 receptor complex in a human pre-T-cell line. 847 26

Human peritoneal mesothelial cells (HPMC) respond to tumor necrosis factor alpha (TNF alpha) by releasing various cytokines that may activate the endothelium and induce recruitment of leukocytes during peristonitis. We characterized the receptors for TNF on HPMC to elucidate their functions in peritonitis. Scatchard analysis determined the presence of 70 x 10(3) TNF receptors/cell with a kDa of 0.44 nM. TNF receptor 1 (TNF-R1, p55) and TNF-R2 (p75) mRNA were demonstrated by reverse-transcriptase-PCR (RT-PCR). TNF-R1 protein was solely detected by flow cytometry (FCM). Interleukin-1 alpha (IL-1 alpha) induced down-regulation of TNF-R1. This was concomitant with accumulation of soluble TNF-R1 (sTNF-R1) detected by specific ELISA. LPS had a lower TNF-R1-shedding activity while TNF alpha did not induce shedding. The IL-1-induced-sTNF-R1-shedding was suppressed by the protein-kinase-A (PKA) inhibitor, H-8, or by H-7, the inhibitor of both PKC and PKA, but not by the specific PKC inhibitor GF. These experiments suggest a role for PKA in the IL-1-shedding signal. No change in TNF-R1 mRNA levels was observed after IL-1 alpha or TNF alpha stimulation while TNF-R2 (p75) mRNA basal levels transiently increased three to fivefold, reaching a peak after four hours followed by an accumulation of sTNF-R2 in the supernatant. Our data suggest that the main receptor expressed on HPMC is TNF-R1. Down-regulation and shedding of TNF-R1 induced by IL-1, and the transient expression of TNF-R2 induced by IL-1 and TNF, may regulate the responses to TNF by HPMC. These results may be important in understanding the inflammatory process of peritonitis were TNF plays a major role.
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PMID:TNF-receptors on human peritoneal mesothelial cells: regulation of receptor levels and shedding by IL-1 alpha and TNF alpha. 880 91

Tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of several retinal diseases. Soluble forms of the TNF receptors, p55 (55 kDa) and p75 (75 kDa), have recently been identified in biological fluids and may regulate TNF activity. The potential biological significance of these receptors for the human retina was examined by determining their presence in human vitreous and their release from eye cup explants in which the retina has been removed leaving an intact retinal pigment epithelium (HRPE). Normal human vitreous and conditioned medium from eye-cup HRPE explants demonstrated the presence of soluble p55 and p75. Soluble p55 was significantly more abundant than p75 in all vitreous samples (P < 0.03). Conditioned medium from eye-cup HRPE explants contained significantly more soluble p55 than p75 (P < 0.00002). Enzyme-linked immunosorbent assay showed the presence of soluble p55, and not p75, in conditioned medium from primary cultured HRPE cells. Activation of the protein kinase C pathway in these cells with the phorbol ester PMA significantly increased the release of soluble p55 (P < or = 0.001); whereas, pharmacological inhibition of protein kinase C with calphostin-C significantly decreased the shedding of p55 (P < or = 0.001). The results indicate that primary cultured HRPE cells shed p55 and regulate this shedding in part through the protein kinase C pathway. The presence of soluble TNF receptors within normal human vitreous and within conditioned medium from the eye-cup HRPE explant model suggests that these soluble receptors may have a biological function in the eye.
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PMID:Soluble tumor necrosis factor receptors are present in human vitreous and shed by retinal pigment epithelial cells. 894 4

We have examined the role of the p75 neurotrophin receptor in survival-promoting effects of nerve growth factor (NGF) and neurotrophin-3 (NT-3) on cultured Purkinje cells. Previously, we showed that NGF promotes Purkinje cell survival in conjunction with (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), an agonist of metabotropic excitatory amino acid receptors, whereas NT-3 by itself increases cell number. We now present evidence that p75 plays different roles in Purkinje cell responses to the two neurotrophins. A metabotropic receptor of the mGluR1 subtype may interact with p75 function, so as to regulate Purkinje cell responsiveness to neurotrophins. When cerebellar cultures were grown for 6 days in the presence of ACPD and a mutant form of NGF that does not bind to p75, no increase in Purkinje cell number was observed. Moreover, the survival-promoting effect of wild-type NGF and ACPD could be inhibited by a neutralizing antiserum to p75 or by a pyrazoloquinazolinone inhibitor of neurotrophin binding to p75. In contrast, the response to NT-3 was potentiated by anti-p75 treatment and by the quinazolinone. These data indicate the mediation of p75 in the trophic response to NGF-ACPD and a negative modulatory role of p75 in the action of NT-3. To probe the role of ACPD in the p75-dependent response to NGF, metabotropic receptor subtype-specific ligands were tested. The pattern of agonist specificity implicated the mGluR1 subtype, a receptor that is expressed at high levels by Purkinje cells and linked to activation of protein kinase C (PKC). Down-regulation or blockade of PKC abolished the response to NGF-ACPD. Consistent with the opposite roles of p75 in effects of the two neurotrophins, blockade of mGluR1 or PKC potentiated the survival response elicited by NT-3. In sum, our data suggest that afferent excitatory transmitters activate specific metabotropic receptors to elicit a p75-mediated action of NGF. NT-3 acts on Purkinje cells by a different mechanism that is not absolutely p75-dependent and that is reduced by neurotrophin access to p75 and metabotropic receptor activity.
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PMID:Differential involvement of metabotropic and p75 neurotrophin receptors in effects of nerve growth factor and neurotrophin-3 on cultured Purkinje cell survival. 948 24

