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)

This article describes investigations of several aspects of the molecular biology of the human renin gene and the three-dimensional structure of renin and its precursor, prorenin. Because of the importance of the RAS in hypertension, heart failure, renal failure, and possibly other disorders such as atherosclerosis, it is critical to understand the detailed control of this system. This control involves regulation at the transcriptional level, folding of prorenin, sorting of prorenin to a regulated pathway where it is proteolytically cleaved to renin and released in response to secretogogues, constitutive release of uncleaved prorenin, and nonproteolytic activation of prorenin. Currently there is great interest not only in the control of renin in the kidney, the sole source of circulating renin, but also at extrarenal sites where RAS activity may regulate cardiovascular functions. The renin gene was found to be expressed significantly in the renal juxtaglomerular cells and several other cell types. Most tissue culture cells did not express the gene; exceptions were cultured SK-LMS-1 cells and cAMP-stimulated human lung fibroblasts. Cultured human uterine-placental cells expressed the human renin gene at levels higher than in other cell types assessed. Renin mRNA had the same start site in the placental cells as the kidney and was regulated by calcium ionophores and cAMP. Thus, these cells provide primary nontransformed human cells to study the homologous human promoter. Transfected renin promoters showed cell type-specific expression and cAMP responsiveness in these cells in constructs containing as few as 102 bp of 5'-flanking DNA. DNA upstream from this appears to contain an inhibitory element(s) that may have some tissue specificity in its distribution. The cAMP response is not due to cAMP induction of a transcription factor that secondarily affects the renin promoter. A novel element may be involved, since the promoter does not contain a CRE element that mediates many cAMP responses, and the cells do not appear to respond to another known cAMP-responsive transcription factor, AP-2. Studies with transfected vectors expressing a mutant cAMP-responsive protein kinase A regulatory subunit suggest that cAMP is not responsible for basal renin promoter activity in the placental cells. By contrast, cAMP induces in essence gene activation in WI26VA4 transformed human lung fibroblasts in which renin mRNA levels increase by up to 150-fold in response to forskolin. Thus, cAMP may activate renin gene expression under certain circumstances and tissue-specific renin gene expression may be directed by more than one mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Molecular biology of human renin and its gene. 174 21

In multidrug-resistant mouse J774.2 cells, the differential overproduction of functionally distinct phosphoglycoprotein isoforms reflects the amplification or transcriptional activation or both of two mdr gene family members, mdr1a and mdr1b. The mdr1a gene is a complex transcriptional unit whose expression is associated with multiple transcript sizes. Independently selected multidrug-resistant J774.2 cell lines differentially overexpress either 4.6- and 5.0-kilobase (kb) or 4.7- and 5.1-kb mdr1a transcripts. However, abundant overproduction of the mdr1a gene product was observed only in cell lines which overexpressed the 4.6- and 5.0-kb mRNAs. In order to determine the basis for mdr1a transcript heterogeneity and the relationship between transcript size and steady-state mdr1a protein levels, genomic and cDNA sequence analyses of the 5' and 3' ends of the mdr1a gene were carried out. Promoter sequence analysis and primer extension mapping indicated that mdr1a transcripts were differentially initiated from two putative promoters to generate either 5.1- and 4.7-kb or 5.0- and 4.6-kb transcripts in four multidrug-resistant J774.2 cell lines. Sequence analysis of 3' cDNA variants and a 3' genomic fragment revealed that the 5.1- and 5.0-kb mRNAs had identical 3'-untranslated regions which differed from those of the 4.7- and 4.6-kb mRNAs as a result of the utilization of a more downstream alternative poly(A) addition signal. Transcript initiation from the putative upstream promoter correlated with a 70 to 85% decrease in steady-state mdr1a protein levels relative to transcript levels. In addition, the identification of putative AP-1 and AP-2 promoter elements suggests a possible role for protein kinase A and protein kinase C in the regulation of mdr1a. The implications of these findings for mdr gene expression and regulation are discussed.
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PMID:Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells. 197 47

Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase are ubiquitous kinases conserved from fungi to mammals. Their activity is regulated by phosphorylation on both threonine and tyrosine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determination of its intron/exon boundaries, and the characterization of its promoter. The gene spans approximately eight kilobases (kb) and can be divided into nine exons and eight introns, each coding region exon containing from one to three of the highly conserved protein kinase domains. Primer extension analysis reveals the existence of two major start sites of transcription located at -183 and -186 base pairs (bp) as well as four discrete start sites for transcription located at -178, -192, -273, and -292 bp of the initiation of translation. However, the start site region lacks TATA-like sequences but does contain initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start sites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, one half serum-responsive element, and putative binding sites for Sp1 (five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (two), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyte nuclear factor-5 (one). To determine the sites critical for the function of the p44 MAPK promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarcoma virus promoter. Progressive deletions of the approximately 1 kb (-1200/-78) promoter region allowed us to define a minimal region of 186 bp (-284/-78) that has maximal promoter activity. Within this context, deletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity. This result implicates a major role of this region that contains the Sp1 sites.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The mouse p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1) gene. Genomic organization and structure of the 5'-flanking regulatory region. 759 46

