Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C alpha and C beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C alpha or C beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.
 ...
PMID:Cyclic AMP-dependent protein kinase controls basal gene activity and steroidogenesis in Y1 adrenal tumor cells. 156 25

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.
 ...
PMID:Suppression of cAMP-mediated signal transduction in mouse adrenocortical cells which express apolipoprotein E. 165 49

In our previous report we showed cytochrome b5 to be a competitive inhibitor of cAMP-dependent protein kinase (PKA) for interaction with cytochrome P450 (P450). While P450 was phosphorylated, cytochrome b5 was not. The phosphorylation of P450 resulted in an inhibition of its catalytic activity. In this report we attempt to determine the relationship between phosphorylation of P450 from phenobarbital-induced rat and its destruction. The results indicate there is a considerable alteration of P450 IIB1 when it is put into the phosphorylation medium. This includes destruction, i.e., loss of the hemoprotein nature (Soret peak), as well as denaturation, conversion of a proportion of the P450 to P420. The extent of phosphorylation correlated best with the amount of destroyed hemoprotein, and not with the formation of P420. There did not appear to be phosphorylation-dependent formation of apo-P450. Further, prior conversion of the P450 to P420 using sodium deoxycholate showed the same extent of phosphorylation as before the conversion. Thus, intact P450 is not required for phosphorylation nor is phosphorylation a prerequisite for hemoprotein destruction. P450 CAM (CIA1), which has the PKA substrate recognition sequence internalized, likewise undergoes conversion to P420 but this denaturation does not result in phosphorylation. Destruction of CIA1 with 6 M urea, however, did permit phosphorylation by PKA. P450 IIB1 destruction was greatly diminished by cytochrome b5. This stabilization resulted in a decreased degree of phosphorylation as well as an increase in negative ellipticity in circular dichroism, indicative of an increase in the proportion of alpha-helical content in the P450. Suggestions are made that this structural modification caused by cytochrome b5 stabilizes the P450 against denaturation as well as against destruction and phosphorylation. Further, when the P450 IIB1 was kept stable as P450 in the absence of cytochrome b5 and without loss of hemoprotein during the incubation period, using phosphate-glycerol buffer containing 0.4% Emulgen 911, the phosphorylation of the P450 was greatly diminished, with only minor effects on the protein kinase reaction itself. These results suggest that the protein kinase reaction itself. These results suggest that the protein kinase substrate recognition sequence is not readily accessible to PKA in mammalian P450 IIB1 but requires a destabilization of the protein for phosphorylation to take place.
 ...
PMID:Relationship between phosphorylation and cytochrome P450 destruction. 227 44

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.
 ...
PMID:Phosphorylation of cytochrome P450: regulation by cytochrome b5. 254 94

P450c17 is a single microsomal enzyme that catalyzes two distinct steroid biosynthetic activities: 17 alpha-hydroxylase and 17,20 lyase. Human beings have only one gene that encodes only one form of P450c17. Three clinical observations indicated that these were independently regulated activities. First, several cases of isolated 17,20 lyase deficiency were reported, in which 17 alpha-hydroxylase activity was spared. Second, most adrenal steroidogenesis in children stops after 17 alpha-hydroxylation, thus permitting the synthesis of cortisol, whereas most gonadal steroidogenesis proceeds to C19 sex steroids as a result of both activities. Third, the 17,20 lyase activity of the human adrenal is developmentally activated during adrenarche. To catalyze these two activities, P450c17 must receive reducing equivalents from electron donors (redox partners). Previous observations showed that the molar ratio of P450 oxidoreductase to P450c17 was 3-fold higher in the testis than in the adrenal, and that increasing the molar ratio of the redox partner to P450c17 would increase the ratio of 17,20 lyase activity to 17 alpha-hydroxylase. We have recently shown that P450c17 must be phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase to acquire 17,20 lyase activity. We have also recently found two cases of isolated 17,20 lyase deficiency that have mutations of residues in the proposed redox partner binding site. Together, these studies suggest a unified view of the regulation of 17,20 lyase activity. The ratio of 17,20 lyase to 17 alpha-hydroxylase activity of P450c17 is regulated by the availability of reducing equivalents flowing to the enzyme. This can be increased by increasing the molar concentration of electron-donating redox partners, such as P450 oxidoreductase or possibly cytochrome b5, as appears to be the case in the gonads. Alternatively, the affinity of P450c17 for redox partners may be selectively increased by Ser/Thr phosphorylation, or selectively decreased by certain mutations in the redox partner binding site, in either case altering an electrostatic interaction between P450c17 and the redox partner. This model is consistent with all present observations about the biochemistry, genetics, enzymology, and clinical phenomenology of P450c17.
 ...
PMID:The regulation of 17,20 lyase activity. 902 28

Our laboratory has proposed that phenobarbital (PB), a typical lipophilic agent that induces some members of the supergene family of liver microsomal cytochromes P450 (e.g., CYP2B1/2 and CYP3A23), acts through a complex process inhibitable by the presence of growth hormone (GH), the absence of some components of the extracellular matrix, or a disrupted cytoskeleton. To verify that these manipulations of the culture environment block specific steps in the PB induction pathway rather than simply exerting nonspecific or toxic effects on CYP2B1/2 gene transcription, we have now examined PB induction of CYP3A23, a gene known to also be transcriptionally activated by dexamethasone (DEX) through a "nonclassical" pathway apparently involving the glucocorticoid receptor. We found that in primary cultures of adult rat hepatocytes treated with PB, induction of CYP3A23 mRNA, just as we reported for induction of CYP2B1/2 mRNA, required the use of Matrigel (a reconstituted basement membrane) and was blocked by the presence of cytoskeletal inhibitors (colchicine or cytochalasins) or of physiologic concentrations of GH in the culture medium. Moreover, PB induction of CYP3A23 and of CYP2B1/2 mRNAs was greatly diminished by inhibitors of cAMP-dependent protein kinase (PKA). In striking contrast, induction of CYP3A23 mRNA by DEX was unaffected by any of these alterations of the culture conditions that block its induction by PB. We conclude that the effects of extracellular matrix, GH, disruption of the cytoskeleton, and activation of cAMP-dependent protein kinase, pharmacologically define multiple, pretranscriptional steps in the pathway(s) for PB induction of liver cytochromes P450.
 ...
PMID:Characterization of a pretranscriptional pathway for induction by phenobarbital of cytochrome P450 3A23 in primary cultures of adult rat hepatocytes. 918 22

