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)

Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a lambda EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6%) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40% upon treatment with 10 microM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.
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PMID:Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene. 153 68

Wild-type Chinese hamster ovary (CHO) cells were transfected with a DNA clone (MT-REV, site A) carrying a mouse gene for a dominant mutant regulatory subunit (RI) gene of cAMP-dependent protein kinase (PKA) from S49 cells along with a marker for G418 resistance. G418-resistant transfectant clone R-2D1 was resistant to 8-Br-cAMP-induced growth inhibition and morphological changes. The cells also did not phosphorylate a 50-kDa protein after cAMP stimulation and had decreased PKA activity, both characteristics of PKA mutants. Northern blot analysis indicated that clone R-2D1 was actively transcribing the MT-REV (site A)-specific RNA. We also tested clone R-2D1 for sensitivity to certain natural product hydrophobic drugs and found increased sensitivity to several drugs including adriamycin. Hypersensitivity to these drugs has previously been shown by us to be a characteristic of a CHO PKA mutant cell line. Expression of the mutant RI gene is also associated with a decrease in expression of the multidrug resistance associated P-glycoprotein (gp170) mRNA and protein. These results show that the PKA mutant RI gene from S49 cells acts as a dominant mutation to reduce the total PKA activity in the CHO transfectants as it does in mouse S49 cells. This study also confirms that reduced PKA activity modulates the basal multidrug resistance of these cells, apparently by causing decreased expression of the mdr gene at the protein and mRNA level.
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PMID:Transfection of a mutant regulatory subunit gene of cAMP-dependent protein kinase causes increased drug sensitivity and decreased expression of P-glycoprotein. 197 96

A cAMP-resistant mutant (Kin-8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP-dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP-resistant phenotype of the Kin-8 mutant was reverted by transformation with DNA from cAMP-responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA-mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2-neo (an SV40-neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin-resistant transformants were recovered from Y1 cells at a frequency of approximately one per 10(3) cells per 10 micrograms of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin-8, then was transformed with pSV2-neo DNA plus high mw DNA prepared from cAMP-responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin-8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin-8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP-responsive transformants, cAMP-dependent protein kinase activity was recovered and approached the activity seen in cAMP-responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP-resistant phenotype of Kin-8 cells. In other cAMP-responsive transformants, protein kinase activity was not appreciably affected by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Recovery of hormonal regulation in protein kinase defective adrenal cells through DNA-mediated gene transfer. 300 21

Plasmids encoding the amino terminal portion of an influenza virus hemagglutinin (HA) fused to polyoma virus middle T (mT) or large T (lT) sequences have been constructed. Stable expression of the chimeric proteins was obtained in established rat embryo fibroblasts following plasmid co-transfection and selection for G418 resistance. The synthesis and localization of the proteins was followed by metabolic labeling with [35S]methionine and [3H]mannose, cell fractionation, and immunoprecipitation with anti-polyoma T antibody. The HA leader and amino terminal peptide direct the synthesis of the lT and mT proteins into the endoplasmic reticulum where they undergo glycosylation, but this occurs with a very low efficiency. Most of the HA-mT and HA-lT fusion protein molecules do not enter completely into the endoplasmic reticulum, but rather achieve their normal locations in the cell as slightly higher molecular weight proteins, presumably due to the extra sequences derived from HA at their amino termini. HA-mT fusion protein is found to have associated tyrosine-specific protein kinase activity precipitable with anti-src as well as anti-T antibody, and cells expressing this fusion protein have a transformed phenotype.
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PMID:Expression of influenza hemagglutinin-polyoma T-antigen fusion proteins in a rat embryo fibroblast cell line. 303 93

Uncontrolled proliferation of acute myeloid leukemia (AML) cells is an important step during leukemogenesis. However, little is known about the mechanisms leading to growth autonomy. Studies using immortalized murine hematopoietic cell lines have suggested that autocrine production of growth factors, or the constitutive activation of molecules in growth factor signalling pathways, are involved. We have established six spontaneous factor-independent cell lines from the human growth factor-dependent TF-1 cell line. The factor-independent cells showed no detectable growth factor activity. Immunoblotting analyses of tyrosine phosphorylation, Raf-1 and extracellular signal-regulated kinase 2 (ERK-2) showed a similar pattern in all the cell lines including TF-1 cells. Furthermore, somatic-cell hybrids between TF-1 and the factor-independent cells grew in absence of growth factor. Taken together this data demonstrates that the factor independence in this system is dominant and suggests that the molecular event is located either downstream of the Raf-1 and MAP kinases pathway or on an alternative pathway. Finally, the karyotype analysis of one factor-independent cell line TF-1i1 and TF-1H- (G418 resistant, HAT sensitive TF-1 cells) and their hybrids demonstrated an unstable derivative chromosome [der(19) t(19;?) (q13.1;?)] which seemed to correlate with the factor-independence capacity. This model may help in our understanding of autonomous proliferation by human myeloid leukemias.
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PMID:Characterization of spontaneous factor-independent cell lines derived from the human leukemic cell line TF-1: a dominant event. 805 74

