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 used co-cultures of porcine ovarian granulosa cells and mouse adrenocortical tumor cells (Y-1) to examine the kinetics of contact-dependent intercellular signal transfer and to assess the molecular mechanisms employed by this process. Exposure to follicle-stimulating hormone (FSH) caused cAMP-dependent protein kinase dissociation in granulosa cells and, with time, in Y-1 cells if, and only if, they contacted a responding granulosa cell. Y-1 cells close to a granulosa cell but not touching it failed to respond similarly. In reciprocal experiments, co-cultures were stimulated with adrenocorticotropic hormone (ACTH). Y-1 cells dissociated protein kinase as did granulosa cells in contact with Y-1 cells; however, granulosa cells that were not in contact with Y-1 cells failed to respond to the hormone. Fluorogenic steroids were secreted by Y-1 cells cultured alone and stimulated with ACTH, but were not secreted by cultures exposed to FSH. Neither hormone caused fluorogenic steroid production by granulosa cells. On the other hand these steroids were secreted in co-cultures stimulated with ACTH and to a lesser degree in co-cultures exposed to FSH. Autoradiography revealed that I125-FSH bound only to granulosa cells, never to Y-1 cells, even if they were in contact with an ovarian cell. The possibility of cell fusion was tested by experiments in which Y-1 cell membranes were labeled with cationized ferritin. These cells were then placed in co-culture with ovarian granulosa cells that had previously been allowed to ingest latex spheres. At regions of gap junctions between Y-1 and granulosa cells ferritin remained attached to the adrenal cell membrane and was never observed to migrate to the granulosa cell membrane. From these data, we conclude that hormone specific stimulation of one cell type leads to protein kinase dissociation in heterotypic partners only if they contact a hormone responsive cell. This signal transfer is bidirectional, exhibits temporal kinetics and occurs in the absence of apparent cell fusion. The only structural feature connecting Y-1 and granulosa cells were gap junctions implying they provided the communication channels; however, alternative mechanisms cannot be excluded. We have not established the identity of the signal being transferred although cAMP is a logical candidate.
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PMID:Hormone-induced intercellular signal transfer dissociates cyclic AMP-dependent protein kinase. 632 20

A recent study from this laboratory has shown that the inflammatory mediator, interleukin-1 alpha (IL-1 alpha), stimulates protein kinase A (PKA) activity and adrenocorticotropic hormone (ACTH) secretion from AtT-20 cells without any detectable increase in intracellular cAMP accumulation. The present studies were conducted to determine if cAMP is involved in IL-1 alpha activation of PKA and if PKA is responsible for IL-1 alpha-induced ACTH release from AtT-20 cells. The data are consistent with a novel mechanism of PKA activation that does not involve cAMP. Inhibition of adenylate cyclase with 2'5'-dideoxyadenosine (2'5'-DDA) did not affect IL-1 alpha-induced increases in PKA activity and ACTH secretion. In contrast, CRF-stimulated PKA activity and ACTH secretion were inhibited by 2'5'-DDA. Additional evidence was obtained using the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). IBMX did not alter IL-1 alpha-induced PKA activity or ACTH secretion, yet IBMX potentiated CRF-induced cAMP accumulation. Inhibition of PKA with the PKA inhibitor, H-8, blocked activation of PKA and ACTH secretion by both IL-1 alpha and CRF in AtT-20 cells. These observations demonstrate that 1) the mechanism of IL-1 alpha activation of PKA is independent of adenylate cyclase or cAMP and 2) PKA is used by IL-1 alpha to induce ACTH secretion from AtT-20 cells.
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PMID:Interleukin-1 increases protein kinase A activity by a cAMP-independent mechanism in AtT-20 cells. 750 95

The effects of Ca2+ on lipolysis and protein kinase activity in adipocytes from exercise-trained rats were investigated. Chronic exercise significantly increased lipolytic responses to norepinephrine and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP). The inhibitory effects of N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a calumodulin inhibitor, on norepinephrine- and dibutyryl cAMP-stimulated lipolysis were significantly greater in trained than in sedentary rats. Training did not alter cAMP-dependent protein kinase activity. However, the inhibitory effect of W-7 on cAMP-dependent protein kinase activity was much greater in trained than in sedentary rats. The basal intracellular free Ca2+ concentration ([Ca2+]i) was significantly higher in trained than in sedentary rats. The rapid and transient increases in [Ca2+]i due to adrenocorticotropic hormone and phenylephrine from basal levels were significantly lower in trained than in sedentary rats. However, the higher basal [Ca2+]i level in trained rats led to increases in sustained [Ca2+]i levels after stimulation. We concluded that in trained rats the regulation of protein kinase activity by cAMP depends to a greater degree on Ca(2+)-calmodulin complex than it does in sedentary rats and that training alters adipocyte intracellular Ca2+ homeostasis, including [Ca2+]i responsiveness to hormones.
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PMID:Ca2+ and lipolysis in adipocytes from exercise-trained rats. 789

