Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.
Mol Cell Biol 1999 Jul
PMID:Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase. 1037 49

Exposure of Saccharomyces cerevisiae to elevated concentrations of hydrogen peroxide induces transcription of several genes involved in the oxidative stress response. Two major transcription factors are involved in this induction, Pos9/Skn7 and Yap1. Fusions of either Yap1 or Pos9/Skn7 with the Gal4 DNA binding domain are active as transcription factors. Gal4-Yap1-dependent reporter gene activity is only weakly regulated by oxidative stress. In contrast, fusion of the Gal4 DNA binding domain to the Pos9/Skn7 protein results in a transcription factor that is independent of the YAP1 gene and is strictly regulated by oxidative stress, indicating that a signaling cascade impinges on the Pos9/Skn7 protein. We have observed that the Ras/PKA (cAMP-dependent protein kinase A) pathway affects this signaling. When PKA activity was low (in the presence of multicopy PDE2 or a cyr1(D822-->A) mutation) maximum reporter gene activity was observed even in the absence of oxidative stress. In contrast, high PKA activity (in strains mutant for either pde2 or bcy1, or expressing the dominant active Ras2Val19) resulted in a complete loss of activation following oxidative stress. The transcription of Pos9/Skn7 target genes was also affected in Ras/PKA pathway mutants. Furthermore, we demonstrated that activated Pos9/Skn7 is necessary for Yap1-dependent reporter gene induction.
Mol Gen Genet 1999 Jun
PMID:The oxidative stress response mediated via Pos9/Skn7 is negatively regulated by the Ras/PKA pathway in Saccharomyces cerevisiae. 1039 11

cAMP initiates the PKA signaling cascade in rat pheochromocytoma PC12 cells, resulting in transcriptional activation of the tyrosine hydroxylase (TH) gene. This effect is mediated primarily through the cAMP responsive element (CRE), located at position -45 to -38 within the TH gene promoter. In this study, we applied an antisense RNA strategy to evaluate the role of the cAMP responsive element binding protein (CREB) in regulating TH gene expression. CREB antisense RNA expression vectors were stably introduced into PC12 cells to generate cell lines deficient in CREB. CREB protein and mRNA levels were diminished up to 90% in the stably transfected cell lines. Promoter analysis experiments demonstrated that cAMP-mediated inducibility of either TH gene proximal promoter activity or the activity of the TH CRE by itself fused upstream of a basal promoter was diminished in CREB-deficient cell lines. PKA activity in the CREB-deficient cell lines was comparable to the activity in control cell lines. In addition, neither ATF1, nor CREM proteins were significantly down-regulated in the CREB-deficient cells. Most significantly, the cAMP-inducibility of endogenous TH mRNA was completely blocked in the CREB-deficient cells, indicating that the response of the endogenous gene to cAMP was dependent on CREB. These results support the hypothesis that CREB (not other CRE-binding proteins) is the key transcription factor that is required for regulating TH gene expression in response to cAMP. Furthermore, our studies indicate that these CREB-deficient PC12 cells are excellent tools to study the participation of CREB in gene regulation.
Brain Res Mol Brain Res 1999 Jul 05
PMID:CREB mediates the cAMP-responsiveness of the tyrosine hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell lines. 1040 70

Whole-cell [(32)P]-protein phosphorylation assays and two-dimensional gel electrophoresis (2-DGE) were applied to the analysis of the beta-adrenoceptor (betaAR)-linked signal transduction pathway. Rat C6 glioma cells were stimulated with isoproterenol and the protein lysates were resolved by 2-DGE. Two dimensional [(32)P]-phosphoprotein 'maps' were generated depicting the modulation of intracellular proteins after isoproterenol stimulation versus unstimulated cells. A total of 274 distinct phosphoprotein spots were detected, of which 200 were up-regulated, 69 were down-regulated, and 5 remained unchanged. An evaluation of isoproterenol's activity across several kinase pathways was performed using a computer-generated 2-DGE template incorporating the location and identification of individual signaling phosphoprotein intermediaries. The template served as a 'reference map' for drug treatment comparisons. We observed a significant increase in the phosphorylation states of several nuclear transcription factors, notably CREB-1, ATF-1, NFkappaB/IkappaBalpha and ELK-1, but not c-Jun. A parallel series of radioimmunoprecipitation studies confirmed our 2-DGE findings. Moreover, isoproterenol increased the phosphorylation state of PKC and of several MAPK-dependent pathway kinases which correlated with a significant increase in their endogenous kinase activity. Isoproterenol's effects on PKA, PKC and ERK-dependent activities were blocked by propranolol, a betaAR antagonist. In conclusion, an acute isoproterenol stimulus induced multiplex pathway modulation via the betaAR in the C6 glioma cell indicating that signaling pathway cross-talk is an essential feature for the regulation of cellular function. Moreover, the immediate advantages of the 2-DGE analytical approach were apparent, and further development of the protein database will provide a valuable tool to screen for broad-based drug-mediated signaling activities.
Brain Res Mol Brain Res 1999 Jul 23
PMID:Probing for drug-induced multiplex signal transduction pathways using high resolution two-dimensional gel electrophoresis: application to beta-adrenoceptor stimulation in the rat C6 glioma cell. 1040 86

