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Query: UNIPROT:P06889 (Mol)
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Spontaneous mutants of mouse FM3A cells (AC1, AC2, and AC3), highly resistant to aphidicolin (3000-, 2500-, and 300-fold increase in resistance, respectively), were isolated by multistep selection. The DNA synthesizing activity in permeabilized cells of all three mutants was substantially resistant to aphidicolin, like that in intact cells. The DNA polymerase activity in nuclear extracts in AC1 and AC3, but not AC2, was resistant to aphidicolin. Partially purified DNA polymerase alpha from AC3, but not from AC1 or AC2, showed resistance to aphidicolin. The apparent Ki value for aphidicolin of AC3 polymerase alpha was three to four times that of the enzyme from the parent cells, but the apparent Km values of the enzyme for dCTP and dTTP were normal. All the mutants showed cross-resistance to both arabinofuranosyladenine and arabinofuranosylcytosine. The AC3 mutant had expanded deoxyribonucleoside triphosphate pools. On two-dimensional polyacrylamide gel electrophoresis, AC1 gave a new protein (mol wt 40 kDa). The aphidicolin-resistance trait was reversible in AC2, unlike in AC1 and AC3. These results show that in mammalian cells there are at least two mechanisms of aphidicolin-resistance that involve an altered DNA polymerase alpha that is resistant to aphidicolin and simultaneous expansion of the four DNA-precursor pools.
Somat Cell Mol Genet 1990 Sep
PMID:High level of aphidicolin resistance with multiple mutations in mouse FM3A cell mutants. 212 28

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl(2)-responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr(1802)GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.
Mol Biol Cell 2000 Apr
PMID:The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments. 1074 28

Activation of adenylyl cyclase (AC), of which there are 10 diversely regulated isoforms, is important in regulating pulmonary vascular tone and remodeling. Immunohistochemistry in rat lungs demonstrated that AC2, AC3, and AC5/6 predominated in vascular and bronchial smooth muscle. Isoforms 1, 4, 7, and 8 localized to the bronchial epithelium. Exposure of animals to hypoxia did not change the pattern of isoform expression. RT-PCR confirmed mRNA expression of AC2, AC3, AC5, and AC6 and demonstrated AC7 and AC8 transcripts in smooth muscle. Western blotting confirmed the presence of AC2, AC3, and AC5/6 proteins. Functional studies provided evidence of cAMP regulation by Ca(2+) and protein kinase C-activated but not G(i)-inhibited pathways, supporting a role for AC2 and a Ca(2+)-stimulated isoform, AC8. However, NKH-477, an AC5-selective activator, was more potent than forskolin in elevating cAMP and inhibiting serum-stimulated [(3)H]thymidine incorporation, supporting the presence of AC5. These studies demonstrate differential expression of AC isoforms in rat lungs and provide evidence that AC2, AC5, and AC8 are functionally important in cAMP regulation and growth pathways in pulmonary artery myocytes.
Am J Physiol Lung Cell Mol Physiol 2001 Jun
PMID:Characterization of adenylyl cyclase isoforms in rat peripheral pulmonary arteries. 1135 Aug 17

