Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Quiescent Swiss 3T3 cells can be stimulated to reenter the cell cycle by various mitogens used in synergistic combinations with insulin-like growth factors (IGFs). The cells constitutively secrete an IGF-binding protein (IGFBP), which can modulate the interaction of IGFs with their receptors and could, therefore, alter cellular responsiveness to IGFs. We have now characterized the IGFBP secreted by Swiss 3T3 cells and tested whether its secretion is regulated by heterologous mitogens. Ligand blotting using [125I]IGF-I revealed a major IGFBP of 40,000 mol wt, and treatment of the cells with tunicamycin reduced the mol wt of this protein to about 32,000. mRNA from Swiss 3T3 cells hybridized to a 32P-labeled oligonucleotide (50-mer) complementary to rat IGFBP-3. Taken together, these results indicate that the principal IGFBP secreted by Swiss 3T3 cells is probably the N-glycosylated IGFBP-3. Production of this IGFBP by Swiss 3T3 cells was stimulated by 50-150% by the mitogens bombesin, vasopressin, platelet-derived growth factor, epidermal growth factor, and 12-O-tetradecanoylphorbol 13-acetate and also by IGF-I. The increased production of IGFBP was first detected after 4-6h of incubation and was then maintained for 48-72 h. Agents that elevate intracellular cAMP and the glucocorticoid dexamethasone reduced IGFBP output. In cells in which protein kinase-C had been down-modulated, the stimulation of IGFBP output by 12-O-tetradecanoylphorbol 13-acetate was abolished, but the stimulation induced by the other mitogens was not prevented. Thus, the production of IGFBP by Swiss 3T3 cells can be regulated by a number of different signalling pathways.
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PMID:Mitogens regulate the production of insulin-like growth factor-binding protein by Swiss 3T3 cells. 170 79

The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha-Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A-mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport.
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PMID:Identification of Ser-55 as a major protein kinase A phosphorylation site on the 70-kDa subunit of neurofilaments. Early turnover during axonal transport. 171 55

HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (greater than 6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibit the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca(++)-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions.
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PMID:HMG 14 and protamine enhance ligation of linear DNA to form linear multimers: phosphorylation of HMG 14 at Ser 20 specifically inhibits intermolecular DNA ligation. 184 50

The role of protein kinase-C-dependent mechanisms in steroidogenic enzyme gene expression was studied in primary cultures of human fetal and adult adrenals. Cells were first cultured for 7-10 days and then stimulated with ACTH or 12-O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase-C activator, for 1-2 days. Cytoplasmic RNA was extracted and analyzed by Northern and dot blotting with 32P-labeled cDNA probes for P450scc (cholesterol side-chain cleavage enzyme/20,22-desmolase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase); for P450c11 (11 beta-hydroxylase/18-hydroxylase/18-methyl oxidase), a 30-mer oligonucleotide was used as a probe. ACTH (200 ng/ml) increased the accumulation of all of the studied steroidogenic enzyme mRNAs in both fetal and adult cultures by several-fold. TPA inhibited this accumulation in a dose-dependent manner (0.01-100 ng/ml), whereas the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate was without effect. On the other hand, in the absence of ACTH, TPA slightly increased all steroidogenic P450 mRNAs in adult cultures. In fetal cultures TPA slightly increased P450scc, P450c11, and P450c21 mRNA levels, whereas it decreased P450c17 mRNA. (Bu)2cAMP and cholera toxin increased steroidogenic enzyme mRNAs such as ACTH. TPA down-regulated (Bu)2cAMP- and cholera toxin-induced P450mRNAs in the same way as ACTH-induced mRNAs. The secretion of ACTH-stimulated cortisol, dehydroepiandrosterone sulfate, and aldosterone was decreased by TPA in both fetal and adult cultures. The basal steroid production was slightly increased by TPA in both culture types. The changes in steroid production correlated well with the alterations in the steroidogenic enzyme gene expression. Our results show that the inhibitory effect of TPA on ACTH-stimulated adrenal steroidogenesis is mediated at the mRNA level of steroidogenic enzymes. Thus, it seems likely that both protein kinase-C- and cAMP-dependent mechanisms are involved in the long term maintenance of steroidogenic enzymes and hormone production in adrenocortical cells.
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PMID:Interaction of phorbol ester and adrenocorticotropin in the regulation of steroidogenic P450 genes in human fetal and adult adrenal cell cultures. 184 59

