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)

We have established mutant SaOS-2 cell lines that express a cyclic AMP (cAMP)-resistant phenotype to investigate the regulation and functional importance of orthophosphoric-monoester phosphohydrolase alkaline optimum (ALPase) in the action of parathyroid hormone (PTH). Cells were stably transfected with a plasmid that directs the synthesis of a mutant form of the type I regulatory subunit of protein kinase A (PKA) under the control of the metallothionein promotor. There was no significant difference between parental SaOS-2 cells and the mutant lines in the affinity or number of receptors for 125I-Nle8,18Tyr34bPTH1-34NH2, either in the absence or presence of Zn2+. When cAMP-dependent gene transcription was examined using transient transfection with a somatostatin promoter-chloramphenicol acetyl transferase (CAT) reporter plasmid, CAT activity stimulated by human PTH and dibutyryl cAMP (DBcAMP) was inhibited by greater than 90% in the presence of Zn2+ in the mutant cell lines. In contrast, activation by a phorbol ester of a pentameric collagenase promoter/CAT construct containing five tandem copies of the AP-1 response element (5x-TRE-CAT) was unaffected in Zn(2+)-treated mutant cells. The inhibitory actions of PTH and DBcAMP on ALPase release were blunted by up to 80-90% in the mutant cell lines in the presence of Zn2+; there were no significant differences in the magnitude of inhibitory effects between these agonists. We conclude that the inhibitory action of PTH on ALPase release in SaOS-2 cells is mediated via activation of PKA. These cAMP-resistant cell lines will be especially useful in elucidating signal transduction mechanism(s) for PTH in human osteoblastic cells.
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PMID:Protein kinase A-dependent inhibition of alkaline phosphatase release by SaOS-2 human osteoblastic cells: studies in new mutant cell lines that express a cyclic AMP-resistant phenotype. 166 91

Metalloproteinases, such as collagenase or gelatinase, and their associated inhibitors appear to control connective tissue remodeling during follicular rupture. We examined the regulation of metalloproteinase inhibitor activity by various treatments in cultured rat granulosa cells. Granulosa cells were harvested from immature PMSG-primed rats and cultured with LH, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), cAMP, or forskolin. Inhibitor activity was measured in the medium. Increasing concentrations of either LH (0.1-1000 ng/ml) or TPA (2.5-100 nM) resulted in a dose-dependent increase in metalloproteinase inhibitor activity (2.9- and 2.4-fold increases above control, respectively). There was also a time-dependent induction of inhibitor activity in cells incubated in the presence of LH (100 ng/ml) for 6, 12, 18, or 24 h. Forskolin (0.1 mM) or cAMP (1 mM) treatment increased inhibitor activity 2.8- and 1.6-fold above that in control cultures. LH and TPA treatment in combination resulted in an additive increase in inhibitor activity compared to LH or TPA treatment alone. This finding suggested that the granulosa cell inhibitor activity might be induced through separate intracellular pathways. The inhibitor present in conditioned medium was isolated by chromatographic separation on a Sepharose 6B mol wt exclusion column. The inhibitor present was approximately 28,000 mol wt, which is consistent with the size of tissue inhibitor of metalloproteinase (TIMP). In addition to the granulosa cell experiments, changes in ovarian mRNA levels for TIMP were determined. There was a preovulatory increase in TIMP mRNA from whole rat ovaries, with the highest levels detected 12 h after hCG administration. The present study establishes that metalloproteinase inhibitor activity from rat granulosa cells is induced through separate pathways: a LH-cAMP-dependent protein kinase-A pathway and a cAMP-independent protein kinase-C pathway. Furthermore, a TIMP-like protein is observed in granulosa cell-conditioned medium, while TIMP mRNA is present in rat ovaries and increases before ovulation, suggesting that the granulosa cell metalloproteinase inhibitor is TIMP. We propose that TIMP acts in part to control the site and extent of follicular connective tissue remodeling associated with ovulation.
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PMID:Hormonal regulation of matrix metalloproteinase inhibitors in rat granulosa cells and ovaries. 184 3

