Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat adrenocortical cells in culture respond to stimulation by ACTH alone (15 fold over basal) and to ACTH + added lipoproteins (as an exogeneous source of cholesterol), with an additional 25-30 fold rise in steroidogenesis. With the addition of okadaic acid (OKA, 100 nM), a potent protein phosphatase inhibitor, the lipoprotein-induced rise in steroidogenesis is blocked. If 20 alpha-hydroxycholesterol is provided instead of lipoprotein-cholesterol, OKA has no effect suggesting that OKA affects only actively transported cholesterol. Since the OKA block is preceded by specific morphological changes in the cell (i.e., the loss of Golgi-associated microtubules followed by the disruption of the Golgi apparatus itself), it is hypothesized that some OKA-sensitive phosphoprotein associated with the microtubule/Golgi network of adrenocortical cells is critical for lipoprotein-derived cholesterol uptake and/or transport during steroidogenesis.
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PMID:Okadaic acid interferes with lipoprotein-supported corticosterone production in adrenal cells. 165

We have identified a highly active Ca2+ calmodulin-dependent protein kinase in the cytoskeletons of normal (bovine fasciculata) and transformed (Y-1 mouse tumor) adrenal cells. In view of evidence for the involvement of calmodulin and microfilaments in the regulation of cholesterol transport and hence steroidogenesis, it is likely that this kinase is important in this process. The kinase activity was examined for its capacity to phosphorylate endogenous proteins analyzed by one- and two-dimensional gel electrophoresis, in the presence of saturating amounts of Ca2+ (5 mM) and calmodulin (5 microM). Three inhibitors of calmodulin (trifluoperazine, pimozide and W-7) inhibit steroidogenesis and Ca2(+)-calmodulin-dependent phosphorylation kinase activity with similar values for EC50 for the two processes. All three inhibitors inhibit the increased transport of cholesterol to mitochondria in response to ACTH. Two substrates for the kinase (alpha-spectrin and beta-tubulin) were identified and two others (51,000 and 60,000 molecular weight) were tentatively identified as the subunits of the kinase itself in cytoskeletons of both cell types. Calmodulin-binding proteins analyzed by [125I]iodocalmodulin overlay and calmodulin-Sepharose affinity chromatography were also identified in the same cytoskeletons including alpha-spectrin, the Ca2+ calmodulin-dependent phosphatase calcineurin and three that were tentatively identified as the two subunits of the kinase itself and myosin light chain kinase. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the cytoskeleton and of forming complexes with spectrin, actin and tubulin. These events may be involved in the regulation of the rate-limiting step of steroidogenesis, i.e. transport of cholesterol to mitochondria.
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PMID:Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells. 196 7

The guinea pig adrenal cortex consists of a steroidogenic ACTH-responsive outer zone and an ACTH-unresponsive inner zone. It has been suggested that calmodulin plays an important role in ACTH-stimulated steroidogenesis. Thus, in an effort to examine the calmodulin 'system' in the guinea pig adrenal cortex model, Ca2+-dependent binding of calmodulin to proteins in subcellular fractions of the outer and inner zones was examined by the [125I]iodocalmodulin overlay technique and compared to similar studies utilizing pancreas, brain and liver tissue. Although the general pattern of calmodulin-binding proteins was similar for the two adrenocortical zones, quantitatively there was a striking difference with greater binding in the outer zone; this was particularly noteworthy for the mitochondrial fraction. The two most prominent calmodulin-binding proteins isolated from cytosol by calmodulin-Sepharose column chromatography had Mr of 60,000 and 47,000. The size of these two proteins suggested the presence of Ca2+/calmodulin-dependent protein kinase II. Western blot analysis, however, failed to demonstrate calmodulin kinase II in either zone, although it was clearly detectable in brain cytosol. The 60 K calmodulin-binding protein in the adrenal cortex also suggested the presence of the calmodulin-binding A subunit of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin. Western blot analysis did reveal the presence of calcineurin in the outer adrenocortical zone; it was not detectable, however, in the inner adrenocortical zone. The relation between the striking zonal differential for calmodulin-binding proteins and the zonal differential in ACTH-stimulated steroidogenesis in the guinea pig adrenal cortex will require further investigation.
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PMID:Calmodulin-binding proteins in subcellular fractions of zones of the adrenal cortex. 277 27

