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 responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.
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PMID:Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells. 1044 6

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.
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PMID:Molecular mechanism for cooperation between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A gene expression. 1045 66

Transforming growth factor-beta (TGF-beta)can induce the cyclin-dependent kinase inhibitors p21 and p15 in a variety of cell types. We have shown previously that Smad3 is required for the growth inhibitory activity of TGF-beta, whereas overexpression of Smads is not sufficient to activate the expression of p21 in HaCaT cells. These data suggest that an additional signaling pathway may be involved in stimulating p21 in HaCaT cells. Given the recent finding that the mitogen-activated protein kinase (MAPK) pathway can cause p21 induction and arrest cells, we examined the involvement of this pathway for p21 and p15 induction by TGF-beta. We found that TGF-beta can regulate the MAPK pathway, leading to the increased transactivation ability of transcription factor Elk. Constitutively active components in the MAPK pathway activate p21 expression, and inhibitors or dominant negative constructs for the MAPK pathway significantly decrease p21 induction by TGF-beta. Both constitutively active MEK and inhibitors for MEK have no effect on Smad activity, including DNA binding, localization, and interaction with coactivator p300/CBP. These findings suggest that the MAPK pathway may be an independent pathway that is involved in p21 and p15 induction by TGF-beta.
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PMID:The MEK pathway is required for stimulation of p21(WAF1/CIP1) by transforming growth factor-beta. 1058 6

Cyclic AMP (cAMP) stimulates the expression of numerous genes via the protein kinase A (PKA)-mediated phosphorylation of CREB at Ser133. Ser133 phosphorylation, in turn, promotes recruitment of the coactivator CREB binding protein and its paralog p300, histone acetyltransferases (HATs) that have been proposed to mediate target gene activation, in part, by destabilizing promoter bound nucleosomes and thereby allowing assembly of the transcriptional apparatus. Here we show that although histone deacetylase (HDAC) inhibitors potentiate target gene activation via cAMP, they do not stimulate transcription over the early burst phase, during which CREB phosphorylation and CBP/p300 recruitment are maximal. Rather, HDAC inhibitors augment CREB activity during the late attenuation phase by prolonging CREB phosphorylation on chromosomal but, remarkably, not on extrachromosomal templates. In reconstitution studies, assembly of periodic nucleosomal arrays on a cAMP-responsive promoter template potently inhibited CREB phosphorylation by PKA, and acetylation of these template-bound nucleosomes by p300 partially rescued CREB phosphorylation by PKA. Our results suggest a novel regulatory mechanism by which cellular HATs and HDACs modulate the phosphorylation status of nuclear activators in response to cellular signals.
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PMID:The phosphorylation status of a cyclic AMP-responsive activator is modulated via a chromatin-dependent mechanism. 1066 37

Previous studies have shown that human T-cell leukemia virus type 1 (HTLV-1) Tax is a key molecule of synoviocyte activation in HTLV-1 associated arthropathy (HAAP). To clarify the molecular mechanism of HTLV-1 Tax-induced transcriptional activation in synoviocytes from HAAP, we investigated the role of cyclicAMP (cAMP)-regulated enhancer (CRE) binding protein (CREB)-binding protein (CBP), as a target molecule of HTLV-1 Tax. Activation of cyclic AMP (cAMP)/protein kinase-A (PK-A) pathway resulted in a significantly high response of CRE promoter in synoviocytes from patients with HAAP as well as in Tax-transiently transfected synoviocytes from patients with rheumatoid arthritis (RA). Mammalian two-hybrid analysis showed that the recruitment of CBP was responsible for CREB activation. Furthermore, PK-A activation induced CBP-Tax complex in synoviocytes from HAAP and the complex contained CREB. These findings demonstrated that complex formation of CBP and Tax is critical for enhanced CREB activity in synoviocytes from HAAP.
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PMID:CBP: A target molecule of HTLV-1 Tax in synoviocyte activation. 1070 98