Ovarian granulosa cells are the primary site of estrogen and progesterone synthesis and play an essential role in the maturation of the developing ovum. Freshly isolated granulosa cells are often used to study the regulation of steroid and protein biosynthesis, but the small number of cells available for these cultures has proven inadequate for many detailed gene regulatory studies. The goal of this study was to develop human granulosa (HG) cell lines that maintain differentiated function. The E6 and E7 open reading frames of high risk strains of human papillomavirus have been used to produce immortalized cell lines. Primary cultures of human luteinized granulosa cells were infected with defective retroviruses containing the E6 and E7 regions of human papillomavirus 16 and with the neomycin phosphotransferase gene to confer G418 resistance. Three of eight clones that were isolated after selection in medium containing G418 were found to produce progesterone following treatment with forskolin or dibutyryl cAMP for 48 h. Forskolin caused these cells to retract in the characteristic rounding response, as described in primary HG cultures. One clone, HGL5, was used for a detailed characterization of differentiated function. HGL5 cells retained the ability to increase progesterone production and convert exogenously added androstenedione to estradiol in response to agonists of the protein kinase-A pathway (forskolin and dibutyryl cAMP), but were not responsive to FSH or LH treatment. A key enzyme in the production of estradiol, cytochrome P450 aromatase, has proven difficult to maintain in long term cultures of granulosa cells. For that reason, we examined the expression of aromatase in the transformed HGL5 clone by monitoring mRNA levels. Aromatase mRNA increased by 4- to 5-fold after forskolin treatment, as determined by Northern analysis. This human granulosa cell culture line maintains many of the functions of normal cells and should provide an important model to study the molecular events controlling granulosa cell differentiation and function.
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PMID:Transformation of human granulosa cells with the E6 and E7 regions of human papillomavirus. 812 45

Phosphodiesterases (PDEs) play a critical role in the regulation of intracellular cyclic nucleotide concentration and, consequently, regulate the state of cellular differentiation. We have reported that the Src-selective tyrosine kinase inhibitor, herbimycin A, potentiates luteinizing hormone (LH)-stimulated cAMP accumulation in culture media by ovarian thecal-interstitial cells (TIC; see Taylor, C and Terranova, P.F. (1995) Lipopolysaccharide inhibits rat ovarian thecal-interstitial cell steroid secretion in vitro. Endocrinology 136, 5527-5532). The present study was conducted to investigate the effects of herbimycin, and changes in Src tyrosine kinase activity, on PDE activity in rat TIC an in the mouse TM3 Leydig cell line. Treatment of TIC with herbimycin (1 microM) for 24 h inhibited basal and LH-stimulated PDE activity (approximately 50 and 70%, respectively) and was associated with an increase in cAMP and progesterone accumulation in culture media. Treatment of TM3 cells with herbimycin inhibited PDE activity and increased cAMP accumulation in a dose- and time-dependent manner. TM3 cell cultures challenged with herbimycin had lower Src tyrosine kinase activity than controls (approximately 50%); however, protein kinase A activity was unaffected. TM3 cells stably transfected with a dominant negative Src tyrosine kinase (TM3Srck-) had lower PDE activity than cells transfected with a G418 resistance gene alone (TM3pSV2neo) which served as control cells. Conversely, TM3 cells expressing a temperature-sensitive Src kinase had significantly greater PDE activity at the Src active temperature (35 degrees C; the temperature at which the enzyme is active) than TM3pSV2neo control cells grown at the same temperature. TM3 cell lysates hydrolyzed minimal amounts of cGMP, indicating a cAMP-specific PDE. Phosphodiesterase activity in both TM3 and rat TIC was sensitive to the PDE4-selective inhibitor RO20-1724, indicating the predominant active enzyme is probably a member of the cAMP-specific PDE4 family. From the present data, we conclude that a tyrosine kinase of the Src family may play an important role in regulating phosphodiesterase activity in thecal and Leydig cells, and thus regulate intracellular cAMP and the state of cellular differentiation.
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PMID:Src tyrosine kinase activity in rat thecal-interstitial cells and mouse TM3 Leydig cells is positively associated with cAMP-specific phosphodiesterase activity. 902 67

