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 reported previously that expression of the human apolipoprotein E (apoE) gene in mouse Y1 adrenocortical cells suppresses basal and adrenocorticotropin (ACTH)-stimulated steroidogenesis. To understand the mechanism of this suppression, we have examined the integrity of cAMP regulated events required for adrenal steroidogenesis. Both acute and chronic responses to ACTH or cAMP are suppressed in Y1 cells which express apoE (Y1-E cells) as compared with parental Y1 cells. Acute morphologic changes in response to cAMP and acute induction of steroidogenesis by cAMP are suppressed in the Y1-E cell lines. Constitutive expression of P450-cholesterol side chain cleavage enzyme mRNA, the rate-limiting enzyme in steroid hormone synthesis, is reduced up to 11-fold in the Y1-E cell lines. The level of mRNA encoding P450-cholesterol side chain cleavage correlates directly with the reduction in basal steroid production observed in the individual Y1-E cell lines. Expression of P450-11 beta-hydroxylase mRNA, although readily detectable in Y1 parent cells, is absent or reduced in the Y1-E cell lines. Inhibition of cAMP-regulated gene expression is not restricted to genes required for steroid synthesis, since cAMP induction of ornithine decarboxylase mRNA is also inhibited in the Y1-E cell lines. These data indicate that suppression of steroidogenesis in Y1-E cells is due, at least in part, to inhibition of cAMP-regulated gene expression. These effects are not due to a defective cAMP-dependent protein kinase, since kinase activity in vitro and activation in vivo are unaltered in the Y1-E cell lines. These results suggest that expression of apoE in Y1 cells blocks cAMP-mediated signal transduction at a point distal to activation of cAMP-dependent protein kinase.
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PMID:Suppression of cAMP-mediated signal transduction in mouse adrenocortical cells which express apolipoprotein E. 165 49

Inhibin subunit expression has recently been shown to occur in rat and sheep adrenals. We now show the presence of inhibin subunit mRNAs in human fetal and adult adrenal tissue specimens and cultured adrenocortical cells. Northern blot analysis revealed that inhibin alpha-subunit gene is as strongly expressed in fetal adrenals as in fetal testes, whereas adult adrenals expressed alpha-subunit mRNA to a lesser extent. beta A-Subunit mRNA was detectable in placenta, and beta B mRNA was found in testes. With reverse transcription-polymerase chain reaction analysis all inhibin subunit mRNAs (alpha, beta A, and beta B) could be found in fetal adrenal samples. In cultured fetal and adult adrenal cells ACTH and dibutyryl cAMP increased inhibin alpha-subunit mRNA 3- to 4-fold. beta A mRNA was spontaneously induced in cultured adrenal cells. 12-O-Tetradecanoyl phorbol-13-acetate, a protein kinase-C regulator, increased beta A mRNA levels 9.6- and 3.3-fold in fetal and adult adrenal cultures, respectively. 12-O-Tetradecanoyl phorbol-13-acetate treatment abolished ACTH-induced alpha-subunit mRNA accumulation in both fetal and adult cultures. Our results show that inhibin genes are expressed in human fetal adrenals and testes during the second trimester of gestation. Adult adrenals also express inhibin genes, although to a lesser extent than fetal adrenals. Both cAMP- and protein kinase-C-dependent pathways regulate inhibin subunit gene expression in adrenocortical cells. These findings suggest that inhibins/activins are produced locally in human adrenals, where they could function as paracrine or autocrine regulators of adrenal growth and steroidogenesis.
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PMID:Hormonally regulated inhibin gene expression in human fetal and adult adrenals. 165 31

We have investigated the ability of amino acid analogues of serine and threonine to inhibit the increase in steroidogenesis elicited by addition of ACTH or cAMP in cells isolated from the rat adrenal cortex. We have found that the serine analogues, D, L-isoserine, alpha-methyl-D, L-serine and L-homoserine, are almost totally ineffective in inhibiting this process but that the threonine analogue, D, L-beta-hydroxynorvaline, at a concentration of 300 microM inhibits stimulated steroid hormone biosynthesis by ca 95%, while inhibiting overall protein synthesis by only ca 40%. This inhibition was found to occur in a dose-dependent manner and to be reversible by a stoichiometric concentration of threonine. These studies suggest that beta-hydroxynorvaline is functioning as a threonine analogue in our experimental system. Both the onset of inhibition by analogue and reversal of this inhibition by the natural amino acid occurred rapidly, without detectable lag. Since results obtained using cAMP as stimulant parallel those obtained using ACTH, the inhibitory effect of the analogue seems to occur subsequent to the synthesis of cAMP. Additionally, the analogue does not inhibit the conversion of pregnenolone to corticosterone, suggesting the site of action of analogue occurs prior to the synthesis of pregnenolone from cholesterol. Thus, the analogue may be exerting its effect on a protein that is synthesized subsequent to ACTH addition and is important in the acute phase of stimulated steroid hormone biosynthesis. Further, since ACTH action on adrenal cortex cells causes the activation of protein kinase A, which phosphorylates serine and threonine residues, it is possible that the effect of the analogue is to prevent the phosphorylation of a newly-synthesized protein.
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PMID:Inhibition of steroidogenesis in rat adrenal cortex cells by a threonine analogue. 165 80

