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)

1. beta-Alanyl-L-histidinato zinc (AHZ), in which zinc is chelated to beta-alanyl-L-histidine, is a new zinc compound. beta-Alanyl-L-histidine can uniquely chelated zinc ion in various essential trace metals. More recently, it has been demonstrated that this compound has more intensive effect than zinc sulfate on bone metabolism, suggesting a role as pharmacological tool in osteoporosis. This review describes mainly the action of AHZ on bone resorption as summarized in the following. 2. The prolonged oral administration of AHZ (10-100 mg/kg/day) can completely prevent bone loss in the femur of ovariectomized rats, indicating the preventive effect of AHZ on bone resorption in vivo. 3. The decrease in bone calcium content induced by various bone resorbing factors was completely inhibited by the presence of AHZ (10(-6)-10(-4) M) in bone tissue culture system in vitro. 4. Many bone resorbing agents can stimulate the formation (differentiation) of osteoclasts from marrow cells. AHZ (10(-6)-10(-4) M) clearly inhibited osteoclast-like cell formation in mouse marrow culture in vitro. 5. AHZ may act on the process of parathyroid hormone-induced protein kinase C activation which is involved in Ca(2+)-signaling in osteoclastic cells.
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PMID:beta-Alanyl-L-histidinato zinc and bone resorption. 759 Jan 5

This study reports a direct effect of TRH on amylase secretion from isolated rat exocrine pancreatic acinar cells. TRH inhibited carbachol (10(-5) M)-stimulated amylase secretion by a maximum of 24% at a concentration of 10(-11) M (p < 0.05), but did not affect basal amylase release in concentrations from 10(-13) M to 10(-8) M. Ceruletide (3 x 10(-10) M)-stimulated amylase secretion was maximally reduced by 23% at a TRH concentration of 10(-10) M (p < 0.05). Direct stimulation of protein kinase C-mediated secretion by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was not altered by TRH. The TRH metabolite cyclo (His-Pro) did not influence basal or stimulated pancreatic secretion in vitro. These findings point to a TRH-mediated modulation of exocrine pancreatic secretion at the receptor site.
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PMID:Inhibitory effect of thyrotropin-releasing hormone on enzyme secretion from isolated rat pancreatic acinar cells. 759 Jun 25

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.
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PMID:Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro. 762 7

In order to identify cDNAs that can induce oncogenic transformation, a retroviral vector was used to transfer a library of cDNAs from the murine 32D hemopoietic cell line into NIH 3T3 fibroblasts. We have identified and recovered a provirus containing a 1.8-kilobase pair cDNA whose expression causes morphological transformation in NIH 3T3 cells. The transforming cDNA contains a complete open reading frame that encodes a protein (designated Lfc) with a region of sequence similarity to the product of the lbc oncogene. This region includes a domain that is characteristic of the CDC24 family of guanine nucleotide exchange factors in tandem with a pleckstrin homology (PH) domain. The Lfc protein is distinguished from Lbc by a 150-amino acid NH2-terminal extension that contains a cysteine- and histidine-rich domain similar to the diacylglycerol-binding site (zinc butterfly) found in protein kinase C. NH2- and COOH-terminal deletion analysis revealed that both the PH and putative guanine nucleotide exchange factor domains are required, but the zinc butterfly is dispensable, for transformation. Although the removal of the PH domain of the Lfc protein completely eliminated its ability to transform NIH 3T3 cells, replacement of this domain with an isoprenylation site restored all of its transforming activity. This suggests that a PH domain-dependent recruitment of the Lfc protein to the cellular membrane is a necessary step for cellular transformation. The lfc gene is expressed in a broad range of tissues as well as in a variety of hemopoietic and non-hemopoietic cell lines. Lfc appears to be a new member of a growing family of proteins that are likely to act as activators of Ras-like proteins in a developmental or cell-lineage specific manner.
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PMID:Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C. 762 63

In this study, the recently identified human protein kinase C-theta (PKC-theta) isoform has been biochemically characterized in detail. An antiserum raised against the unique V3 domain of PKC-theta identified an 80-kDa protein in all human T-cell lines tested, in erythroleukemia K562 cells and in histiocytic lymphoma U-937 cells, but not in a B-lymphoma line (Raji) or in several melanoma, carcinoma, schwanoma or astrocytoma lines, confirming, at the protein level, its predominant expression in hematopoietic cell lines, in particular T cells. Immunoreactive PKC-theta was detected almost exclusively in the cytosolic compartment of unstimulated Jurkat T cells. Stimulation with phorbol ester, however, caused rapid translocation to the membrane. In order to compare the properties of PKC-theta with a representative member of the Ca(2+)-dependent PKC enzymes, full-length cDNAs encoding PKC-theta or PKC-alpha were transiently expressed in COS-1 cells, and recombinant enzymes were partially purified via a six-histidine peptide tag. The catalytic activity of these PKC enzymes was assayed against distinct substrates in the absence and presence of known PKC cofactors. Significant differences were found with respect to activation requirements and substrate preferences between PKC-theta and PKC-alpha. Both enzymes were stimulated by phospholipid and phorbol ester, and were active towards a PKC-derived substrate peptide corresponding to the pseudosubstrate site of PKC. In contrast to PKC-alpha, however, full activation of PKC-theta did not require Ca2+, and its basal activity towards histone H1 was not stimulated by lipid cofactors. Additionally, a myelin-basic-protein-(MBP)-derived peptide, which was readily phosphorylated by PKC-alpha, was a poor substrate for PKC-theta. Similar to PKC-alpha, transient PKC-theta overexpression in murine EL4 thymoma cells caused an approximately 2.5-fold increase in the phorbol-12-myristate-13-acetate-induced transcriptional activation of an interleukin-2 promoter-reporter gene construct. The unique expression and functional properties of PKC-theta suggest that it may play a specialized role in T-cell signaling pathways.
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PMID:Expression and biochemical characterization of human protein kinase C-theta. 792 38