The isoforms of adenylyl cyclase that mediate cyclic AMP signaling pathways in the retina are, for the most part, unknown. Therefore, the protein expression patterns of adenylyl cyclase isoforms in the rodent retina were characterized immunocytochemically using antibodies directed against Ca(2+)-stimulated (AC1, AC3 and AC8), Ca(2+)-inhibited (AC9) and Ca(2+)-insensitive (AC2, AC4, AC7) isoforms of adenylyl cyclase. The ganglion cell layer and the inner nuclear layer (INL) were immunoreactive for both Ca(2+)-sensitive (AC1, AC3) and Ca(2+)-insensitive (AC2, AC4) isoforms of adenylyl cyclase. Antibodies against isoforms from all three classes of adenylyl cyclase labeled the inner plexiform layer. In the outer retina, antibodies against Ca(2+)-insensitive isoforms labeled photoreceptors and the outer plexiform layer (OPL). Radial elements in the ONL and INL were AC4-immunoreactive and the nerve fibre layer and optic nerve were AC2-, AC4- and AC9-immunoreactive. Antibodies against AC7 did not label rodent neural retina. These data indicate that there is a heterogeneous distribution of adenylyl cyclase isoforms throughout the rodent retina. Nonetheless, there is a general indication of a greater expression of Ca(2+)-insensitive adenylyl cyclase isoforms in the outer retina, particularly within photoreceptors.
Brain Res Mol Brain Res 2002 May 30
PMID:Localization of adenylyl cyclase proteins in the rodent retina. 1200 33

In addition to a bicarbonate-regulated soluble adenylyl cyclase (sAC), mammalian spermatozoa, like somatic cells, appear to contain receptor/G protein-regulated AC activity that contributes to the modulation of specialized cell processes. This study provides evidence that agents, known to influence somatic membrane-associated AC (mAC) but apparently not germ cell sAC, can modulate cAMP production and functional state in mouse spermatozoa. Specifically, forskolin significantly enhanced cAMP production and capacitation, while inclusion of 2',5'-dideoxyadenosine significantly blocked these responses. Furthermore, GTPgammaS and NaF stimulated cAMP, but GDPbetaS and mastoparan had no apparent effect, consistent with recent evidence that G(s), but not G(i), contributes to AC/cAMP regulation in uncapacitated cells. In addition, intact mouse spermatozoa were screened for all known mAC isoforms by immunolocalization, using commercially available specific antibodies. The most abundant isoforms appeared to be AC2, AC3, and AC8, each with distinct distributions in the acrosomal and flagellar regions; AC1 and AC4 also appeared to be present, although less abundantly, in the midpiece and acrosomal cap regions, respectively. Intriguingly, however, Western blotting revealed that the major immunoreactive proteins in mouse sperm lysates were considerably smaller (approximately 50-60 kDa) than their somatic cell counterparts, suggesting that mature spermatozoa contain multiple mACs which may function in a shortened form. Of particular interest were AC3 and AC8, located in the same regions as, and hence possibly directly associated with, specific cell surface receptors and G proteins that are able to regulate the spermatozoon's acquisition and maintenance of fertilizing ability via changes in AC/cAMP.
Mol Reprod Dev 2003 Oct
PMID:Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa. 1295 Jan 6

Long-term infusion of prostacyclin, or its analogs, is an effective treatment for severe pulmonary arterial hypertension. However, dose escalation is often required to maintain efficacy. The aim of this study was to investigate the mechanisms of prostacyclin receptor desensitization using the prostacyclin analog cicaprost in rat pulmonary artery smooth muscle cells (PASMCs). Desensitization of the cAMP response occurred in 63 nM cicaprost after a 6-h preincubation with agonist. This desensitization was reversed 12 h after agonist removal, and resensitization was inhibited by 10 microg/ml of cycloheximide. Desensitization was heterologous since desensitization to other G(s)alpha-adenylyl cyclase (AC)-coupled agonists, isoproterenol (1 microM), adrenomedullin (100 nM), or bradykinin (1 microM), was also reduced by preincubation with cicaprost. The reduced cAMP response to prolonged cicaprost exposure appeared to be due to inhibition of AC activity since the responses to the directly acting AC agonist forskolin (3 microM) and the selective AC5 activator NKH-477 were similarly reduced. Expression of AC2 and AC5/6 protein levels transiently decreased after 1 h of cicaprost exposure. The PKA inhibitor H-89 (1 microM) added 1 h before cicaprost preincubation (6 h, 63 nM) completely reversed cicaprost-induced desensitization, whereas the PKC inhibitor bisindolylmaleimide (100 nM) was only partly effective. Desensitization was not prevented by the G(i) inhibitor pertussis toxin. In conclusion, chronic treatment of PASMCs with cicaprost induced heterologous, reversible desensitization by inhibition of AC activity. Our data suggest that heterologous G(s)alpha desensitization by cicaprost is mediated predominantly by a PKA-inhibitable isoform of AC, most likely AC5/6.
Am J Physiol Lung Cell Mol Physiol 2004 Aug
PMID:Mechanism of cicaprost-induced desensitization in rat pulmonary artery smooth muscle cells involves a PKA-mediated inhibition of adenylyl cyclase. 1510 93