cAMP-dependent protein kinase appears to play a role in cAMP-induced gene expression in mammalian cells. There exist two major types of cAMP-dependent protein kinase, type I and type II, which are distinguished by their regulatory subunits, RI and RII, respectively. We investigated the role of type I and type II protein kinase in the cAMP-induced gene expression by either stable or co-transfection of RI alpha, RII alpha, or RII beta gene in an expression vector together with somatostatin-chloramphenicol acetyltransferase (SS-CAT) fusion gene using a cAMP-unresponsive mutant pheochromocytoma cell line (A126-1B2). Introduction of the RII beta gene restored the capability of these cells to induce the SS-CAT gene expression in response to forskolin stimulus and induced a changed morphology which resembled that of wild type. The RII alpha gene also induced SS-CAT gene expression but to a lesser degree than that achieved by the RII beta gene, whereas the RI alpha gene had no effect. The induction of SS-CAT gene expression by the RII beta gene was specifically blocked by the 21-mer RII beta antisense oligodeoxynucleotide. These results show for the first time that type II but not type I regulatory subunit of cAMP-dependent protein kinase is essential for a cAMP-induced gene transcription.
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PMID:Type II regulatory subunit of protein kinase restores cAMP-dependent transcription in a cAMP-unresponsive cell line. 197 35

A marked decrease in the type I cAMP-dependent protein kinase regulatory subunit (RI alpha) and an increase in the type II protein kinase regulatory subunit (RII beta) correlate with growth inhibition and differentiation induced in a variety of types of human cancer cells, in vitro and in vivo, by site-selective cAMP analogs. To directly determine whether RI alpha is a growth-inducing protein essential for neoplastic cell growth, human HL-60 promyelocytic leukemia cells were exposed to 21-mer RI alpha antisense oligodeoxynucleotide, and the effects on cell replication and differentiation were examined. The RI alpha antisense oligomer brought about growth inhibition and monocytic differentiation, bypassing the effects of an exogenous cAMP analog. These effects of RI alpha antisense oligodeoxynucleotide correlated with a decrease in RI alpha receptor and an increase in RII beta receptor level. The growth inhibition and differentiation were abolished, however, when these cells were exposed simultaneously to both RI alpha and RII beta antisense oligodeoxynucleotides. The RII beta antisense oligodeoxynucleotide alone has been previously shown to specifically block the differentiation inducible by cAMP analogs. These results provide direct evidence that RI alpha cAMP receptor plays a critical role in neoplastic cell growth and that cAMP receptor isoforms display specific roles in cAMP regulation of cell growth and differentiation.
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PMID:Differentiation of HL-60 leukemia by type I regulatory subunit antisense oligodeoxynucleotide of cAMP-dependent protein kinase. 200 Apr 8

Two intracellular signal transduction mechanisms such as cAMP-protein kinase a and phosphatidylinositol (PI) turnover-protein kinase c are known to be dually involved in the regulation of luteinizing hormone-releasing hormone (LHRH) release. However, it is not yet evident that the activation of two intracellular pathways affects the LHRH gene expression. The present study aims, therefore, to determine whether the activation of two intracellular pathways affects changes in LHRH mRNA. To this end, we took advantage of an in vitro superfusion system, where rat hypothalamic tissues were superfused with media containing forskolin (FKN) and/or phorbol-12-myristate-13-acetate (PMA). Superfusates were collected at 10-min intervals and LHRH release was determined by radioimmunoassay. After a 2-h superfusion period, the post-superfusion hypothalami were recovered and poly (A) RNA fractions were isolated. Alterations in LHRH mRNA in response to FKN and/or PMA were determined by an RNA-blot hybridization assay using a 32P-end-labeled LHRH oligonucleotide (29-mer) probe. In vitro perfusion of hypothalamic fragments with PMA and/or FKN stimulated LHRH release as well as LHRH mRNA. The combined infusion of FKN and PMA did not produce an additive effect on the LHRH mRNA levels, but it was effective in synergistically increasing LHRH secretion in vitro. These data clearly demonstrate that the biosynthetic machinery of LHRH is influenced by activation of two intracellular pathways, both cAMP-protein kinase a and phosphatidyl-inositol turnover-protein kinase c, indicating the transsynaptic regulation of hypothalamic LHRH gene expression.
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PMID:Activation of intracellular pathways with forskolin and phorbol ester increases LHRH mRNA level in the rat hypothalamus superfused in vitro. 217 Jul 97