The protein kinase C (PKC) activation domain of the parathyroid hormone (PTH) was believed to be the 28-34 region of the molecule. We have now shown that PTH-(29-32) is the smallest PTH fragment that can stimulate significantly membrane-associated PKC activity in ROS 17/2 rat osteosarcoma cells. As was previously shown for full-length PTH-(1-84) and the fully bioactive PTH-(1-34) fragment, there were two peaks in the PKC response to PTH-(29-32): one peak was obtained with low picomolar concentrations and the other with much higher nanomolar concentrations of the fragment. The PKC-activating ability was unaffected by the loss of Asn33 and Phe34, but it was abolished by removing His32. Thus, the PTH-(28-31) and PTH-(29-31) fragments did not stimulate membrane-associated PKC activity. The much larger PTH-(1-31) fragment also did not stimulate membrane-associated PKC activity, although it stimulated adenylyl cyclase as strongly as PTH-(1-34). This functional sensitivity to the loss of the polar His32 was not caused by a specific need for His or another polar amino acid in this position because replacing it with the apolar Leu did not abolish adenylyl cyclase or PKC activation. It is concluded that the minimum, fully functional PKC activation domain of the PTH molecule is Gln29-Asp30-Val31-His32.
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PMID:Further definition of the protein kinase C activation domain of the parathyroid hormone. 807 68

The mechanism by which GH-releasing peptides elicit GH secretion has remained largely unknown. In this study, the effects of a second generation GH-releasing peptide, Ala-His-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2(GHRP-1), on cAMP, intracellular Ca2+ ([Ca2+]i), and GH release were examined using rat pituitary gland static monolayer cell cultures. It was found that GHRP-1 increased GH release in a dose-dependent manner up to 3-fold, while having no effect on cAMP levels. In contrast, simultaneous elevations of cAMP and GH were observed after treatment with GHRH. To further define the underlying mechanism of GHRP-1-mediated GH release, its effect on [Ca2+]i was determined using a fluorescent Ca2+ indicator, fura-2. GHRP-1 dose dependently increased [Ca2+]i up to 45.5 nM +/- 5.6 nM. A similar elevation of [Ca2+]i was observed after GHRH treatment. Similar to GHRH, GHRP-1-induced increases in [Ca2+]i and GH release were inhibited by somatostatin. Furthermore, the GHRP-1-induced increases in [Ca2+]i and GH were also suppressed by nifedipine. The interaction between the voltage-dependent Ca2+ channels and GHRP-1 was investigated in cells maximally stimulated by KCl. The addition of GHRP-1 had no effect on the KCl-stimulated GH release. To investigate the possible interaction between the adenylyl cyclase pathway and GHRP-1, cells were maximally stimulated with forskolin or (Bu)2cAMP. Addition of GHRP-1 stimulated GH release beyond that observed using cAMP elevating agents. Similar results were obtained in the presence of a protein kinase C, 4 beta-phorbol 12-myristate 13-acetate (PMA). The GHRP-1-stimulated GH release was additive to that observed with PMA stimulation. Based on these findings, it was concluded that 1) GHRP-1 treatment leads to an increase in [Ca2+]i; 2) unlike GHRH, GHRP-1 releases GH via a Ca(2+)-dependent, cAMP-independent mechanism; 3) GHRP-1-induced increases in [Ca2+]i and GH release are sensitive to somatostatin inhibition; and 4) cAMP-elevating agents and PMA have an additive effect on the GHRP-1-stimulated GH release, indicating these agents stimulate GH release via a mechanism separate from that of GHRP-1.
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PMID:Mechanisms of action of a second generation growth hormone-releasing peptide (Ala-His-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2) in rat anterior pituitary cells. 809 15