Short periods of water deprivation can stimulate the growth of seminal and lateral roots in rice, and inhibit the emergence of adventitious roots. Identification of genes in the different tissues that respond to a water deficit may help us to understand the mechanism underlying root growth under conditions when water is scarce. cDNA-amplified fragment length polymorphism (AFLP) analysis was used to profile gene expression upon imposition of water deficit in three types of root tissue from the upland rice variety Azucena: seminal root tips, lateral root zones and adventitious root primordial zones. In all, 121 unique transcript-derived fragments (TDFs) were cloned, and Northern analysis was carried out for 30 TDFs to confirm their expression patterns. Sixty-six TDFs were differentially expressed in all three root samples. Four (AC2, D6, L22 and T23) were up-regulated by water deficit in seminal root tips and lateral root zones, and down-regulated in adventitious root primordial zones, an expression pattern which reflects the phenotypic changes observed in the different root sectors. In contrast, T17 and T37 showed the opposite expression pattern in Azucena: up-regulation in adventitious roots and repression in the other two zones. Functions could be assigned to five of these six TDFs on the basis of homology: they encode an expansin (T37), a fruit-ripening protein similar to ASR (T23), submergence-induced protein 2A (T17), a dehydrin (D6) and a 9- cis -epoxycarotenoid dioxygenase1 (L22), respectively. AC2 did not show a significant match to any known gene. Northern analysis showed that these six clones exhibited expression patterns that differed between the two cultivars tested (Azucena and the lowland variety IR1552) with respect to regulation by water limitation. Furthermore, T17, T37, D6 and T23 mapped within intervals known to contain QTLs (quantitative trait loci) for root growth in rice under water deficit. These genes may regulate or co-regulate the growth and development of the three root zones in a tissue-specific manner, and may play a role in the processes that underlie the early changes in root architecture under conditions of water deprivation.
Mol Genet Genomics 2004 Nov
PMID:Analysis of transcripts that are differentially expressed in three sectors of the rice root system under water deficit. 1548 Jul 89

The conserved late element (CLE) was originally identified as an evolutionarily conserved DNA sequence present in geminiviral intergenic regions. CLE has subsequently been observed in promoter sequences of bacterial (T-DNA) and plant origin, suggesting a role in plant and plant viral gene regulation. Synthetic DNA cassettes harboring direct repeats of the CLE motif were placed upstream from a -46 to +1 minimal CaMV 35S promoter-luciferase reporter gene and reporter activity characterized in Nicotiana species during both transient and stable expression. A single direct-repeat cassette of the element (2x CLE) enhances luciferase activity by 2-fold, independent of the element's orientation, while multiple copies of the cassette (4-12x CLE) increases activity up to 10- to 15-fold in an additive manner. Transgenic tobacco lines containing synthetic CLE promoter constructs enhance luciferase expression in leaf, cotyledon and stem tissues, but to a lesser extent in roots. Single nucleotide substitution at six of eight positions within the CLE consensus (GTGGTCCC) eliminates CLE enhancer-like activity. It has been previously reported that CLE interacts with the AC2 protein from Pepper Huasteco Virus (PHV-AC2). PHV-AC2 (also called AL2 or C2) is a member of the transcriptional activator protein, or TrAP, gene family. In transient and stable expression systems PHV-AC2 expression was found to result in a 2-fold increase in luciferase activity, irrespective of the presence of CLE consensus sequences within the reporter's promoter. These data suggests that the PHV-AC2 protein, instead of interacting directly with CLE, functions as either a general transcriptional activator and/or a suppressor of post-transcriptional gene silencing.
Plant Mol Biol 2005 Jul
PMID:Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco. 1602 33