Type beta transforming growth factors represent a family of polypeptides that modulate growth and differentiation. TGF-beta exerts its effects on target cells through interaction with specific cell surface receptors, but the signal transduction pathways are largely unresolved as yet. In this study we report that TGF-beta 1 induces a rapid phosphorylation of the cyclic AMP responsive element binding protein (CREB) in mink lung CCl64 cells. Phosphorylation induced by TGF-beta 1 is not mediated by the cAMP-dependent protein kinase. Parallel to the increase in phosphorylation of CREB, an increase in binding to the collagenase TPA responsive element was observed. CREB participates in the binding to this element, probably as a heterodimer with another as yet unknown protein. The modification imposed on CREB and its involvement in an enhanced TRE-binding could be a mechanism by which TGF-beta 1 induces the TRE-mediated transcriptional activation.
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PMID:TGF-beta 1 induces phosphorylation of the cyclic AMP responsive element binding protein in ML-CCl64 cells. 185 Jun 93

Previous studies identified synapsin I as a potential substrate for a newly discovered growth factor-sensitive, proline-directed protein kinase originally isolated from rat pheochromocytoma. The present study describes the site-specific phosphorylation of synapsin I by highly purified preparations of proline-directed protein kinase. The incorporation of [32P]phosphate into bovine brain synapsin I was dependent upon both the amount of kinase present and the time of incubation. The maximum stoichiometry of phosphorylation approached 1 mol of phosphate/mol of synapsin I protein. When analyzed by sodium dodecyl sulfate-gel electrophoresis and autoradiography, [32P]phosphate was found to be incorporated into both synapsin Ia and Ib. Phosphoamino acid analysis demonstrated that serine residues were phosphorylated exclusively. Digestion of phosphorylated synapsin I with trypsin followed by high performance liquid chromatography (HPLC) phosphopeptide analysis indicated that the tryptic peptide containing the major phosphorylation site eluted as a single peak at approximately 17% acetonitrile. The primary structure of this phosphopeptide, determined by gas-phase sequencing, was found to be Gln-Ser-Arg-Pro-Val-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Pro-Ala-Thr-Arg-Pro-Pro- Ala-Ser-Pro-Ser-Pro-Gln-Arg. Sequential Edman degradation of this HPLC-purified tryptic phosphopeptide revealed that serine 20 of this peptide was the major phosphorylated residue. This phosphoacceptor site is immediately flanked by a carboxyl-terminal proline residue, an observation that further verifies the proline-directed nature of this protein kinase. The tryptic phosphopeptide corresponds exactly to a sequence in the collagenase-sensitive, proline-rich "tail" region of bovine synapsin I. This novel phosphorylation site is close to but distinct from phosphorylation sites 2 and 3, which are known to be phosphorylated by calcium/calmodulin-dependent protein kinase II and are considered to be of regulatory importance.
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PMID:Phosphorylation of synapsin I at a novel site by proline-directed protein kinase. 210 63

We describe a method of culturing intact porcine thyroid follicles for physiological de novo thyroid hormone formation; the roles of cAMP and protein kinase-C in thyroid hormone formation were also studied. Thyroid follicles were obtained by digesting minced porcine thyroid tissue with 0.04% collagenase and cultured in Coon's Modified Ham's F-12 medium supplemented with 0.5% calf serum, 0.5 mU/ml TSH, other standard hormones, and 3 antibiotics (6H medium). On the fourth day of culture, 6000-8000 follicles/well were plated in 12-well culture dishes. On the sixth day, thyroid hormone formation was carried out by incubating thyroid follicles with 0.5 microM KI in the presence of 6H medium for 2 days in a 5% CO2-95% air incubator at 37 C. To examine the effects of cAMP and protein kinase-C on de novo thyroid hormone formation, follicles were incubated with KI in the presence of 1-2.5 mM (Bu)2cAMP, 10 microM forskolin, 2 microM prostaglandin E2 (PGE2), or 0.5-1 microM 12-O-tetradecanoylphorbol-13-acetate in TSH-free medium for 2 days. The amount of newly formed thyroid hormone was measured by RIA of T3 content in the Pronase digest of thyroid follicular cells. Thyroid follicles cultured in 6H medium had normal polarity of the membrane, determined by electron microscope, and thyroid cAMP was responsive to the alteration of TSH. In this culture system cAMP alone was sufficient to form thyroid hormone. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase-C stimulator, disrupted thyroid follicles and inhibited cAMP-mediated thyroid hormone formation. The integrity of follicular structure was also required for thyroid hormone formation in this culture system. This study introduces perhaps the most physiological culture system for de novo thyroid hormone formation. Our data provide direct evidence that thyroid hormone formation is linked to cAMP and that the protein kinase-C system acts as an inhibitor of thyroid hormone formation.
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PMID:Physiological de novo thyroid hormone formation in primary culture of porcine thyroid follicles: adenosine 3',5'-monophosphate alone is sufficient for thyroid hormone formation. 215 8