The heat shock transcription factor Hsf1p and the stress-responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae. Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild-type strains using [35S]-methionine labelling and two-dimensional gel electrophoresis. Among 52 proteins induced by a shift from 25 degrees C to 38 degrees C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p. The two sets of proteins overlapped only slightly. Three proteins were induced independently of these transcription factors, suggesting the involvement of other transcription factor(s). The Ras/cAMP/PKA signalling pathway cAMP had a negative effect on the induction of the Msn2p/Msn4p regulon, but did not affect the Hsf1p regulon. Thus, the two types of transcription factor are regulated differently and control two sets of functionally distinct proteins, suggesting two different physiological roles in the heat shock cellular response.
Mol Microbiol 1999 Jul
PMID:The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons. 1041 44

The role of human Sex Hormone-Binding Globulin (SHBG), the plasma carrier of sex steroids, and its membrane receptor, SHBG-R, in estrogen-dependent breast cancer has been investigated in our laboratory in the past few years. SHBG-R is expressed in MCF-10 A cells (not neoplastic mammary cells), MCF-7 cells (breast cancer, ER positive) and in tissue samples from patients affected with ER positive breast cancer, but not in estrogen-insensitive MDA-MB 231 cells. The SHBG/SHBG-R interaction, followed by the binding of estradiol to the complex protein/receptor, causes a significant increase of the intracellular levels of cAMP, but does not modify the amount of estradiol entering MCF-7 cells. The estradiol-induced proliferation of MCF-7 cells is inhibited by SHBG, through SHBG-R, cAMP and PKA. Similarly, the proliferation rate of tissue samples positive for SHBG-R was significantly lower than the proliferation rate of negative samples. SHBG and SHBG-R could thus trigger a 'biologic' anti-estrogenic pathway. In order to get a more detailed knowledge of this system, we first examined the frequence of the reported mutated form of SHBG in 255 breast cancer patients. The mutated SHBG is characterized by a point mutation (Asp 327 --> Asn) causing an additional N-glycosylation site, which does not affect the binding of steroids to SHBG. The frequence of the mutation was significantly higher (24.5%) in estrogen-dependent breast cancers than in healthy control subjects (11.6%). This observation confirms the close relationship between SHBG and estrogen-dependent breast cancer and suggests that the mutation could modify SHBG activity at cell site. Lastly, the possibility of using SHBG to modulate the estradiol action in breast cancer was further studied by transfecting MCF-7 cells with an expression vector carrying the SHBG cDNA (study in collaboration with G.L. Hammond). Transfected cells are able to produce significant amount of SHBG in their medium, but their SHBG-R is reduced to undetectable levels. The SHBG produced by transfected MCF-7 cells is, however, able to inhibit estradiol-induced proliferation of MCF-7 cells expressing a functional receptor. Thus, the local production of SHBG obtained with transfection could be a useful tool to control cell growth in estrogen-dependent breast cancer.
J Steroid Biochem Mol Biol
PMID:Sex hormone-binding globulin, its membrane receptor, and breast cancer: a new approach to the modulation of estradiol action in neoplastic cells. 1041 27

Bovine cholesterol side-chain cleavage cytochrome P450 (P450scc; product of the CYP11A gene) gene expression is regulated by gonadotropins via cAMP in the ovary, and by ACTH via cAMP in adrenal cortical cells. Previously, we characterized response elements located at -57/-32 and at -111/-101 bp in the 5'-flanking region of the bovine CYP11A gene required for cAMP-stimulated transcription in both mouse Y-1 adrenal tumor cells and bovine ovarian cells in primary culture, which bind SF-1 (or Ad4-BP) and Sp1, respectively. The role of these transcription factors in CYP11A transcription was further confirmed by deletion and mutation analyses. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites and a mammalian two-hybrid system indicate that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y-1 cells. Here we report that SF-1 and Sp1 are able to associate with one another in vitro and in vivo. The NH2-terminal region of SF-1, especially the DNA-binding domain, is the binding site for Sp1. In addition, as CBP is a common coactivator required for the transcriptional activity of numerous transcription factors including nuclear receptors, we investigated whether CBP functions as a cofactor for the regulation of bovine CYP11A promoter activity. We show here that CBP enhanced the PKA-induced CYP11A promoter activity, while a double mutation of both Sp1 and SF-1 sites within the CYP11A promoter region abolished CBP-induced activity. Furthermore, CBP stimulated Sp1-dependent transactivation, and a CBP/Sp1 complex in vivo was demonstrated by a co-immunoprecipitation assay. Also, CBP potentiated the transcriptional activity of GAL4-SF-1 in the presence of PKA. Thus, the cooperation between SF-1 and Sp1, required for the regulation of bovine CYP11A gene expression, is mediated by a direct protein-protein interaction and/or the common coactivator CBP.
Mol Cell Endocrinol 1999 Jul 20
PMID:Molecular mechanism for cooperation between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A gene expression. 1045 66