We identified dexamethasone-induced Ras protein 1 (Dexras1) as a negative regulator of protein kinase C (PKC) delta, and the consequences of this regulation have been examined for adenylyl cyclase (EC 4.6.1.1) type 2 (AC2) signaling. Dexras1 expression in human embryonic kidney 293 cells completely abolished dopamine D2 receptor-mediated potentiation of AC2 activity, which is consistent with previous reports of its ability to block receptor-mediated Gbetagamma signaling pathways. In addition, Dexras1 significantly reduced phorbol 12-myristate 13-acetate (PMA)-stimulated AC2 activity but did not alter Galpha(s)-mediated cAMP accumulation. Dexras1 seemed to inhibit PMA stimulation of AC2 by interfering with PKCdelta autophosphorylation. This effect was selective for the delta isoform because Dexras1 did not alter autophosphorylation of PKCalpha or PKCepsilon. Dexras1 disruption of PKCdelta autophosphorylation resulted in a significant blockade of PKC kinase activity as measured by [gamma-32P]ATP incorporation using a PKC-specific substrate. Moreover, Dexras1 and PKCdelta coimmunoprecipitated from whole-cell lysates. Dexras1 did not alter the membrane translocation of PKCdelta; however, the ability of Dexras1 to interfere with PKCdelta autophosphorylation was isoprenylation-dependent as determined using the farnesyltransferase inhibitor methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and a CAAX box-deficient Dexras1 (C277S) mutant. PMA-stimulated AC2 activity was also not affected by Dexras1 C277S. Taken as a whole, these data suggest that Dexras1 functionally interacts with PKCdelta at the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKCdelta activity. Moreover our study suggests that Dexras1 acts to modulate the activation of AC2 in an indirect fashion by inhibiting both Gbetagamma- and PKC-stimulated AC2 activity. The current study provides a novel role for Dexras1 in signal transduction.
Mol Pharmacol 2006 May
PMID:Dexamethasone-induced Ras protein 1 negatively regulates protein kinase C delta: implications for adenylyl cyclase 2 signaling. 1648 24

The Begomovirus transcriptional activator protein (TrAP/AC2/C2) is a multifunctional protein which activates the viral late gene promoters, suppresses gene silencing, and determines pathogenicity. To study TrAP-mediated transactivation of a stably integrated gene, we generated transgenic tobacco plants with a Mungbean yellow mosaic virus (MYMV) AV1 late gene promoter-driven reporter gene and supertransformed them with the MYMV TrAP gene driven by a strong 35S promoter. We obtained a single supertransformed plant with an intact 35S-TrAP gene that activated the reporter gene 2.5-fold. However, 10 of the 11 supertransformed plants did not have the TrAP region of the T-DNA, suggesting the likely toxicity of TrAP in plants. Upon transformation of wild-type tobacco plants with the TrAP gene, six of the seven transgenic plants obtained had truncated T-DNAs which lacked TrAP. One plant, which had the intact TrAP gene, did not express TrAP. The apparent toxic effect of the TrAP transgene was abolished by mutations in its nuclear-localization signal or zinc-finger domain and by deletion of its activation domain. Therefore, all three domains of TrAP, which are required for transactivation and suppression of gene silencing, also are needed for its toxic effect.
Mol Plant Microbe Interact 2007 Dec
PMID:The mungbean yellow mosaic begomovirus transcriptional activator protein transactivates the viral promoter-driven transgene and causes toxicity in transgenic tobacco plants. 1799 Sep 62


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