Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cAMP-dependent protein kinase was compared in normal and Rous Sarcoma Virus transformed chicken embryo fibroblasts. Total cAMP binding activity and cAMP-dependent histone kinase activity were unaltered by RSV transformation. The apparent Km for activation of histone kinase activity by cAMP was 35 nM in both normal and transformed cells. Using 8-N3-cAMP photoaffinity labeling, normal and transformed cells were also found to contain equal quantities of a single 42,000 Mr regulatory sub-unit isoenzyme of A-kinase. This isoenzyme corresponded to the lower molecular weight isoenzyme of the two enzymes found in normal chicken skeletal muscle. Both avian isoenzymes were about 4,000 Mr smaller than the corresponding bovine type I and type II regulatory subunits. Rous Sarcoma Virus transformation does not directly alter the amount or activity of cAMP-dependent protein kinase.
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PMID:Comparison of cAMP-dependent protein kinase in normal and Rous sarcoma virus transformed chick embryo fibroblasts. 627 Feb

A nontransformed line of Chinese hamster ovary (CHO) cells (Pollard and Stanners, 1979) has been transformed by the Schmidt-Ruppin subgroup D strain of Rous sarcoma virus (SR-RSV). SR-RSV transformed CHO cells are shown to differ from spontaneously transformed cells in that the virally transformed cells are more resistant to growth inhibition or changes in cell shape by 8-Br-cyclic AMP or cholera toxin. SR-RSV transformed rat (NRK) cells also have a reduced sensitivity to growth inhibition by 8-Br-cyclic AMP. Cyclic AMP-dependent protein kinase was examined in SR-RSV transformed CHO cells, but no differences in enzyme level, activation by cyclic AMP, chromatographic behavior, or its ability to phosphorylate endogenous proteins in whole cells could be detected. It is concluded that transformation of CHO and NRK cells by SR-RSV alters the cells in a manner different from spontaneous transformation, and that this alteration does not affect cAMP-dependent protein kinase activity.
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PMID:Rous sarcoma virus transformed cells are resistant to cyclic AMP. 628 4

We have constructed plasmids that direct the synthesis of the Rous sarcoma virus transforming gene (src) product (p60src) in Escherichia coli. A 203-base-pair lac promoter-operator DNA encoding the first eight amino acids of beta-galactosidase was ligated to the 5' end of the src gene from the Prague A strain of Rous sarcoma virus (PrA-RSV) which had been cloned in pBR325. Antiserum, from a tumor-bearing rabbit, directed against pp60src was used to screen bacteria containing the recombinant plasmid for a protein of approximately 60,000 daltons, and several colonies producing a protein immunologically related to pp60src were detected. Partial proteolytic cleavage analysis revealed that the src-related protein produced in bacteria is structurally similar to pp60src immunoprecipitated from PrA-RSV-infected chicken cells. Partially purified src protein from E. coli can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. Tryptic phosphopeptide analysis demonstrated that the catalytic subunit phosphorylated a serine-containing tryptic peptide in the bacterial src protein that comigrated with the phosphoserine-containing tryptic peptide of pp60src immunoprecipitated from 32P-labeled PrA-RSV-infected chicken cells.
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PMID:Construction of plasmids for expression of Rous sarcoma virus transforming protein, p60src, in Escherichia coli. 628 67

The phosphotransferase activity of the Rous sarcoma virus src gene product, pp60src, was inhibited both in vitro and in vivo by the bioflavonoid quercetin. The Ki for the inhibitory effect was in the range of 6-11 microM under conditions in vitro. The inhibitory effect of quercetin was competitive towards the nucleotides ATP and GTP as substrates for pp60src and was non-competitive towards alpha-casein as the protein substrate of this kinase activity. In contrast, studies in vitro of the phosphotransferase activity of the catalytic subunit of the cAMP-dependent protein kinase showed that this flavonoid did not inhibit the phosphorylation of physiological substrates of this enzyme. In cultured cells the half-maximal inhibition of tyrosine phosphorylation of pp60src as well as the phosphorylation of the Mr = 34000 protein, a physiological substrate of pp60src, was in the range 0.06-0.08 mM.
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PMID:The effect of quercetin on the phosphorylation activity of the Rous sarcoma virus transforming gene product in vitro and in vivo. 631 42

A rapid reverse-phase high performance liquid chromatography (HPLC) method is presented for isolating the alpha, beta, gamma, and delta subunits of rabbit muscle phosphorylase kinase. The HPLC separation allows micropreparative purification of all the subunits with 66-88% recoveries. Relative molecular weights of the subunits as determined by sodium dodecyl sulfate gel electrophoresis in 4, 5, 7, and 10% acrylamide are alpha 132,000, alpha' 127,000, beta 113,000 and gamma 43,000. Amino acid compositions are reported for the HPLC purified subunits. alpha contains about 2 mol of endogenous phosphate/mol of protein and beta, gamma, and delta each contain about 1 mol of phosphate/mol of protein. Despite the identity of delta and calmodulin, essentially no protein-bound phosphate was found associated with bovine brain calmodulin. Holophosphorylase kinase contains about 20 mol of endogenous phosphate/mol of protein. The first NH2-terminal sequence analyses of the alpha, beta, and gamma subunits were determined by Tarr manual Edman degradation. Within the NH2-terminal 23 residues of gamma ( TRDAALPGSHSTHGFYENYESKE . . . ) there are six identities and one conservative interchange with the catalytic subunit of bovine cAMP-dependent protein kinase. The first 17 residues of the NH2-terminal sequence of alpha ( MRSRSNSGVRLDSYARL . . . ) exhibit six identities and one conservative interchange with the transforming protein from the Rous sarcoma virus (Schmidt- Rupin strain) provided a single gap is inserted in the src gene product. Further structural information is required to evaluate the significance of these sequence similarities. The beta subunit has a blocked NH2 terminus.
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PMID:High performance liquid chromatography purification and structural characterization of the subunits of rabbit muscle phosphorylase kinase. 672 54