Adrenocorticotropin (ACTH) acts via protein kinase A and the putative phosphorylation of a regulatory protein(s). We have examined a role in this process for inhibitor-1 which, following phosphorylation by protein kinase A, inhibits a phosphoprotein phosphatase activity. In the tissues we have examined inhibitor-1 was found primarily in the cytosol (90%) with the rest in the mitochondrial pellet. The highest concentration was in the adrenal cortex. Using adrenal cortex slices, the stimulation of steroidogenesis by ACTH and dibutyryl cAMP is paralleled by a corresponding increase in the phosphorylation of inhibitor-1 and this is not affected by inhibitors of protein synthesis which inhibit the steroidogenic response. The increase in the phosphorylation of inhibitor-1 occurs in the cytosol, while that in the mitochondrial pellet is not affected. Exogenous phosphorylated inhibitor-1, however, was found to inhibit phosphoprotein phosphatase activity in the mitochondrial pellet. The results suggest that the ACTH-induced increase in phosphorylated inhibitor-1 in the cytosol can affect susceptible phosphoprotein phosphatase activity both in the cytosol and the mitochondrial pellet and, hence, the level of phosphorylation of regulatory protein(s) involved in steroidogenesis.
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PMID:The stimulation by adrenocorticotropin of the phosphorylation of adrenal inhibitor-1: a possible role in steroidogenesis. 285 Sep 49

Inhibition of the phosphorylation of the synaptic plasma membrane (SPM) protein B50 by [D-Trp8]-somatostatin in vitro is time-dependent. Increasing the time of incubation of hippocampal synaptic plasma membranes with the peptide from 15 sec to 30 min prior to addition of 7.5 microM [gamma-32P]ATP results in a complete reduction of B50 phosphorylation. Incubation of synaptic plasma membranes for 30 min in the absence of peptide does not alter basal B50 phosphorylation. Neither ACTH nor beta-endorphin produces similar effects--inhibition of B50 phosphorylation by ACTH is maximal at 15 sec and beta-endorphin produces only a small inhibition, even after 30 min. [D-Trp8]-somatostatin is not activating a membrane-bound protease, since maximal inhibition of B50 phosphorylation by the peptide is seen in the presence of leupeptin or bacitracin. Hippocampal synaptic plasma membranes contain protein phosphatase activity. Assays of B50 phosphorylation in synaptic plasma membranes done under conditions that favor either net phosphorylation or dephosphorylation are consistent with inhibition of protein phosphatase activity by [D-Trp8]-somatostatin. This evidence suggests that [D-Trp8]-somatostatin interacts with SPM binding sites in the hippocampus, which may alter the activity of an endogenous protein phosphatase to determine the degree of B50 phosphorylation.
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PMID:Characteristics of [D-Trp8]-somatostatin-sensitive B50 phosphorylation. 287 46

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.
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PMID:Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase. 300 26

Cholesterol metabolism and steroidogenesis in the outer (zona fasciculata/glomerulosa) and inner (zona reticularis) zones of the adrenal cortex were examined in the guinea pig. It is known from previous studies that the content of cholesterol in the inner zone is considerably lower than that in the outer zone, although basal low density lipoprotein (LDL) receptor activity is similar in the two zones. To further explore cholesterol metabolism in the guinea pig adrenal cortex, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol synthesis, has been examined for which this paper forms the initial report. It was found that the basal specific activity of HMG-CoA reductase was similar in the outer and inner adrenocortical zones (approximately 230 pmol mevalonate formed/min X mg microsomal protein). The administration of ACTH caused 4- and 5-fold increases in HMG-CoA reductase activity in the outer and inner zones, respectively. In fact, the increase in HMG-CoA reductase activity with ACTH treatment was always greater for the inner zone than for the outer zone. This is in contrast to LDL receptor activity, which does not increase in the inner zone as it does in the outer zone with ACTH treatment. When dexamethasone was administered, HMG-CoA reductase activity decreased in the outer zone by about 50%, while there was no change in reductase activity in the inner zone. The latter finding is similar to what happens with LDL receptor activity during dexamethasone administration. Why suppression of endogenous ACTH had no effect on HMG-CoA reductase activity in the inner zone while exogenous ACTH administration caused a marked increase in enzyme activity is not clear, but may be related to phosphorylation/dephosphorylation mechanisms. Based on the use of sodium fluoride in solutions to block HMG-CoA reductase phosphatase, evidence is presented which indicates that a pharmacological dose of ACTH alters the phosphorylation/dephosphorylation status of HMG-CoA reductase in the inner adrenocortical zone, but not in the outer cortical zone.
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PMID:Differential activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in zones of the adrenal cortex. 302 27