The beta-3 adrenoceptor protein lacks most of the potential phosphorylation sites for beta adrenoceptor kinase and protein kinase A. In addition, it exhibits a lower affinity toward norepinephrine than beta-1 or beta-2 adrenoceptors. It is thus expected that beta-3 adrenoceptors could be less implicated in desensitization processes than the beta-1 or beta-2 adrenoceptors. An attempt to demonstrate the physiological relevance of this prediction was performed by using fat cells having a beta-3 adrenergic responsiveness or not (hamster and guinea pig). The influence of prolonged in vivo exposure to norepinephrine on the beta adrenoceptor-mediated lipolytic responses was investigated in both species. In control guinea pigs, isoproterenol, norepinephrine and epinephrine were fully lipolytic, whereas BRL 37344 [(R',R')-4-2[2-((2[(3-chlorophenyl)-2-hydroxyethyl]amino] propyl)phenyl]phenoxyacetic acid], CGP 12177](+-)-4-(3-tertiarybutylamino-2-hydroxypropoxy)- benzimidazole-2-on hydrochloride] and other beta-3 agonists were inefficient, whereas hamster adipocytes exhibited maximal response to the beta-3 agonists. Blockade of the lipolytic effect of isoproterenol in the guinea pig gave a rank order of beta antagonists [CGP 20712A (1-[2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino]-3-4-(1-methyl-4-tr i-fluoro-methyl-1H-imidazol-2-yl)phenoxy-2-propanol methanesulfonate] > bupranolol > or = propranolol >> ICI 118551 [erythrodl-1-(7- methylindan-4-yloxy)-3-isopropylaminobutan-2-ol)] in agreement with that of a beta-1 effect. In contrast, the selective beta-1 antagonist CGP 20712A did not counteract the effect of BRL 37344 in hamsters and bupranolol was the best beta antagonist tested; a result arguing for the predominance of a beta-3 component in the adrenergic activation of lipolysis, as in rat fat cells. In treated guinea pigs (6-day treatment with osmotic minipumps delivering norepinephrine at the rate of 5 micrograms/min/kg), the adrenocorticotropic hormone dose-response curve was identical to that of controls, but the curves for isoproterenol, norepinephrine and epinephrine were flattened and shifted to the right. A down-regulation of beta-1 and beta-2 adrenoceptors was evidenced by a reduction in [3H]CGP 12177 high-affinity binding sites. In treated hamsters, compared to the controls, there was no change in the lipolytic response to the beta adrenergic agonists. Other protocols of chronic exposure to norepinephrine (e.g., daily injections) at different doses were also unable to reduce the beta-lipolytic effect in the hamster fat cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Desensitization of beta-1 and beta-2, but not beta-3, adrenoceptor-mediated lipolytic responses of adipocytes after long-term norepinephrine infusion. 809 43

We undertook these studies to explore the intracellular signaling mechanisms activated by a newly described human brain melanocortin receptor (hMC3R). Hepa cells transfected with the hMC3R gene responded to stimulation with alpha-melanocyte stimulation hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) with dose-dependent increases in cellular content of cyclic 3',5'-adenosine monophosphate (cAMP) reaching a maximum of over 1500% of control cells at the 10(-8) M dose (EC50 = 10(-11) M). In contrast, the production of [3H]inositol phosphates in cells prelabeled with myo-[2-3H]inositol exhibited a biphasic dose-response curve with increases as high as 155% of basal at 10(-11) M alpha-MSH or ACTH, but beyond that a dose-dependent decrease was observed. The inhibitory component of the dose-response curve could be abolished by pretreatment of transfected cells with the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate (Rp-cAMP) or the protein kinase A inhibitor H-89. Increases in intracellular calcium induced in transfected cells by alpha-MSH in doses ranging from 10(-11) to 10(-7) M could not be observed unless the cells were pretreated with H-89. By replacing the third intracytoplasmic loop of the canine H2-histamine receptor with that of hMC3R the biphasic characteristic of agonist-induced production of [3H]inositol phosphates was conferred to the chimeric receptor. These data indicate that the hMC3R is coupled to both cAMP and inositol phospholipid/Ca(2+)-mediated post-receptor signaling systems and that the latter response is regulated by protein kinase A activity.
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PMID:Interaction of dual intracellular signaling pathways activated by the melanocortin-3 receptor. 817 43