The interaction between plasma sex hormone-binding globulin (SHBG) and its receptor (SHBG-R) inhibits estradiol-induced proliferation of MCF-7 cells (human estrogen-dependent breast cancer) through cAMP and PKA. Thus, SHBG can modulate estradiol action in breast cancer, but the implications of this require a more detailed knowledge of the SHBG-R. To this end, we have transfected MCF-7 cells with an expression vector carrying the human SHBG cDNA (S-MCF-7) and studied the effects of this on both SHBG-R binding and cell proliferation. Control cells were parental MCF-7 (P-MCF-7) and MCF-7 cells transfected with the beta-galactosidase gene (B-MCF-7). Transfections were mediated by lipofectin followed by selection of transfected cells with G418. The amounts of SHBG in culture medium were evaluated by IRMA assay, with only S-MCF-7 cells shown to secrete SHBG; SHBG-R levels were evaluated by tracer binding technique. In P-MCF-7 and B-MCF-7 cells, SHBG-R was detectable as a two-binding site receptor, but no binding of SHBG was observed in S-MCF-7 cells. Proliferation of cells treated with estradiol was evaluated by [3H]thymidine incorporation in the three cell lines and in cells pretreated with SHBG (1 nM) purified from human serum or with conditioned medium from S-MCF-7 cells (medium S). In all three lines, cell proliferation increased after estradiol treatment. Preincubation with purified SHBG was effective in reducing estrogen-induced cell proliferation to basal levels in P-MCF-7 and B-MCF-7 but not in S-MCF-7 cells. The estradiol effect was also inhibited in P-MCF-7 cells treated with medium S. In conclusion, 1) SHBG inhibits estradiol-induced proliferation in cells containing a functional SHBG-R, whereas it has no detectable effect in cells in which the SHBG-R is either absent or not available to bind SHBG; and 2) S-MCF-7 cells are insensitive to SHBG (locally produced or exogenous) because their SHBG-R is occupied by SHBG.
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PMID:Control of the membrane sex hormone-binding globulin-receptor (SHBG-R) in MCF-7 cells: effect of locally produced SHBG. 961 86

The entire ACTH receptor (ACTH-R) cDNA was amplified by RT/PCR from mouse Y-1 adrenocortical cells, subcloned into the pMOSBlue T vector, sequenced and inserted into the pSVK3 mammalian vector to obtain pSVACTHR. Balb 3T3 fibroblasts were co-transfected with pSVACTHR plus pSV2-neo and the transfectants were selected with G418 and cloned. Genomic integration of pSVACTHR and transcription of ACTH-R cDNA were checked by Southern blot and RT/PCR respectively. Expression of active ACTH-R protein was tested by measuring cAMP production in response to ACTH. Two ACTH-R expressing transfectants (clones 03 and 07) increased cAMP accumulation in response to ACTH. They were morphologically identical to parental 3T3 cells, but required 10-20% FCS to grow. In these transfectants, ACTH induced c-FOS protein expression, but did not activate the ERK isoforms of MAP Kinase and did not stimulate DNA synthesis. Apparently, the ACTH-R in Balb 3T3 cells induces the c-fos gene by a pathway independent of cAMP/protein kinase A and ERK/MAP Kinase.
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PMID:ACTH induces c-fos proto-oncogene in fibroblasts expressing the ACTH receptor. 988 21

cAMP-dependent protein kinase (cAK) regulates the activity of several membrane-bound ion channels and carriers. The role of cAK in regulating the transport of osmoprotective amino acids in the distal tubule is unknown. We examined the regulation of Na(+)- and Cl(-)-dependent proline transport in MDCK cells expressing a mutant murine regulatory subunit (RIalpha(AB)) of cAK. For this purpose, MDCK cells were transfected with an expression vector encoding RIalpha(AB) driven by the metallothionein 1 promoter together with neomycin-resistance (NEO) gene. Stable G418-resistant colonies were isolated that expressed RIalpha(AB) as demonstrated by Northern hybridization analysis using a cDNA probe for RIalpha and cAK assay that showed decreased enzyme activity. A clone constitutively expressing high levels of RIalpha(AB) (M(AB)) in a Zn-independent manner and a control clone transfected with the NEO gene alone (M(neo)) were selected for transport studies. We examined the effect of the cAMP-stimulating agents forskolin (F) and IBMX on NaCl-dependent uptake of [(3)H]proline by confluent monolayers of transfected MDCK cells. While F/IBMX-induced mean inhibition of proline transport in M(neo) cells was 48 and 45% at 5 and 15 min, respectively, inhibition of proline uptake in M(AB) cells was 9% (5 min) and 0% (15 min). These data demonstrate that the inhibition of NaCl-linked proline transport in response to elevated cAMP is reversed in MDCK clones that express mutant cAK and provide evidence that cAK mediates the modulatory action of cAMP on proline transport. cAK may play an important role in controlling transport of proline and other osmoprotective amino acids in the renal tubule.
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PMID:Proline transport in MDCK cells expressing a mutant regulatory subunit of cAMP-dependent protein kinase. 1116 28


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