The E2F proteins form a family of transcription factors that regulate the transition from the G1 to the S phase in the cell cycle. E2F activity is regulated by members of the retinoblastoma protein (pRb) family, ensuring the tight control of E2F-responsive genes. During the G1 phase, phosphorylation of pRb by cyclin-dependent kinases (CDKs), most notably cyclin D-CDK complexes, releases pRb from E2F, facilitating cell-cycle progression by the timely induction of E2F-targeted genes such as cyclin E. However, it is not known whether E2F proteins are directly targeted by CDKs. Here we show that E2F-5 is phosphorylated by the cyclin E-Cdk2 complex, which functions in the late G1 phase, but not by the early-G1-phase-acting cyclin D-CDK complex. A phosphorylation site in the trans-activation domain of E2F-5 stimulates transcription and cell-cycle progression by the recruitment of the p300/CBP family of co-activators, whose binding to E2F-5 is stabilized upon phosphorylation by cyclin E-Cdk2. These results indicate that E2F activity may be directly regulated by cyclin E-Cdk2, and imply an autoregulatory mechanism for cell-cycle-dependent transcription through the CDK-stimulated interaction of E2F with p300/CBP co-activators.
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PMID:Regulation of E2F transcription by cyclin E-Cdk2 kinase mediated through p300/CBP co-activators. 1078 42

Differentiation of primary villous cytotrophoblasts into syncytia is associated with increasing production of alpha and beta human CG subunits, which is predominantly governed at the level of messenger RNA expression. Here, we present a detailed study on the mechanisms involved in the differentiation-dependent regulation of the trophoblast-specific CGalpha gene promoter. Site-directed mutations in each of the five DNA-elements of the composite enhancer were performed to investigate the contribution of the individual regulatory sequences to the overall transcriptional activity of the promoter at two different stages of trophoblast in vitro differentiation. We show that deletion of one cyclic AMP response element (CRE) did not affect CGalpha promoter activity in cytotrophoblasts; however, it reduced transcription by 33% in differentiating cultures. Removal of both CREs almost abolished transcription at early and later stages of in vitro differentiation. Upon mutation the enhancer elements alphaACT, JRE, and CCAAT significantly decreased luciferase reporter transcription; however their contribution to the total promoter activity did not change during in vitro differentiation. Contrary to that, mutated TSE diminished promoter activity by 19% during 12 and 48 h of cultivation but reduced luciferase expression by 78% between 48 and 84 h of differentiation. In electrophoretic mobility shift assay, the TSE interacted with activating protein (AP)-2alpha in both primary trophoblasts and choriocarcinoma cells. While CRE-interacting proteins were detectable 12 h after isolation, the TSE-binding complex did not appear before 36 h of in vitro differentiation. During syncytium formation increasing protein expression of activating transcription factor (ATF)-1, cAMP response element-binding protein (CREB)-1, and AP-2alpha was observed on Western blots. Moreover, phosphorylated CREB-1 and ATF-1 accumulated between 24 and 78 h of trophoblast cultivation. By fluorescence immunohistochemistry, we show that CREB-1 was predominantly expressed in syncytiotrophoblasts, whereas ATF-1 and AP-2alpha localized to the syncytium and some cytototrophoblasts as well as to stromal and endothelial cells of the placental villus. Phosphorylated CREB-1/ATF-1 and the coactivator protein CBP were primarily detected in syncytial nuclei, suggesting the presence of functional, cAMP-dependent transcriptional complexes in the differentiated tissue. In agreement to the in vivo situation, phosphorylated CREB-1/ATF-1 were observed in nuclei of the differentiated trophoblast cultures. The activity of the CGalpha promoter as well as CREB-1/ATF-1 phosphorylation increased upon elevation of cAMP levels and overexpression of the catalytic subunit of protein kinase A. Additionally, we demonstrate that overproduction of the enzyme enhanced protein expression and binding of AP-2alpha to the TSE. We conclude that differentiation-dependent transcription of the CGalpha gene in villous trophoblasts is mainly governed by increasing expression of AP-2alpha and PKA-dependent phosphorylation of CREB-1 and ATF-1.
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PMID:Promoter elements and transcription factors involved in differentiation-dependent human chorionic gonadotrophin-alpha messenger ribonucleic acid expression of term villous trophoblasts. 1101 30

The conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine by tyrosine hydroxylase (TH) is the first and rate-limiting step in biosynthesis of catecholamine neurotransmitters. TH gene expression is regulated in a cell type-specific and cAMP-dependent manner. Evidence from this laboratory and others indicates that the cAMP response element (CRE), residing at -45 to -38 bp upstream of the transcription initiation site, is essential for both basal and cAMP-inducible transcription of the TH gene. To understand the control mechanisms of TH gene transcription in greater detail, we sought to identify and characterize the transcription factors involved in recognition and activation of the CRE of the TH gene. Remarkably, electrophoretic mobility shift assay and antibody supershift experiments indicated that all three major CRE-binding protein factors, i.e. CREB, ATF1, and CREM, may participate in forming specific DNA/protein complexes with the CRE of the TH gene. To address the transcriptional activation function of individual factors, we replaced the TH CRE with a GAL4-binding site and cotransfected this modified TH promoter-reporter gene with an effector plasmid that encodes GAL4-fused transcription factor. Our results indicate that CREB but not ATF1 can support basal promoter activity while both can robustly induce the promoter activity in response to co-expression of the catalytic subunit of cAMP-dependent protein kinase (PKA). We further show that the coactivator CBP up-regulates PKA-mediated activation of the TH promoter and, if tethered to the TH promoter by a GAL4-fusion, can robustly transactivate the TH promoter even in the absence of PKA. Collectively, our results suggest that multiple CRE-binding factors interact with the CRE and regulate, in conjunction with the coactivator CBP, the transcriptional activity of the TH gene.
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PMID:Regulation of tyrosine hydroxylase gene transcription by the cAMP-signaling pathway: involvement of multiple transcription factors. 1110 36

The glycoprotein hormones, ACTH, TSH, FSH, and LH regulate diverse functions in endocrine cells. Although cAMP and PKA have long been shown to mediate specific intracellular signaling events including the transcription of specific genes via the CREB-CBP complex, recent observations have indicated that PKA does not account for all of the intracellular targets of cAMP. For example, TSH stimulation of thyroid cell proliferation is not completely blocked by PKA inhibitors. TSH and FSH can stimulate PKB phosphorylation by a PKAindependent but PI3-K/PDK1-dependent pathway. An FSH inducible kinase, Sgk, has recently been shown to be a close relative of PKB. Sgk is also a target of PI3-K-PDK1 pathway, indicating that some effects previously ascribed to PKB may be mediated by this inducible kinase. The identification of novel cAMP-binding proteins that exhibit guanine nucleotide exchange (GEF) activity (cAMP-GEFS; Epacs) has open new doors for cAMP action that include activation of small GTPases such as Rap1a, Rap2, and possibly Ras. These GTPases are known activators of downstream kinase cascades, including p38MAPK and Erk1/2 as well as PI3-K. Thus, FSH and TSH activation of PKB and Sgk may occur via this alternative cAMP pathway that involves cAMP-GEFs and the activation of the PI3-K/PDK1 pathway.
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PMID:New signaling pathways for hormones and cyclic adenosine 3',5'-monophosphate action in endocrine cells. 1115 28

We have assessed the potential role of sterol regulatory element-binding protein-1c (SREBP-1c) on the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC ) (PEPCK-C). SREBP-1c introduced into primary hepatocytes with an adenovirus vector caused a total loss of PEPCK-C mRNA and a marked induction of fatty acid synthase mRNA that directly coincided with the appearance of SREBP-1c in the hepatocytes. It also blocked the induction of PEPCK-C mRNA by cAMP and dexamethasone in these cells. In contrast, a dominant negative form of SREBP-1c (dnSREBP-1c) stimulated the accumulation of PEPCK-C mRNA in these cells. SREBP-1c completely blocked the induction of PEPCK-C gene transcription by the catalytic subunit of protein kinase A (PKA), and increasing concentrations of dnSREBP-1c reversed the negative effect of insulin on transcription from the PEPCK-C gene promoter in WT-IR cells. The more than 10-fold induction of PKA-stimulated PEPCK-C gene transcription caused by the co-activator CBP, was also blocked by SREBP-1c. In addition, dnSREBP-1c reversed the strong negative effect of E1A and NF1 on PKA-stimulated transcription from the PEPCK-C gene promoter. An analysis of the possible site of action of SREBP-1c using stepwise truncations of the PEPCK-C gene promoter indicated that the negative effect of SREBP-1c on transcription is exerted at a site between -355 and -277. We conclude that SREBP-1c is an intermediate in the action of insulin on PEPCK-C gene transcription in the liver and acts by blocking the stimulatory effect cAMP that is mediated via an interaction with cAMP-binding protein.
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PMID:Sterol regulatory element-binding protein-1c mimics the negative effect of insulin on phosphoenolpyruvate carboxykinase (GTP) gene transcription. 1144 21


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