Pituitary-derived trophic hormones regulate cell-type-specific expression of VL30 retrotransposons in tissues that are engaged in steroidogenesis. We show that adrenocorticotropic hormone and forskolin induced VL30 transcription in the steroidogenic adrenal cell line Y1 and that the transcriptional activation was cell type- and protein kinase A-dependent. Three novel cAMP-responsive elements (CREs), within the VL30 long terminal repeat, were identified and shown to activate transcription synergistically when templates bearing multiple sites were compared with templates bearing a single site. This type of regulation was evident only in forskolin-treated cells, and the response elements were found to be inactive as mediators of constitutive transcription. In vitro binding analyses indicated that a consensus CRE and the nonconsensus VL30 CREs differ with respect to binding affinity and specificity to a number of nuclear factors that were identified to be related to proteins within the CREB, Jun, and C/EBP families of transcription factors. The relatively low affinity and/or a restricted binding specificity of the VL30 CREs made it possible to detect forskolin-induced binding of CREB- and Jun-related proteins to these sequences. We suggest that cAMP-induced transcription, specific for steroidogenic cells, can be mediated by a novel type of nonconsensus CREs and that the mechanism for this type of gene regulation is distinct from that mediated through a consensus CRE. We also report the identification of a novel factor, distinct from previously characterized CRE-binding proteins, that constitutively binds to the identified CREs.
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PMID:Cooperating nonconsensus cAMP-responsive elements are mediators of adrenocorticotropin-induced VL30 transcription in steroidogenic adrenal cells. 838 84

This review highlights contributions from my laboratory in which the sites and mechanisms of action of the adrenocorticotropic hormone (ACTH) in the adrenal cortex have been explored. Early studies showing that ACTH stimulates adrenal steroidogenesis by interacting with specific receptors at the cell surface are summarized. Next, the development of a strategy of genetic analysis to define the signalling events that follow ACTH interaction with its receptor is described. This strategy involved the isolation and characterization of mutant adrenal cell lines harboring specific defects in the ACTH-responsive steroidogenic pathway. I describe the isolation and characterization of several of these mutants and demonstrate how these mutants have helped to establish obligatory roles for adenylyl cyclase, cyclic AMP (cAMP), and cAMP-dependent protein kinase in the steroidogenic actions of ACTH. Finally, some of our studies on the regulated expression of the steroidogenic cytochrome P450 enzymes in Y1 adrenal cells are reviewed. These latter studies have led to the discovery of a novel promoter element and transcription factor (designated steroidogenic factor 1) that participates in the coordinate expression of these cytochrome P450 enzymes and that is required for their regulated expression by ACTH and cAMP.
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PMID:The 1994 Upjohn Award Lecture. Molecular and genetic approaches to the study of signal transduction in the adrenal cortex. 856 76