The c-Raf-1 protein kinase plays a central role in the mitogenic response of cells to growth factors, cytokines, and many oncogenes. Despite the critical importance of this enzyme, very little is known of its biochemical properties or mechanisms of regulation. In these experiments, we used the only candidate physiologic substrate identified as yet for c-Raf-1, mitogen-activated protein kinase kinase (MAPKK), to examine enzymatic characteristics and candidate modulators of c-Raf-1, c-Raf-1 was purified from Sf9 cells infected with recombinant baculovirus encoding a histidine-tagged c-Raf-1. The Km values of c-Raf-1 for ATP and MAPKK were 11.6 microM and 0.8 microM, respectively, and the stoichiometry of phosphorylation of MAPKK by c-Raf-1 was 1.67 mol of phosphate per mol of MAPKK. In contrast to prior reports, Mg2+ was the preferred cation at Mg2+ and Mn2+ concentrations > 5 mM. c-Raf-1 substrate specificity was extremely restricted, consistent with the identification of only one candidate physiologic substrate to date and highlighting the necessity of using MAPKK rather than artificial substrates in c-Raf-1 activity assays. Of multiple potential substrates tested, the only one phosphorylated to > 20% of the level of MAPKK phosphorylation was myelin basic protein (22%). Heat-denatured MAPKK was phosphorylated at only 2% the level of native MAPKK, indicating that the restricted substrate specificity may be due to tertiary-structural requirements. We also examined whether c-Raf-1 activity is modulated by lipid binding to the cysteine finger region in its regulatory domain. Of multiple mitogen-stimulated or cell-membrane lipids tested, only phosphatidylserine and diacylglycerol in the presence of Ca2+ (2.5 mM) increased c-Raf-1 kinase activity significantly (1.5-fold). The increase is probably not of physiologic significance because it was about two orders of magnitude less than the stimulation of protein kinase C by these lipids. On gel-filtration chromatography, the peak of c-Raf-1 kinase activity and immunoreactivity eluted at a predicted molecular mass of > 150 kDa, suggesting that active c-Raf-1 (but not inactive c-Raf-1) exists as a multimeric complex. This complex may not include p21ras, however, because immunoreactive p21ras was not identified in the active fractions.
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PMID:Enzymatic characteristics of the c-Raf-1 protein kinase. 810

The mechanism of action of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6), a synthetic peptide which specifically induces the secretion of growth hormone (GH) in rat somatotrophs, is still poorly understood. We have studied the effects of GHRP-6 on the cytosolic free calcium concentration ([Ca2+]i) of somatotrophs in primary culture. [Ca2+]i was monitored in individual somatotrophs by dual emission microspectrofluorimetry, using Indo-1 as the intracellular fluorescent Ca2+ probe. A short application of GHRP-6 (10(-5) M, 10 s) induced a biphasic Ca2+ response in most cells (44%), which consisted in a rapid and large rise in [Ca2+]i followed by sustained oscillations. This response is dose dependent in a range of concentrations from 10(-10) to 10(-5) M. The first phase of the GHRP-6 response persisted in the absence of Ca2+ in the extracellular medium, whereas the second phase was inhibited. The application of Ca2+ channel blockers like cadmium chloride (200 microM) or PN-200-110 (200 nM) also prevented the second phase. Conversely, when the cells were pretreated with thapsigargin (TG) (100 nM), the first phase of the GHRP-6 Ca2+ response was abolished, whereas the second phase alone was preserved. When the cells were depleted in PKC by incubation with 10(-6) M PMA for 24 h, the second phase of the GHRP-6 response was inhibited, and only the first phase was maintained. These results were corroborated by using phloretin, a PKC inhibitor. These data show that GHRP-6 induces a biphasic elevation of the [Ca2+]i in rat somatotrophs. The first phase is probably due to mobilization of the intracellular Ca2+ stores, whereas the second phase is a PKC-dependent process.
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PMID:GHRP-6 induces a biphasic calcium response in rat pituitary somatotrophs. 819 4

Cysteine-rich regions of protein kinase C (PKC) are critical for the lipid-dependent regulation of activity and are implicated in the coordination of zinc. A glutathione S-transferase fusion protein containing the second cysteine-rich region, Cys2, of PKC gamma with bound zinc with a stoichiometry of 1.8 +/- 0.1 mol of zinc/mol of protein. Deletion analysis within this cysteine-rich region defined amino acids essential for zinc coordination. An NH2-terminal histidine (His102) and a COOH-terminal cysteine (Cys151) were both critical for the coordination of distinct zinc atoms. Both represent the ultimate residues of a 50-amino acid consensus motif with six conserved cysteines and two conserved histidines present in the cysteine-rich regions of all PKC isoforms. Removal of histidine His102 abolished phorbol ester binding, while deletion of cysteine Cys151 did not. Deletion of valine (Val147) greatly diminished phorbol ester binding, which was completely lost only when valine (Val144) was also deleted. No significant further reduction in zinc stoichiometry below one resulted even when three COOH-terminal conserved cysteines (Cys151, Cys143, and Cys135) and a conserved histidine (His140) were deleted. These results are consistent with a model in which two zinc atoms are tetracoordinated per cysteine-rich region in two independent coordination spheres that are not functionally equivalent. These analyses determine a minimal peptide (residues 102-144) of 43 amino acids capable of [3H]PDBu binding.
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PMID:A phorbol ester binding domain of protein kinase C gamma. Deletion analysis of the Cys2 domain defines a minimal 43-amino acid peptide. 830 Jun 28


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