A subpopulation of cells was derived from the Hs431 connective tissue sarcoma cell line which possessed high affinity (estimated Kd = 0.38-0.55 nM) binding sites for human recombinant [125I]-IL-1 alpha. Binding at 4 degrees C was slow approaching equilibrium by 4 hrs. Dissociation of [125I]-IL-1 alpha was also slow and unaffected by high concentrations of cold ligand. The binding site also underwent ligand-induced internalization at 37 degrees C. An Mr = 83,000 protein was identified in affinity crosslinking studies. Despite these similarities to previously reported IL-1 receptors, Hs431 cells did not exhibit biological responses to IL-1 which have been observed in other cell lines. IL-1 did not induce PGE2 or collagenase synthesis. IL-1 also failed to induce ornithine decarboxylase activity (ODC) or stimulate [3H]-thymidine incorporation. In contrast, the Hs431 cells did contain a functional epidermal growth factor (EGF) receptor as determined from binding studies, protein kinase activity, induction of ODC, and stimulation of [3H]-thymidine incorporation. Thus, the refractoriness of Hs431 cells to IL-1 was fairly specific and did not result from a generalized defect associated with cell transformation.
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PMID:Characterization of a high affinity interleukin-1 (IL-1) specific binding site in a human synovial sarcoma (Hs431) cell line. 216 29

Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids encoding PKI(1-31) inhibit the expression that is stimulated by the addition of cAMP analogs in both cell lines; basal expression, however, is inhibited by PKI(1-31) only in the JEG-3 cell line and not in the CV-1 cells. These observations indicate that, in JEG-3 cells, PKI(1-31) is a specific inhibitor of kinase A-mediated gene transcription, but it does not modify kinase C-directed transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells. 247 35

The effect of the obligatory precursor of prostaglandin biosynthesis, arachidonic acid, on the release of relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells, identified as large luteal cells (LLCs). The rate of development of plaques in time-course studies and the area of plaques were then used as an index of the rate of relaxin release and cumulative amount of hormone released, respectively. Incubation of collagenase-dispersed luteal cells derived from early pregnant pigs with 0.1-100 microM arachidonic acid (AA) resulted in dose-dependent increases in the rate of plaque formation. Despite AA stimulation, however, only 55-65% of all LLCs formed plaques during the experimental incubation period (up to 12 h). Minimally and maximally effective doses were about 1 and 10 microM, respectively. In the presence of 10 microM AA, maximal plaque formation occurred significantly faster (1-2 h) than in controls (4-8 h; P less than 0.05). The percentage of plaque-forming cells (plaque-forming LLCs) was, likewise, significantly greater in 10 microM AA-treated monolayers than in controls during the first 3-4 h of incubation. Similarly, agents that liberate endogenous AA (phospholipase A2 and melittin) also stimulated relaxin release. The stimulatory effect of AA (10 microM) on relaxin release was almost wholly blocked by a cyclooxygenase inhibitor (ibuprofen; 20 microM); but not by a lipooxygenase inhibitor (nordihydroguaretic acid; 20 microM). However, the same dose of ibuprofen (20 microM) failed to modulate the stimulatory effect of prostaglandin E2 (1 microM) or phorbol diester (4 beta-phorbol 12 beta-myristate 13 alpha-acetate; 50 nM) on the rate of relaxin release. These results indicate that a product(s) of the cyclooxygenase pathway of AA metabolism participates in the control of relaxin release, but that this metabolite(s) is not essential to the biological action of at least one stimulatory secretagogue. Moreover, this metabolite failed to influence a subpopulation of nonresponsive LLCs. These data taken in association with our previous demonstration that the pathways of both calcium mobilization and protein kinase-C activation are implicated in the regulation of relaxin release, are consistent with the view that AA liberation may amplify the actions of other signalling mechanisms.
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PMID:Analysis of relaxin release by cultured porcine luteal cells using a reverse hemolytic plaque assay: effects of arachidonic acid, cyclo- and lipooxygenase blockers, phospholipase A2, and melittin. 250 67