Adrenal cortical mitochondria contain a mixed function oxidase capable of converting cholesterol to pregnenolone; this enzyme requires NADPH, oxygen and cholesterol. This cholesterol side chain cleavage enzyme system contains a Flavoprotein, an iron sulphur protein and a specific cytochrome P450 termed cytochrome P450scc. ACTH stimulates the adrenal cortex by activating adenyl cyclase producing an elevated intracellular concentration of cAMP. This in turn increases the activity of a cytosolic cAMP dependent protein kinase. Adrenal cortical cytosol contains a cholesterol ester hydrolase which is activated by ATP and a protein kinase. This enzyme may be deactivated by a phosphoprotein phosphatase. The adrenal cortex contains lipid droplets that are rich in esterified cholesterol. Cholesterol ester hydrolase can release free cholesterol from the lipid droplets. The free cholesterol released may be used to supplement the mitochondrial cholesterol as a pregnenolone precursor. Steroid hormone production by the adrenal cortex exhibits a diurnal rhythm and correlates with the activity of the cytosolic cholesterol ester hydrolase. The acute steroidogenic response to ACTH may be in part attributed to the availability of free cholesterol to the mitochondrial cholesterol side chain cleavage enzyme complex. The intracellular movement of free cholesterol from lipid droplets to mitochondrial inner membranes may be impeded by protein synthesis inhibitors such as cycloheximide. The precise mechanism of this block in steroidogenesis remains to be elucidated. Various drugs and oestrogenic hormones suppress the plasma and adrenal cholesterol concentrations. If adrenal cells are deficient in cholesterol, these cells exhibit a diminished response to ACTH. The response to this hormone can be corrected by supplying cholesterol via exogenous plasma lipoproteins. The route that free cholesterol follows within the adrenal cortical cell and the physiological factors influencing free cholesterol movement in such cells are important issues to be explored in future.
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PMID:Cholesterol metabolism in the adrenal cortex. 631 Feb 52

Angiotensin II (AII) receptors are known to interact with two distinct guanine nucleotide binding proteins, Gq/11 and Gi, in rat adrenal glomerulosa cells to activate phospholipase C and to inhibit adenylate cyclase, respectively. However, in cultured bovine glomerulosa cells AII potentiates rather than inhibits the stimulatory effect of adrenocorticotropin (ACTH) on cAMP levels. This effect of AII was partially mimicked by phorbol 12-myristate 13-acetate (PMA) and was partially inhibited by staurosporine or depletion of protein kinase C but was unaffected by pertussis toxin treatment. No potentiation was detectable in disrupted cells or in membrane preparations. In intact glomerulosa cells, treatment with cyclosporin A or FK506 completely inhibited AII- or PMA-induced potentiation of cAMP production without affecting the response to ACTH. In COS-7 cells transfected with the rat AT1 receptor, AII caused 2-3-fold enhancement of the ACTH-induced cAMP response, an effect that was partially reproduced by PMA. These potentiating actions of AII and PMA were prevented by preincubation with cyclosporin A or FK506, and the latter effect was abolished by rapamycin. These results implicate the Ca2+- and calmodulin-dependent protein phosphatase, calcineurin, in AII-induced enhancement of adenylate cyclase activity in both adrenal glomerulosa and transfected COS-7 cells. The finding that AII enhances ACTH-stimulated production of cAMP by a second messenger-mediated mechanism that involves the participation of calcineurin reveals an additional mode of cross-talk between pathways activated by Ca(2+)-mobilizing and cAMP-generating receptors.
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PMID:Evidence for participation of calcineurin in potentiation of agonist-stimulated cyclic AMP formation by the calcium-mobilizing hormone, angiotensin II. 792 24

The steady state level of most cellular phosphoproteins is dependent on the relative catalytic activities of intracellular protein kinases and phosphatases. In adrenal cortex, ACTH acts through PKA activation and Ser/Tre phosphorylation. Phosphatases involved in this pathway are not completely described, particularly the role of phosphotyrosine protein phosphatase (PTP) activity on ACTH action. We investigated potential changes in PTPs activity in adrenal gland upon in vivo and in vitro PKA activation. In vivo ACTH stimulates cytosolic PTP activity (2-fold). Similar effect is detected by in vitro stimulation. In accordance with the effects of ACTH on PTP activity, cell permeable PTP inhibitors block ACTH stimulation on adrenal zona fasciculata (ZF) cells: ACTH (1 nM) = 108.2 +/- 3.5 ng corticosterone/10(5) cells vs. ACTH + phenylarsine oxide (2 nM) = 60 +/- 4 (P < 0.001) and ACTH + pervanadate (10 mM) = 68 +/- 2 (P < 0.01). These results are reproduced when cells are stimulated with cAMP. The inhibition is not observed when steroidogenesis is supported by 22(R)OH cholesterol. We describe, for the first time, a hormonal regulation of PTP activity. According to the effect of PTP inhibitors on steroid production activated by ACTH we propose that PTP activation is a crucial event in hormone action in the steroidogenic pathway. We also propose that PTP activity is located after PKA activation and prior to cholesterol transport to the inner mitochondrial membrane.
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PMID:Phosphotyrosine protein phosphatases activation by ACTH in rat adrenal gland. 988 10


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