The mitochondria, the microsomes and the cytosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.
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PMID:The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis. 166 Nov 27

CRF is a potent hypophysiotropic factor which stimulates POMC-producing cells in both the intermediate and anterior pituitary. Although its secretagogue effects and its stimulatory action on POMC gene expression are well documented, the mechanisms by which CRF modulates gene regulation are poorly understood. In this study we have investigated the mechanisms by which CRF stimulates the immediate early gene c-fos. Studies were performed in the corticotroph-derived AtT20 cell line. We show that CRF induces a transient increase in c-fos mRNA levels. This induction is reduced by blockade of calcium entry and by calmodulin inhibitors, suggesting that the CRF-induced c-fos increase is mediated in part by the second messenger Ca2+ and the Ca2+/calmodulin kinase. When protein kinase-A (PKA) was inhibited by introduction of a mutated regulatory subunit of PKA that lacks cAMP-binding sites, the stimulation of c-fos mRNA by CRF was abolished. Taken together, these results suggest that CRF activates the c-fos protooncogene via PKA and the Ca2+/calmodulin kinase. These results were confirmed and extended by gene transfer studies using chimera genes containing c-fos promoter sequences coupled to the chloramphenicol acetyl transferase reporter gene. This series of experiments shows that CRF stimulates c-fos transcription by mechanisms requiring PKA activation. Furthermore, cotransfection experiments with the POMC promoter linked to the chloramphenicol acetyl transferase reporter gene along with an expression vector coding for cFOS showed efficient stimulation of POMC gene transcription by cFOS. In summary, c-fos mRNA accumulation is an early genomic signal in pituitary cells in response to CRF, and cFOS may represent a signal controlling POMC gene expression.
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PMID:The protooncogene c-fos is induced by corticotropin-releasing factor and stimulates proopiomelanocortin gene transcription in pituitary cells. 166 13

We have used microspectrofluorometry and video imaging techniques in order to study and compare the changes in intracellular calcium concentrations [( Ca2+]i) of individual Fura-2 loaded glomerulosa cells cultured for three days and stimulated either with angiotensin II (AT), K+, or adrenocorticotropin (ACTH). As previously demonstrated for freshly isolated cells, K+ ion induces an immediate increase in [Ca2+]i, although AT induces a biphasic response, characterized by an initial transient spike, followed by a sustained plateau. In this study, we demonstrate, for the first time, that ACTH is able to induce a [Ca2+]i increase in cultured glomerulosa cells from rat and bovine sources. Moreover, it is clear that the pattern of [Ca2+]i increase elicited by ACTH is different from that observed with AT. In most cases, addition of ACTH leads to a slow increase in [Ca2+]i after a long latency period ranging from 10-15 min, which could be correlated to cAMP time-production. The present results show that: (a) in the absence of extracellular Ca2+, ACTH does not increase [Ca2+]i; (b) the response develops slowly and cases immediately after [Ca2+]e depletion or addition of calcium channel blockers, such as nifedipine or omega-conotoxin; (c) the addition of the calcium channel agonist Bay K 8644 enhances the ACTH response; (d) the cAMP analog, 8-Br-cAMP, induces an increase in [Ca2+]i similar to that observed with ACTH, which is also dependent of the presence of calcium in the extracellular medium; (e) time-production of ACTH-induced cAMP follows quite well the increase in [Ca2+]i; (f) Bay K 8644 also enhances the 8-Br-cAMP induced increase in [Ca2+]i; and (g) ACTH-induced Cai response is inhibited by the specific protein kinase A blocker, HA1004. These observations, combined with previous results obtained on the effects of ACTH on calcium currents and action potentials, suggest that the [Ca2+]i increase induced by ACTH results from a calcium influx through dihydropyridine and omega-conotoxin sensitive calcium channels, which need to be phosphorylated by cAMP for full activation. The use of video-imaging techniques has allowed us to examine the spatial distribution of changes in [Ca2+]i in single cells. The ability to simultaneously record images of a number of cells confirm the heterogeneity of cellular responses, and corroborate results obtained through photocounting only. Our results indicate that ACTH initially increases [Ca2+]i locally beneath the cell membrane and throughout the cell thereafter, whereas angiotensin II elicits a more prominent effect in certain regions of the cell and eventually extends to the entire cell surface.
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PMID:Effects of ACTH and angiotensin II on cytosolic calcium in cultured adrenal glomerulosa cells. Role of cAMP production in the ACTH effect. 172 36