AP-2 is a cell type-specific DNA-binding transcription factor that regulates selected target genes in vertebrate organisms. Here we investigated cell type-specific expression and regulation of AP-2 in neuroectodermal cell lineages. During retinoic acid (RA)-mediated differentiation of P19 embryonal carcinoma cells into neuroectodermal cell types that include immunohistochemically defined neurons and astrocytes, we observed a strong induction of AP-2 transcripts and protein. In contrast, AP-2 mRNA was not induced in P19 cells which undergo mesoendodermal differentiation in response to 1% dimethylsulfoxide or low concentrations of RA, respectively. The potential of both neurons and astrocytes to express AP-2 was ascertained by using cerebellar neurons and astrocytes derived from newborn mice. Unlike these types of cells, microglial cells do not express AP-2. Dibutyryl cyclic AMP further enhanced levels of AP-2 transcripts in both P19 astrocytes and primary astrocytes which also respond to agents elevating intracellular cAMP (noradrenaline, isoproterenol, forskolin). The cAMP-dependent induction of AP-2 could be blocked by inhibitors of protein kinase A. In contrast to its action in P19 cells, RA had no effect on AP-2 mRNA levels in primary astrocytes. Our results indicate that AP-2 may play a role as a retinoic acid-sensitive regulator during differentiation of neurons and glia from an embryonic neural precursor. Furthermore, AP-2 may be involved in gene transcription in both mature neurons and astrocytes.
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PMID:Cell type-specific regulation of expression of transcription factor AP-2 in neuroectodermal cells. 795 25

Insulin induction of acetyl-CoA carboxylase (ACC) and differentiation of 30A5 preadipocytes into adipocytes requires a brief exposure of the cells to cAMP. Using the techniques of DNase I footprinting, DNA band shift, and analysis of the point and deletion mutations, a region from -113 to -95 has been identified as the site through which cAMP sensitizes the cell for the response to insulin. One sequence-specific DNA-protein complex, b3, is formed in the DNA-mobility shift assay when nuclear extract from 30A5 cells is mixed with the oligonucleotide representing this region. Purified human AP-2 also generates the complex corresponding to b3 with the same ACC PII probe or with the AP-2 consensus sequence probe from SV40 promoter. Substitution of A for G in the sequence GGGGCTGGG abolishes the formation of b3 sequence-specific complex. Stably transfected 30A5 cells with the same mutations in the plasmid no longer respond to insulin in spite of their exposure to cAMP. These results establish that the 21 base pair region in ACC promoter II and the binding of AP-2 protein to this sequence are required for cAMP action. cAMP-dependent protein kinase phosphorylates AP-2 both in vitro and in vivo and the phosphorylation of AP-2 does not affect its binding activity.
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PMID:The site of cAMP action in the insulin induction of gene expression of acetyl-CoA carboxylase is AP-2. 810 69

The most potent, physiologic activator of proopiomelanocortin (POMC) gene transcription is corticotropin releasing hormone (CRH) and increased intracellular cAMP is critical for this effect. The 5'-flanking region of the murine POMC gene has several potential binding sites for regulatory proteins. To characterize the region between nucleotides -141 and -106, which includes a TRE-like site and an adjacent AP-2 consensus sequence, and to study its role in signal-transcription coupling, gel mobility shift assays and transient expression of CAT chimeras were performed. In transient transfections of AtT-20 cells with pCATp-141/-106, CRH treatment led to significant increases in CAT expression compared with CRH treatment of cells transfected with the enhancerless vector. However, no response to direct activation of cAMP dependent protein kinase or protein kinase C was detected. Despite the high homology of the sequence -137/-131 to the consensus AP-1 binding site (TRE), the nuclear factor(s) in AtT-20 cells binding to this region appears to be different than authentic AP-1 since neither a competitor oligonucleotide having the authentic TRE sequence nor antibodies against Jun or Fos affected the gel shift pattern of a probe having the -137/-131 sequence. We conclude that the -141 to -106 region of the murine POMC gene contains a functional CRH responsive element and that second messenger systems that transduce the CRH signal to this element do not exert their actions solely through activation of PKA or PKC.
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PMID:Characterization of a corticotropin releasing hormone responsive region in the murine proopiomelanocortin gene. 814

AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data.
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PMID:The 50 kDa protein subunit of assembly polypeptide (AP) AP-2 adaptor from clathrin-coated vesicles is phosphorylated on threonine-156 by AP-1 and a soluble AP50 kinase which co-purifies with the assembly polypeptides. 825 32