We investigated the effects of a protein kinase (PK) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), on the regulation of heat shock protein (hsp)72 gene expression in a human glioblastoma cell line (A-172) using a gel mobility-shift assay and Western blot analysis. Heat shock transcription factor 1 (HSF1) was phosphorylated immediately after heat treatment (44 degrees C, 30 min) and the phosphorylation of HSF1 was suppressed by H-7. The increase in DNA binding ability of HSFI to heat shock element (HSE) by heat shock was significantly suppressed by the addition of H-7 in a dose-dependent manner. Similarly, the accumulation of hsp72 by heat shock was suppressed by the addition of H-7 in a dose-dependent manner. Since H-7 is known to be a potent inhibitor of some PKs, especially calcium-dependent PK (PKC), cyclicAMP-dependent PK (PKA) and cyclicGMP-dependent PK (PKG), it is possible that the activation of HSF1 by phosphorylation and subsequent hsp72 gene expression are dependent on some of those PKs. The nature of H-7 as a non-specific inhibitor for PKs is discussed in relation to its availability for regulation of heat sensitivity of cells depending on cellular level of hsp72.
Mol Cell Biochem 1999 Jul
PMID:The protein kinase inhibitor, H-7, suppresses heat induced activation of heat shock transcription factor 1. 1048 32

In the rat thyroid FRTL-5 cell line calcitriol, the biologically most active of the naturally occurring vitamin D metabolites, attenuates both TSH-stimulated cAMP production and the effects of cAMP. Calcitriol treatment abolishes the upregulation of the TSHR number occurring in cells cultivated in the absence of TSH. In addition, the level of G(i-2)alpha increases, which may further attenuate the transmembrane signaling of TSH and facilitate the effects of IGFs. The effect of cAMP on PKAI stimulation is inhibited by increasing the level of the PKA subunit RIIbeta. Regulation of TSHR, G(i-2)alpha and RIIbeta is associated with altered cell proliferation and differentiation in several cells and tissues. Effects of calcitriol on these proteins indicate how the vitamin D endocrine system may regulate cAMP signaling in both classical and nonclassical target tissues.
Crit Rev Biochem Mol Biol 1999
PMID:Vitamin D: a hormonal regulator of the cAMP signaling pathway. 1056 77

Endothelin-1 (ET-1) is a hypertensive peptide, which is expressed in the rat adrenal gland, where it stimulates aldosterone secretion from zona glomerulosa (ZG) by activating the ETb receptor subtype. A higher effectiveness of ET-1 has been frequently observed when the integrity of adrenal tissue is preserved. Hence, we compared the aldosterone secretagogue action of ET-1 on dispersed rat ZG cells and capsule-ZG strips. ET-1 concentration-dependently raised aldosterone output by both preparations with similar potency. However, the efficacy of the maximal effective concentration of ET-1 (10-8 M) was about 2.7-fold higher in capsule-ZG strips. The ETb-receptor antagonist BQ-788 (10-7 M) abolished aldosterone response to 10-8 M ET-1 in both ZG preparations, while the ETa receptor antagonist BQ-123 was ineffective. The aldosterone secretagogue action of 10-8 M ET-1 on dispersed ZG cells was concentration-dependently suppressed by the protein kinase (PK) inhibitor calphostin-C. Conversely, both calphostin-C and the nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) evoked a concentration-dependent partial reversal of the aldosterone response to 10-8 M ET-1 of capsule-ZG strips. The NO donor L-arginine enhanced basal aldosterone yield of capsular strips, but not dispersed ZG cells. The PKA, cyclooxygenase and lipoxygenase inhibitors H-89, indomethacin and phenidone, as well as the beta-adrenoceptor antagonist l-alprenolol, were ineffective. Collectively, these findings allow us to conclude that in the rat i) the ETb receptor-mediated PKC activation is the main signaling mechanism involved in the direct stimulatory effect of ET-1 on ZG cells; and ii) the higher responsiveness of capsular strips to ET-1 may be accounted for by the ETb receptor-mediated release by stromal elements of NO, which in turn increases aldosterone secretion from ZG cells in a paracrine manner.
Int J Mol Med 2000 Jan
PMID:Comparison of the signaling mechanisms involved in the ETB receptor-mediated secretagogue action of endothelin-1 on dispersed zona glomerulosa cells and capsule-zona glomerulosa preparations of the rat adrenal gland. 1060 72


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