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)

The tumor-promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) caused a time- and dose-dependent morphological change in Y-1 adrenocortical tumor cells. The morphological alteration was apparent 2 hr following addition of 1 microgram/ml TPA to cell cultures and became more striking with longer treatment times. Smaller doses of TPA took a longer time to produce an effect. Cultures grown in the presence of TPA exhibited more rounding and piling up of cells than similar cultures maintained in medium lacking TPA. These TPA-stimulated morphological changes were reversible, and after 24 hr in TPA-free media, the cultured cells began to flatten. After 96 hr in TPA-free media they resembled the control cultures. The reversibility of the morphological change was also dose dependent: cells treated with 1 microgram/ml TPA took a longer time to resume the typical control morphology than did cultures treated with 0.01 microgram/ml TPA. In addition, TPA treatment resulted in a decrease in cell growth rate, an increase in steroid production, and an increase in the localization of free catalytic units of cAMP-dependent protein kinase in the cytoplasm. The steroidogenic effect of ACTH on the cell population was inhibited in cultures maintained in TPA. The results of this study indicate that TPA induces morphological changes in the Y-1 adrenocortical tumor cell population while increasing steroidogenesis and the activation of cAMP-dependent protein kinase and decreasing cell growth rate.
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PMID:Characterization of the morphological, growth, and steroidogenic effect of TPA on mouse Y-1 adrenal cortical tumor cells in culture. 284 97

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

Release of adrenal catecholamine by carbachol has been shown to be coincident with an increase in intracellular cAMP levels. Bovine adrenal medullary (BAM) cells were prepared and maintained in culture and used to examine the role of cAMP in stimulus-secretion coupling. The addition of ACTH to these cells caused a 10- to 50-fold increase in cellular cAMP without an effect on catecholamine secretion, suggesting cortical cell contamination. Percoll density separation of both BAM cells and adrenal cortical cells revealed that the greatest cAMP responses to ACTH corresponded to the catecholamine-containing cell fractions and not to those density layers where cortical cells sedimented. BAM cells isolated on Percoll did not metabolize [14C]cholesterol to steroids as would be expected were the ACTH-stimulated cAMP accumulations due to cortical cell contamination of the cultures. ACTH stimulated protein phosphorylation in 32P-labeled BAM cells in a manner indistinguishable from that induced by carbachol and forskolin. The major soluble phosphoprotein to be affected by these agents had a relative mol wt of 55-57 kdaltons on sodium dodecyl sulfate-gels and corresponded to tyrosine hydroxylase, which is a specific marker enzyme in the adrenal for chromaffin cells. We propose that bovine adrenal chromaffin cells express ACTH receptors which are coupled to adenylate cyclase. While no acute effect of ACTH was found on catecholamine secretion, ACTH may play a direct role in the regulation of catecholamine synthesis by stimulating the phosphorylation of tyrosine hydroxylase by cAMP-dependent protein kinase.
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PMID:Direct effects of adrenocorticotropic hormone on bovine adrenomedullary cells: adenosine 3',5'-monophosphate-dependent phosphorylation of tyrosine hydroxylase. 286 12

The stimulation of steroidogenesis by antimitotic drugs has been studied in wild-type (Y-1) and cAMP-dependent protein kinase-deficient (kin-8) mouse adrenal tumor cell lines. Unlike some other cells, Y-1 cells do not increase their cAMP output upon exposure to antimitotic drugs such as colchicine, vinblastine or podophyllotoxin, which readily increase steroidogenesis. Moreover, no increase in cAMP can be detected over an extended time span. Stabilization of tubulin polymers by taxol or high concentrations of vinblastine blocks ACTH-, cholera toxin- or colchicine-stimulated steroidogenesis without major effects on cAMP levels. Colchicine and podophyllotoxin stimulate steroidogenesis in the cAMP-dependent protein kinase-deficient mutant to the same degree as in the wild-type Y-1 cells, although absolute steroid yields are lower in the mutant cells. We suggest that the antimitotic agents stimulate adrenal steroidogenesis by a cAMP-independent pathway that may involve facilitation of cholesterol access to the mitochondrion.
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PMID:Cyclic AMP-independent stimulation of steroidogenesis in Y-1 adrenal tumor cells by antimitotic agents. 287 35

The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.
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PMID:Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton. 291 25