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

Arginine vasopressin (AVP), oxytocin (OT), and angiotensin-II (AII) elicit a biphasic ACTH secretory response by perifused anterior pituitary cells consisting of an initial transient (less than 3-min) spike phase and a subsequent sustained plateau phase. In contrast, CRF produces a monophasic sustained plateau type of ACTH secretory response. We have previously demonstrated that 1) influx of extracellular Ca2+ (Cae2+) via L-type voltage-sensitive Ca2+ channels is involved in both the response to CRF and the sustained phase of the response to AVP and OT; 2) release of intracellular Ca2+ (Cai2+) is involved in the spike phase of the response to AVP, OT, and AII; and 3) activation of protein kinase-C is required for the sustained phase, but not for the spike phase, of the response to AVP. CRF action is mediated by activation of protein kinase-A. In this study we further examined the role of Cai2+ by exploiting the fact that a low concentration (1 microM) of ionomycin, a potent Ca2+ ionophore, releases Cai2+ from nonmitochondrial inositol-1,4,5-trisphosphate (IP3)-sensitive Cai2+ stores without causing Cae2+ influx. Pretreatment with ionomycin for 10 min decreased the spike phase of the response to 100 nM AVP, OT, and AII, but had no effect on the response to 10 nM CRF or the sustained phase of the responses to the other agonists. The combination of CRF plus AVP induced a biphasic and synergistic release of ACTH. Ionomycin pretreatment reduced the spike phase, especially the first 1 min, without any effect on the sustained phase. These results indicate that Cai2+ release, but not Cae2+ influx, is involved in the spike phase of the response to AVP, OT, and AII and that Cai2+ is not involved in the synergistic effect of the combination of CRF plus AVP. Having established these relationships, we examined the effect of 2-h perifusion with 100 nM dexamethasone (DEX) on stimulated ACTH release. DEX pretreatment reduced the total response to CRF, the sustained phase of the responses to AVP and OT, and the sustained phase of the synergistic response to CRF plus AVP. However, DEX had no effect on the spike phase of the responses to AVP, OT, or AII or the spike phase of the response to CRF plus AVP. These results indicate that DEX inhibits ACTH release mediated by activation of either protein kinase-A or protein kinase-C, but does not affect inositol-1,4,5-trisphosphate/Cai2(+)-mediated ACTH release.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of intracellular Ca2+ depletion and glucocorticoid on stimulated adrenocorticotropin release by rat anterior pituitary cells in a microperifusion system. 184 62

We have examined the distribution and colocalization of protein kinase-C (PKC) in cultured rat anterior pituitary cells by light microscopic immunocytochemistry using monoclonal antibodies to group A rat brain PKC subspecies. Type I (PKC gamma) was not detected in the cells, in line with the assertion that the gamma-enzyme is expressed specifically in central nervous tissues. The other subspecies recognized by the antibodies (PKC beta and PKC alpha) were present throughout the cytoplasm in a diffused pattern, while the nuclei were apparently unstained. The number of cells stained with the antibodies in juvenile animals (12 days old) increased rapidly with age and reached a plateau between adult (5-month-old) and older (1-yr-old) rats. Type II (PKC beta) was the predominant subspecies detected in anterior pituitary cells. Double immunofluorescence staining techniques enabled the colocalization of PKC with various anterior pituitary cell types. Surprisingly, not all of the hormone-producing cells were stained with the PKC antibodies. Moreover, within the different pituitary cell types, the percentage of PKC-stained cells varied, revealing heterogeneity among the various cell populations. Thus, among somatrophs, mammotrophs, thyrotrophs, ACTH-containing cells, and gonadotrophs, only 9%, 22%, 13%, 44%, and 26%, respectively, reacted with the PKC antibodies. We suggest that activation of pituitary PKC might mobilize only a fraction of the hormone-containing cells.
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PMID:Immunocytochemical localization of protein kinase-C subtypes in anterior pituitary cells: colocalization in hormone-containing cells reveals heterogeneity. 193 8

The ubiquitous casein kinase II (CKII) has been shown to accumulate in the cell nuclear compartment, following exposure to extracellular growth stimuli (O. Filhol et al., Biochemistry, 1990, 29, 9928-9936). The aim of the present study was to examine whether intracellular polyamines, whose levels are increased under similar conditions, could be related to this process. It is shown that (i) CKII accumulates in nuclei of adrenocortical cells exposed to their trophic hormone ACTH; (ii) this CKII nuclear translocation is concomitant with an increase in nuclear polyamine content resulting from ACTH-induced polyamine synthesis; (iii) selective inhibition of polyamine biosynthesis by DFMO results in the inhibition of both ACTH-induced cellular polyamine increase and CKII nuclear accumulation. These observations suggest that polyamines may be examined as intracellular messengers in the regulation of CKII activity and subcellular distribution in the cell response to growth factors and trophic hormones.
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PMID:Casein kinase II and polyamines may interact in the response of adrenocortical cells to their trophic hormone. 195 32


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