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that is inhibited by adrenocorticotropic hormone (ACTH) at picomolar concentrations. Inhibition of IAC may be a critical step in depolarization-dependent Ca2+ entry leading to cortisol secretion. In whole-cell patch clamp recordings from AZF cells, we have characterized properties of IAC and the signalling pathway by which ACTH inhibits this current. IAC was identified as a voltage-gated, outwardly rectifying, K(+)-selective current whose inhibition by ACTH required activation of a pertussis toxin-insensitive GTP binding protein. IAC was selectively inhibited by the cAMP analogue 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (8-pcpt-cAMP) with an IC50 of 160 microM. The adenylate cyclase activator forskolin (2.5 microM) also reduced IAC by 92 +/- 4.7%. Inhibition of IAC by ACTH, 8-pcpt-cAMP and forskolin was not prevented by the cAMP-dependent protein kinase inhibitors H-89 (5 microM), cAMP-dependent protein kinase inhibitor peptide (PKI[5-24]) (2 microM), (Rp)-cAMPS (500 microM), or by the nonspecific protein kinase inhibitor staurosporine (100 nM) applied externally or intracellularly through the patch pipette. At the same concentrations, these kinase inhibitors abolished 8-pcpt-cAMP-stimulated A-kinase activity in AZF cell extracts. In intact AZF cells, 8-pcpt-cAMP activated A-kinase with an EC50 of 77 nM, a concentration 2,000-fold lower than that inhibiting IAC half maximally. The active catalytic subunit of A-kinase applied intracellularly through the recording pipette failed to alter functional expression of IAC. The inhibition of IAC by ACTH and 8-pcpt-cAMP was eliminated by substituting the nonhydrolyzable ATP analogue AMP-PNP for ATP in the pipette solution. Penfluridol, an antagonist of T-type Ca2+ channels inhibited 8-pcpt-cAMP-induced cortisol secretion with an IC50 of 0.33 microM, a concentration that effectively blocks Ca2+ channel in these cells. These results demonstrate that IAC is a K(+)-selective current whose gating is controlled by an unusual combination of metabolic factors and membrane voltage. IAC may be the first example of an ionic current that is inhibited by cAMP through an A-kinase-independent mechanism. The A-kinase-independent inhibition of IAC by ACTH and cAMP through a mechanism requiring ATP hydrolysis appears to be a unique form of channel modulation. These findings suggest a model for cortisol secretion wherein cAMP combines with two separate effectors to activate parallel steroidogenic signalling pathways. These include the traditional A-kinase-dependent signalling cascade and a novel pathway wherein cAMP binding to IAC K+ channels leads to membrane depolarization and Ca2+ entry. The simultaneous activation of A-kinase- and Ca(2+)-dependent pathways produces the full steroidogenic response.
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PMID:Adrenocorticotropic hormone and cAMP inhibit noninactivating K+ current in adrenocortical cells by an A-kinase-independent mechanism requiring ATP hydrolysis. 889 75

Incubation of rat adrenal glomerulosa cells with low concentrations (up to 50 nM) of the protein kinase (PKC) inhibitor staurosporine (ST) inhibited aldosterone (ALDO) and cyclic AMP (cAMP) production stimulated by adrenocorticotropic hormone (ACTH) and cholera toxin. Only higher concentrations (1.6 microM) of staurosporine inhibited dibutyryl-cAMP- and forskolin-induced stimulation of aldosterone production. cAMP levels were increased only with low concentrations of the PKC inhibitor. This latter increase was avoided by treatment with a maximal concentration of isobutylmethylxanthine (MIX). Our results suggest that: (1) second messengers other than cAMP are involved in ACTH action; (2) staurosporine inhibits different kinases involved in ACTH action in a dose-dependent manner; (3) the protein kinase inhibited by high concentrations of staurosporine appears to be the cAMP-dependent kinase, PKA; and (4) the protein kinase inhibited by low concentrations of staurosporine remains to be identified. This latter species is suggested as being involved in mediating ACTH-induced activation of Gs.
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PMID:Effects of staurosporine on ACTH-mediated stimulation of aldosterone production. 891 88

The adrenocorticotropic hormone (ACTH) inhibits the growth of Y1 mouse adrenocortical tumor cells as well as normal adrenocortical cells in culture but stimulates adrenocortical cell growth in vivo. In this study, we investigated this paradoxical effect of ACTH on cell proliferation in Y1 adrenal cells and have unmasked a growth-promoting effect of the hormone. Y1 cells were arrested in the G1 phase of the cell cycle by serum starvation and monitored for progression through S phase by measuring [3H]thymidine incorporation into DNA and by measuring the number of nuclei labeled with bromodeoxyuridine. Y1 cells were stimulated to progress through S phase and to divide after a brief pulse of ACTH (up to 2 h). This effect of ACTH appeared to be cAMP independent, since ACTH also induced cell cycle progression in Kin-8, a Y1 mutant with defective cAMP-dependent protein kinase activity. The growth-promoting effect of ACTH in Y1 was preceded by the rapid activation of p44 and p42 mitogen-activated protein kinases and by the accumulation of c-FOS protein. In contrast, continuous treatment with ACTH (14 h) inhibited cell cycle progression in Y1 cells by a cAMP-dependent pathway. The inhibitory effect of ACTH mapped to the midpoint of G1. Together, the results demonstrate a dual effect of ACTH on cell cycle progress, a cAMP-independent growth-promoting effect early in G1 possibly mediated by mitogen-activated protein kinase and c-FOS, and a cAMP-dependent inhibitory effect at mid-G1. It is suggested that the growth-inhibitory effect of ACTH at mid-G1 represents an ACTH-regulated check point that limits cell cycle progression.
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PMID:Unmasking a growth-promoting effect of the adrenocorticotropic hormone in Y1 mouse adrenocortical tumor cells. 936 63


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