Recent work indicates that PTH can stimulate osteoblastic cells to secrete neutral collagenase, an enzyme thought to be linked to bone matrix turnover. Since recent studies suggest that the calcium/protein kinase-C (PKC) message system is involved in signal transduction stimulated by PTH, we examined the role of these putative second messengers of PTH in the regulation of collagenase production by the osteoblastic tumor cell line UMR 106-01. Immunohistochemical staining of cells exposed to PTH (10(-7) M) revealed that about 20% of the entire population was positive for collagenase, compared to less than 3% staining positively in control untreated cells. Incubation with the cAMP analog 8-bromo-cAMP (8BrcAMP) increased the number of collagenase-staining cells in a dose-dependent manner (ED0.5 = 2.5 x 10(-4) M), but to a lower level than PTH, with the maximal effect producing about 15% positive cells. The calcium ionophore ionomycin (10(-7) M) was ineffective, whereas phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased collagenase-specific staining to about 5%, but only at high concentrations (10(-5) M). Incubation of UMR 106-01 cells with ionomycin and PMA did not change the effect of the latter. When the three agents were used in combination, an additive effect was observed, which fully reproduced that of PTH. Similarly, the amount of collagenase released into the medium by cells stimulated with maximal concentrations of 8BrcAMP (10(-3) M) was only 80% of that induced by maximal doses of PTH (10(-7) M). PMA (10(-5) M) was slightly stimulatory, and ionomycin was ineffective alone, but they were synergistic with submaximal doses of 8BrcAMP (10(-4) M). In agreement with the immunohistochemical results, the full hormonal effect was reproduced when the three second messenger analogs were used in combination. In conclusion, signal transduction from PTH receptor to collagenase production is mediated mainly by cAMP; the Ca2+/PKC system appears to have a contributory role necessary for the full expression of hormonal response. These results support the hypothesis of a dual pathway of target cell activation by PTH.
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PMID:Second messenger signaling in the regulation of collagenase production by osteogenic sarcoma cells. 254 4

LH has been shown to be the principal hormone regulating ovarian thecal-interstitial cell (TIC) differentiation. It has been well documented that LH stimulates cAMP production and that cAMP analogs mimick the stimulatory actions of LH, but the mechanisms by which LH and cAMP stimulate TIC differentiation are unknown. The purpose of these studies was to characterize LH-stimulated differentiation of isolated TIC in serum-free medium and examine the role of cAMP-dependent protein kinase (PKA) isoenzymes in TIC differentiation. Highly purified (greater than 90%) TIC which were free from granulosa cell contamination were isolated from collagenase-dispersed ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When the purified TIC (20,000 viable cells/well) were cultured (2 days) in serum-free medium (0.2 ml in 96-well plates), low levels of steroids were produced. LH stimulated a dose-related (ED50 = 2.6 +/- 0.4 ng/ml) increase (50-fold) in androsterone, the principal androgen produced. LH stimulated an immediate dose-related increase in cAMP production, but there was a 20-h lag before LH stimulated an increase in androsterone production, which reached maximum levels at 30 h. LH-stimulated progesterone production increased immediately to a maximum at 10 h, then progesterone levels decreased as androsterone production increased. To determine the role of PKA in stimulating androsterone and progesterone production, TIC were cultured (2 days) with 8-aminohexylamino-cAMP (100 microM) plus N6-benzoyl-cAMP (30 microM) or 8-thiomethyl-cAMP (30 microM) plus N6-benzoyl-cAMP (30 microM) to directly and selectively activate type I or type II PKA, respectively. Selective activation of either isoenzyme increased androsterone and progesterone production by TIC. Immunoblots revealed that either type I or type II PKA increased the contents of P450scc and P45017 alpha in TIC. This is the first demonstration that direct activation of either type I or type II PKA stimulates TIC differentiation. These results indicate that LH stimulates TIC differentiation by a mechanism mediated by activation of one or both PKA isoenzymes.
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PMID:Evidence that luteinizing hormone-stimulated differentiation of purified ovarian thecal-interstitial cells is mediated by both type I and type II adenosine 3',5'-monophosphate-dependent protein kinases. 254 87


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