Oligopeptides predicted from the nucleotide sequence of the oncogene v-fes of feline sarcoma virus (FeSV) were synthesized chemically and used to generate specific antibodies. Antisera against a 12-amino-acid-long oligopeptide (12-mer) located 42 residues from the carboxyl terminus of the v-fes coding sequence efficiently recognized the transforming proteins encoded by Snyder-Theilen (ST) and Gardner-Arnstein (GA) strains of FeSV. This 12-mer also contains 10 amino acid residues homologous in order and position to those predicted from the nucleotide sequence of the oncogene v-fps of avian Fujinami sarcoma virus (FSV). The anti-12-mer immunoprecipitated the FSV-specific transforming protein molecules from FSV-transformed cells. Binding of these antipeptide antibody molecules to the v-fes and the v-fps gene products inhibited their associated tyrosine-specific protein kinase (EC 2.7.1.37) activities. The ability to generate such site-specific antisera to the products of related oncogenes will be valuable in the molecular characterization of retroviral transforming proteins and their normal cellular homologs.
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PMID:Antibodies of predetermined specificity detect two retroviral oncogene products and inhibit their kinase activities. 657 83

Calcitonin (CT) is a peptide hormone that interacts with the cAMP-and phospholipase C-associated CT receptor subtypes. We investigated whether CT modulates the interaction of human tumoral osteoclast-like (GCT23) cells with a protein of the bone matrix, bone sialoprotein-II (BSP-II). Single GCT23 cells loaded with the intracellular Ca2+ indicator fura-2 were treated with the maximal active dose (300 micrograms/ml) of the 18-mer Arg-Gly-Asp (RGD)-containing BSP-IIA fragment, and the cytosolic free Ca2+ concentration ([Ca2+]i) was measured by dual wavelength microfluorometry. BSP-IIA stimulated an elevation in [Ca2+]i, consisting mainly of a peak, followed by a rapid return toward baseline. Pretreatment with CT induced a modest elevation of [Ca2+]i. However, CT significantly inhibited the response to BSP-IIA in a dose-dependent manner. Maximal inhibition (90% vs. untreated) was observed in the micromolar range. The intracellular mechanisms leading to this effect were investigated by pretreatment of GCT23 cells with the cAMP permeant analog, (Bu2)cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 micrograms/ml BSP-IIA. The effect induced by CT was specific, because an increase in the extracellular Ca2+ concentration, which is also known to inhibit bone resorption, failed to modify the ability of BSP-IIA to alter [Ca2+]i in GCT23 cells. To investigate whether the CT-induced alteration of BSP-IIA-dependent cell signals was due to a modification in the synthesis of cell surface receptors (integrins) for the extracellular matrix macromolecules, 1-h CT-treated [35S]methionine metabolically labeled GCT23 cell lysates were immunoprecipitated with anti-alpha 3-, -alpha v-, -beta 1-, and -beta 3-integrin subunit antibodies. Autoradiography demonstrated that 10(-7)-10(-6) M CT did not alter new synthesis of the alpha v beta 3 and the alpha 3 beta 1 receptors. Similarly, CT did not affect surface expression of these receptors, assessed by enzyme-linked immunosorbent assay. Finally, no alteration of the adhesion rate and spreading of GCT23 cells onto BSP-IIA-coated substrates was observed. This indicates that CT-induced down-regulation of immediate cell signals prompted by BSP-IIA in GCT23 cells is a postintegrin receptor event.
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PMID:Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism. 786 71

Enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type I has been correlated with cancer cell growth. We provide evidence that RI alpha is a growth-inducing protein that may be essential for neoplastic cell growth. Human colon, breast, and gastric carcinoma and neuroblastoma cell lines exposed to a 21-mer human RI alpha antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) exhibited growth inhibition with no sign of cytotoxicity. Mismatched sequence (random) S-oligodeoxynucleotides of the same length exhibited no effect. The growth inhibitory effect of RI alpha antisense oligomer correlated with a decrease in the RI alpha mRNA and protein levels and with an increase in RII beta (the regulatory subunit of protein kinase type II) expression. The growth inhibition was abolished, however, when cells were exposed simultaneously to both RI alpha and RII beta antisense S-oligodeoxynucleotides. The RII beta antisense S-oligodeoxynucleotide alone, exhibiting suppression of RII beta along with enhancement of RI alpha expression, led to slight stimulation of cell growth. These results demonstrate that two isoforms of cyclic AMP receptor proteins, RI alpha and RII beta, are reciprocally related in the growth control of cancer cells and that the RI alpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI alpha, is a powerful biological tool toward suppression of malignancy.
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PMID:An antisense oligodeoxynucleotide that depletes RI alpha subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells. 842 67


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