Insect molting hormone (ecdysteroid) inactivation occurs by several routes, including 26-hydroxylation and further oxidation to the 26-oic acids. Thus, the ecdysteroid 26-hydroxylase is a critical enzyme involved in precise regulation of ecdysteroid titers during insect development. Administration of the ecdysteroid agonist, RH-5849 (1,2-dibenzoyl, 1-tert-butyl hydrazone), or 20-hydroxyecdysone to the tobacco hornworm, Manduca sexta, results in induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes. The biochemical and kinetic properties of the ecdysteroid 26-hydroxylase were investigated. The mitochondrial enzyme was found to have optimal activity at a pH of 7. 5 in a Hepes or sodium phosphate buffer at 30-37 degrees C. The apparent K(m) of the microsomal 26-hydroxylase for 20-hydroxyecdysone substrate was lower than that of the mitochondrial enzyme for either 20-hydroxyecdysone or ecdysone substrate. The V(max) of the 26-hydroxylase in both subcellular fractions was slightly higher using 20-hydroxyecdysone as substrate compared to ecdysone. Demonstration that activity of the mitochondrial 26-hydroxylase was inhibited by incubation in a CO (or N(2)) atmosphere, taken together with the requirement for reducing cofactor and the efficacy of the P450 inhibitors, ketoconazole and fenarimol, provided strong evidence that the hydroxylase is cytochrome P450-dependent. Indirect evidence suggested that the mitochondrial and microsomal ecdysteroid 26-hydroxylase(s) could exist in a less active dephosphorylated state or more active phosphorylated state. Using Escherichia coli alkaline phosphatase to remove covalently bound phosphate groups, the activity of the 26-hydroxylase was decreased and, conversely, activity was enhanced using a cAMP-dependent protein kinase with appropriate cofactors. In addition, the protein kinase was shown to reactivate the 26-hydroxylase activity in alkaline phosphatase-treated fractions.
 ...
PMID:Characterization of ecdysteroid 26-hydroxylase: an enzyme involved in molting hormone inactivation. 1077 27

In this study, primary serum-free cultured rat granulosa cells (rGCs) were used as a cellular model to investigate the effects of fenvalerate on progesterone production. Various concentrations (0, 1, 5, 25, 125 and 625 microM) of fenvalerate were added to the cell cultures for 24 h. rGCs were stimulated by compounds such as follicle-stimulating hormone (FSH), 8-bromo-cAMP or 22(R)-hydroxycholesterol (22R-HC). Progesterone production and intracellular cAMP content were measured in control and treated groups. Expression of P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR) were monitored by real-time PCR and Western blotting. Results showed that fenvalerate inhibited basal progesterone production in rGCs in the absence of stimulators. This inhibition was stronger in the presence of FSH and was not fully reversed by 8-bromo-cAMP or 22R-HC. The increase of cAMP content, stimulated by FSH, was inhibited by fenvalerate implicating that the intracellular cAMP-dependent signal pathway was involved. Fenvalerate reduced mRNA and protein expression of P450scc. These results suggested that multi-site inhibition of progesterone production by fenvalerate including a cAMP-dependent protein kinase pathway and reduction on P450scc gene expression and/or its enzymatic activity in rGCs.
 ...
PMID:Effects of fenvalerate on progesterone production in cultured rat granulosa cells. 1590 53

Short-term nutritional supplementation stimulates folliculogenesis in ewes probably by insulin-mediated actions of glucose in the follicle. The aim of this study was to determine the effect of glucose on follicle number and granulosa levels of Aromatase P450 and phosphorylated Akt and AMPK. Twelve Ile-de-France ewes were allocated to two groups; one (n=7) infused with saline and the other (n=5) with glucose (10mM/h) for 72h in the luteal phase. At the end of infusion, ovaries were collected and all follicles >1mm in diameter were dissected to recover granulosa cells. Aromatase P450 and phosphorylated Akt and AMPK were analysed by Western blotting of granulosa cell lysates. Blood plasmas collected before and during the infusions were analysed for progesterone, oestradiol, LH, FSH, glucose, insulin and IGF-I. The infusion of glucose significantly increased follicle number but, significantly reduced Aromatase P450 and phosphorylated Akt and AMPK in granulosa cells. The circulating concentration of glucose rose significantly 3h after the start of the glucose infusion and remained elevated until 27h then fell; the circulating concentration of insulin rose significantly by 3h and remained elevated. The circulating concentration of oestradiol fell significantly by 32h and remained low; the circulating concentrations of LH and FSH were unaffected. These data show that short-term infusion of glucose stimulated follicular growth but decreased Aromatase P450 in granulosa cells. The reduced levels of phosphorylated Akt and AMPK suggest that the phosphatidylinositol 3-kinase pathway has been inhibited by high concentrations of glucose. These data also suggest that there may be functional cross-talk between FSH and insulin signalling in granulosa cells.
 ...
PMID:The infusion of glucose in ewes during the luteal phase increases the number of follicles but reduces oestradiol production and some correlates of metabolic function in the large follicles. 2194 3