Diversion of membrane proteins from the trans-Golgi network (TGN) or the plasma membrane into the endosomal system occurs via clathrin-coated vesicles (CCVs). These sorting events may require the interaction of cytosolic domain signals with clathrin adaptor proteins (APs) at the TGN (AP-1) or the plasma membrane (AP-2). While tyrosine- and di-leucine-based signals in several proteins mediate endocytosis via cell surface CCVs, segregation into Golgi-derived CCVs has so far only been documented for the mannose 6-phosphate receptors, where it is thought to require a casein kinase II phosphorylation site adjacent to a di-leucine motif. Although recently tyrosine-based signals have also been shown to interact with the mu chain of AP-1 in vitro, it is not clear if these signals also bind intact AP-1 adaptors, nor if they can mediate sorting of proteins into AP-1 CCVs. Here we show that the cytosolic domain of the lysosomal membrane glycoprotein lamp-1 binds AP-1 and AP-2. Furthermore, lamp-1 is present in AP-1-positive vesicles and tubules in the trans-region on the Golgi complex. AP-1 binding as well as localization to AP-1 CCVs require the presence of the functional tyrosine-based lysosomal targeting signal of lamp-1. These results indicate that lamp-1 can exit the TGN in CCVs and that tyrosine signals can mediate these sorting events.
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PMID:The tyrosine-based lysosomal targeting signal in lamp-1 mediates sorting into Golgi-derived clathrin-coated vesicles. 889 68

The rat angiotensin II type 2 receptor (AT2-R) gene was isolated, and cis-regulatory regions in its 5'-flanking area were analyzed. Primer extension and RNase protection analyses revealed a single transcriptional initiation site at the position 24 bp downstream of the TATA box. The 5'-flanking region of AT2-R contained several cis-regulatory elements, such as AP-1, AP-2, C/EBP, NF-1, NF-IL6, NF-kappa B, and glucocorticoid- and cAMP-responsive elements (CRE). The treatment of PC12 cells with dibutyryl cAMP caused a marked decrease (90%) in the AT2-R mRNA level, which was blocked by the inhibitor of protein kinase A and did not require new protein synthesis. The protein level was also reduced 84% after a 24-h exposure to cAMP and the binding affinity was unchanged. The half-life of the AT2-R mRNA decreased -66% by cAMP as compared with control (18.4 +/- 0.4 h). Deletion and mutation analyses of the 5'-flanking region (1.2 Kb) revealed that there were one negative (-1,199 to -739) and two positive cis-regulatory regions (-739 to -436 and -59 to +45), and that the CRE motif located at -426 repressed (-23%) the promoter activity of the rat AT2-R gene. The region between -59 and +45 containing TATA box and AP-2 site accounted for 70% of the promoter activity. These findings indicate that the promoter activity of the rat AT2-R gene is modulated by several cis-regulatory regions and that cAMP markedly downregulates the expression of the AT2-R mainly by inducing AT2-R mRNA destabilization rather than CRE-mediated inhibition of the gene transcription. Thus, humoral factors that transduce cAMP as an intracellular signal may modulate AT2-R-mediated function of Ang II by reducing AT2-R expression.
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PMID:Down-regulation by cAMP of angiotensin II type 2 receptor gene expression in PC12 cells. 898 58

Oxytocin receptor (OTR) gene transcription has predominantly been thought to be regulated by estrogen. However, the continuous presence of receptors in certain brain regions after gonadectomy suggests the existence of alternate mechanisms of regulation. We have cloned and sequenced 4 kb of 5'-flanking DNA of the rat OTR gene and identified an internal segment which was absent in the initial publication of this promoter sequence. Sequence analysis of this segment, as well as of a novel upstream region, revealed the presence of a CRE as well as several other potential regulatory elements, including AP-1, AP-2, AP-3, AP-4 sites, an ERE, and a half-SRE (SRE/2). The effects of phorbol 12-myristate 13-acetate (PMA), forskolin, and NGF treatment on this promoter were tested in transfection experiments in MCF7 and SK-N-SH cells. Transcription of the full-length OTR promoter was induced by forskolin and by the phorbol ester PMA, and a synergistic (17-fold) effect was observed in MCF7 cells treated with both agents. Receptor binding studies using the OTR antagonist 125I-labeled ornithine vasotocin, and Western blot analyses of OTRs in MCF7 cells, showed that PMA and forskolin also increased the density of endogenous human oxytocin receptors. Mutational analyses of the CRE and half-SRE sites in this promoter indicated that these elements function as enhancers and support forskolin and NGF effects, respectively, on transcription. These studies have identified a novel region of the rat OTR promoter containing elements which impart cAMP and/or phorbol ester inducibility of OTR gene transcription. A potential role of the PKA and/or PKC pathways in OTR gene regulation is suggested.
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PMID:NGF, cyclic AMP, and phorbol esters regulate oxytocin receptor gene transcription in SK-N-SH and MCF7 cells. 947 29


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