Adult rat chromaffin cells may proliferate or extend neurites when stimulated by nerve growth factor (NGF) but their response is predominantly proliferative, making them a unique model for studying how mitogenic specificity is achieved. We examined contributions of the NGF receptors trk and p75 and of the major NGF signaling pathways to proliferation versus neurite outgrowth. The type of initial NGF response does not correlate with intensity of immunoreactivity for trk or p75. However, proliferation is initiated at lower NGF concentrations than neurite outgrowth, suggesting that it requires a less intense signal. Mitogenic cooperativity between receptors at low NGF concentrations is suggested by inhibitory effects of p75-blocking antibodies, but responses to trk-agonist antibody indicate that trk activation alone can induce proliferation. NGF-induced phosphorylation of ras-mediated mitogen-activated protein kinases (MAPK) Erk1 and Erk2 is as prolonged in normal chromaffin cells as in PC12 cells, where NGF is neuritogenic. Trk-agonist antibody, which is as mitogenic as NGF but less neuritogenic, causes equally prolonged but less intense ERK phosphorylation. The MAPK kinase(MEK-1) inhibitor PD98059 partially inhibits Erk phosphorylation and does not inhibit chromaffin cell proliferation, while depolarization selectively inhibits proliferation without blocking Erk phosphorylation. Proliferation is markedly reduced by the phosphoinositol-3 (PI-3) kinase inhibitor LY294002 while downregulation of protein kinase C (PKC) causes no change. These findings suggest that low-level, rather than short-duration, stimulation of NGF signaling pathways causes NGF to be mitogenic. Ras-mediated MAPK activation may be more critical in neurite outgrowth than in proliferation and PI-3 kinase may be the major mitogenic determinant.
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PMID:Nerve growth factor receptor signaling in proliferation of normal adult rat chromaffin cells. 993 50

K-252b, a member of the staurosporine family of protein kinase inhibitors, selectively potentiates the activation of the nerve growth factor receptor, TrkA, by a nonpreferred ligand, neurotrophin-3 (NT-3), in a variety of cell types. At higher (micromolar) concentrations of K-252b, an inhibitory effect occurs because of the inhibitory action of K-252b on the Trk kinase. By examining analogs of K-252b, we identified the compound L-753,000 (NB-506), which potentiates the action of NT-3 on TrkA but is devoid of the inhibitory action of K-252b. L-753,000 was effective at nanomolar concentrations in a Chinese hamster ovary cell line that expressed TrkA but was devoid of p75, the low-affinity neurotrophin receptor. L-753,000 also potentiated the activation of mitogen-activating protein kinase signaling (downstream from Trk activation) by NT-3 in this cell line. Although L-753,000, like K-252b, had a negligible effect in the absence of NT-3, the compound was found to potentiate NT-3-induced survival in both rat and chick primary cultures of dissociated dorsal root ganglia (DRG) and on neurite outgrowth of chick DRG explants. Unlike K-252b, which at micromolar concentrations inhibits the survival response of NT-3 in dissociated rat DRG, L-753,000 continued to potentiate the actions of NT-3 up to a concentration of 10 microM. Furthermore, the compound, unlike K-252b, did not inhibit an unrelated protein kinase, protein kinase C, at concentrations up to 10 microM. Because L-753, 000 selectively potentiates the NT-3-induced stimulation of TrkA without inhibiting Trks and other protein kinases, it represents a novel class of selective modifiers of neurotrophin actions.
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PMID:The staurosporine-like compound L-753,000 (NB-506) potentiates the neurotrophic effects of neurotrophin-3 by acting selectively at the TrkA receptor. 1038


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