Tyrosine kinase are important mediators of signal transduction in eukaryotic cells. In order to better understand the mechanism of catalysis we studied a set of mutants of the prototype tyrosine kinase, the c-Src protein, a homologue of the Rous Sarcoma virus oncogene. Based on an X-ray structure of cAMP-dependent protein kinase (cAPK) we mutated an arginine residue conserved in subdomain VI of all known kinases to a non-charged residue. This residue coordinates phosphate of the autophosphorylation site located in subdomain VII of cAPK and this interaction has been proposed to be crucial for substrate binding. The mutant R385A of c-Src had low kinase activity towards exogenous substrates yet was able to autophosphorylate at tyrosine 416. When introduced into an activated v-src gene the R385A mutation totally blocked cell transformation. Our data suggest that the function of the conserved arginine 385 is to coordinate the phosphate of the autophosphorylation site and to provide in this way a stable template for substrate binding.
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PMID:Catalytic activity and transformation potential of v-Src require arginine 385 in the substrate binding pocket. 752 17

gamma-PAK, originally designated PAK I and subsequently identified as a member of the p21-activated protein kinase family, has been shown to have cytostatic properties and to be involved in maintaining cells in a nondividing state [Rooney, R. D., et al., (1996) J. Biol. Chem. 271, 21498-21504]. The determinants for phosphorylation of substrates by gamma-PAK have been identified by examining the kinetics of phosphorylation of a series of synthetic peptides patterned after the sequence KKRKSGL, which is the site phosphorylated by gamma-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro. With these peptides, the recognition sequence for gamma-PAK has been shown to contain two basic amino acids in the -2 and -3 positions, as represented by (K/R)RXS, in which the -2 position is an arginine, the -3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino acid. A basic amino acid in the -1 or -4 position improves the rate of phosphorylation by increasing the Vmax and decreasing the Km. An acidic amino acid in the -1 position increases the rate (2.5-fold), as does an acidic residue in the -4 position, although to a lower extent (1.6-fold). Proline in the -1 or +1 position has a deleterious effect and inhibits phosphorylation by gamma-PAK. The substrate requirements of protein kinases that recognize basic amino acids on the N-terminal side of the phosphorylatable residue such as cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) have been compared with gamma-PAK using the same peptides. An acidic residue in the -1 position negatively affects PKA and PKC; thus, peptides containing the sequence KRES can be used to identify gamma-PAK.
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PMID:Determinants for substrate phosphorylation by p21-activated protein kinase (gamma-PAK). 940 39

The steroidogenic acute regulatory (StAR) protein is indispensable for maximal trophic hormone-stimulated steroidogenesis by the adrenal gland, testis, and ovary. Recently, our laboratory developed an in vitro primary culture system of porcine granulosa-luteal cells that retain responsiveness to LH and show LH and insulin [or insulin-like growth factor (IGF-I)] synergy in stimulating StAR messenger RNA accumulation. Here, we examine the mechanisms subserving this LH-insulin (IGF-I) augmentation. We corroborate LH's amplification of insulin as well as IGF-I-stimulated granulosa-luteal cell progesterone and cAMP accumulation (P < 0.001). Insulin or IGF-I elevated LH receptor transcript accumulation, and LH did not alter this effect. To determine the hormonal responsiveness of StAR promoter, truncated regions of the -1423 to +130 bp upstream sequence of the porcine gene were ligated into a firefly luciferase reporter plasmid. Transient transfection of the StAR plasmid containing the full-length porcine 5'-flanking region of StAR (pStAR1423/luc) showed superadditive stimulation by LH and insulin or IGF-I after 24 h. LH, but not insulin or IGF-I alone, stimulated pStAR1423/luc activity. Deletion of the proximal putative steroidogenic factor-1 (-48 to -41) site abolished hormonally driven StAR promoter activity. A stable cAMP analog, 8-bromo-cAMP (1 mM), and insulin/IGF-I also evoked supraadditive StAR promoter expression. To further explore the role of cAMP in LH-insulin (or IGF-I) actions, we cotransfected a Rous sarcoma virus (RSV)-driven minigene encoding the heat-stable inhibitor of the cAMP-dependent protein kinase (RSV/PKI) or a mutant plasmid (RSV/PKImut) along with the pStAR1423/luc promoter construct. Cotransfection of PKI, but not PKImut, with pStAR1423/luc significantly attenuated LH's stimulation of luciferase activity and also reduced the magnitude of the transcriptional amplification exerted by LH and insulin or IGF-I. In corollary analyses of the protein kinase A (PKA) pathway, cotransfection of full-length pStAR1423/luc and a complementary DNA encoding a constitutively activated PKA catalytic subunit elevated basal and insulin (or IGF-I)-stimulated StAR promoter expression. LH and insulin (or IGF-I) also augmented steady state StAR transcript levels, as assessed by homologous RT-PCR, and StAR protein concentrations, as evaluated by Western blotting. Together, these investigations document a significant role for insulin or IGF-I in enhancing LH-stimulated progesterone and cAMP biosynthesis and endogenous StAR message and protein accumulation and in augmenting cAMP-PKA-dependent transcriptional activation of the exogenous StAR promoter.
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PMID:Concerted regulation of steroidogenic acute regulatory gene expression by luteinizing hormone and insulin (or insulin-like growth factor I) in primary cultures of porcine granulosa-luteal cells. 1108 28


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