To define the role of cAMP in the actions of ACTH, the dissociation of cAMP-dependent protein kinase and the subsequent intracellular location of its free catalytic units were monitored after exposure of Y-1 cells to ACTH, FSH, or cyclic nucleotide analog. To accomplish this, a fluorescinated cytochemical probe was used that complexes specifically with free catalytic units from cAMP-dependent protein kinase. Also, the effects of hormone or nucleotide on secretion of fluorogenic steroids and DNA synthesis were examined. Y-1 cells dissociated protein kinase in a dose-dependent fashion when exposed to ACTH or cAMP analog, but did not respond to FSH, which was one of the control agents used. After 30 min of treatment with 1.5 X 10(-10) M ACTH, free catalytic units were observed only in the cytoplasm of Y-1 cells, whereas a similar time of exposure to 3 X 10(-10) M ACTH led to the appearance of catalytic units in nucleolus as well as in cytoplasm. ACTH (6 X 10(-10) M) caused a rise in cytoplasmic and nucleolar protein kinase dissociation proportionally greater than that seen in cultures exposed to 3 X 10(-10) M ACTH. Upon treatment with 6 X 10(-10) M ACTH, the amount of free catalytic units in cytoplasm and nucleolus was detectably greater than that in controls within 1 min of stimulation and continued to rise with increasing time of exposure to hormone. The nuclear, mostly nucleolar, content of free catalytic unit appeared to peak after 15 min of stimulation, while cytoplasmic enzyme levels continued to rise up to 60 min. Exposure of Y-1 cells to nucleotide analog caused cAMP-dependent protein kinase dissociation with temporal kinetics and a subcellular distribution similar to that seen after ACTH stimulation. We conclude that actions of ACTH are mediated by cAMP-dependent protein kinases. Further, there appear to be two intracellular pools of protein kinase, one nucleolar, the other cytoplasmic, and these may be independently regulated, with the nucleolar enzyme requiring higher concentrations of ACTH for dissociation than those needed for cytoplasm protein kinase. These observations may be relevant to the fact that more ACTH is required to inhibit DNA synthesis than is necessary to enhance steroid production.
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PMID:Intracellular kinetics of free catalytic units dissociated from adenosine 3',5'-monophosphate-dependent protein kinase in adrenocortical tumor cells (Y-1). 298 Oct 71

To explore the role of calmodulin (CaM) in lipolysis, studies were carried out on effects of CaM inhibitors on hormone-stimulated lipolysis, the activity of cAMP-dependent protein kinase, and phosphorylation of endogenous substrate proteins. When adipocytes were incubated with trifluoperazine (TFP) and W-7 but not with W-5, stimulation of lipolysis by epinephrine was blunted. W-7 also inhibited lipolysis induced by ACTH, 1-methyl-3-isobutylxanthine (MIX) or (Bu)2 cAMP. The binding of 3H-cAMP to its receptor protein (the regulatory subunit of protein kinase) as well as the activity of cAMP-dependent protein kinase was suppressed by W-7, and the anti-CaM antibody, but not by W-5. The CaM-dependence of the protein kinase was also proved by the fact that the protein kinase activity that was markedly reduced in CaM-depleted cell extracts, was significantly restored by addition of exogenous CaM to them. Furthermore, W-7 decreased cAMP-stimulated phosphorylation of endogenous substrate proteins (mol wt 230k, 200k, 130k, 85k, 75k, and 50kdalton), among which the one of 85kdalton is most likely to be the hormone-sensitive lipase. These findings suggest that CaM is involved in the mechanism of hormone-induced lipolysis by exerting stimulatory effects on the activation of cAMP-dependent protein kinase in cell extracts capable of phosphorylating substrate proteins including hormone-sensitive lipase.
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PMID:The role of calmodulin in hormone-stimulated lipolysis. 298 17

In microsomes of bovine fasciculata reticularis cells incubated with or without 10(-8) M ACTH during 20 min, we measured covalent and non covalent cAMP binding under exchange or non-exchange conditions and cAMP-kinase activity. ACTH induced a decrease in cAMP-kinase activity and in the number of free cAMP binding sites. These results indicate an activation by ACTH of a part of microsomal cAMP-dependent protein kinase. Photoaffinity labeling of microsomal protein with 8-azido-cAMP revealed the presence of both cAMP-kinase isoenzyme I and II in this cellular fraction. Using this method, it was demonstrated that ACTH1-24 caused a preferential and nearly complete activation of microsomal protein kinase I.
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PMID:Activation of microsomal cAMP-dependent protein kinase isoenzyme I by ACTH1-24 in bovine adrenal cells. 299 46

Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF-stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression.
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PMID:Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes. 299 99

Highly purified plasma membranes from Y-1 adrenal tumor cells were incubated with [gamma-32P]ATP with and without cAMP to determine whether endogenous protein substrates are phosphorylated by a cAMP-dependent protein kinase. Three membrane proteins (mol wt 270,000, 35,000, and 17,000) were phosphorylated without cAMP and, to a greater extent, with the nucleotide (0.05-20 microM). Phosphorylation was rapid (less than 60 sec), specific for cAMP, and occurred exclusively at serine residues. Two of the three proteins (35,000 and 17,000) were phosphorylated in whole cells under the influence of cAMP when the cells were incubated with [32P]orthophosphate. The cAMP-dependent protein kinase of these plasma membranes was not extracted by Triton X-100 (0.5% wt/vol) nor by KCl (0.4 M) but was almost completely extracted by the two agents together. By means of photoactivation of 8-azido-[32P]cAMP, it was found that both regulatory subunits RI and RII are present in the membranes. To the extent that the second messenger acts only by way of protein kinase enzymes, these changes in the three proteins are likely to be important in the responses of the plasma membranes of adrenal cells to ACTH.
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PMID:Phosphorylation of three proteins in the plasma membrane of Y-1 adrenal cells by a membrane-bound adenosine 3',5'-monophosphate-dependent protein kinase. 300 63


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