Hepatitis C virus RNA-dependent RNA polymerase (NS5B polymerase).
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Amino Acid Sequence↗
Biomedical subjects
C H Hagedorn
Publications and source records attributed to C H Hagedorn.
Expression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli.
The hepatitis C virus (HCV) represents a major public health problem that can produce liver failure and hepatocellular carcinoma in chronically infected patients. Our goal was to express the HCV non-structural protein 5B (NS5B) protein of HCV genotype 1a in Escherichia coli and initiate studies of its role in HCV genomic replication. In this report we demonstrate that a recombinant NS5B protein with an amino terminal sequence of ASMSYSWTG has RNA-dependent RNA polymerase (RDRP) activity. This recombinant enzyme was active in poly(U) polymerase assays and produced template-sized RNA products when globin mRNA was used as a template. The polymerase activity of recombinant NS5B was primer-dependent and was active for at least 60 min of incubation at 30 degrees C. Deletion of the carboxyl terminal region of HCV NS5B resulted in a loss of RDRP activity indicating that the enzymatic activity observed was due to the full-length recombinant enzyme. Recombinant NS5B (RDRP) should assist in understanding the mechanism of HCV replication and the identification of specific enzyme inhibitors.
Escherichia coli↗
Identification of the cap binding domain of human recombinant eukaryotic protein synthesis initiation factor 4E using a photoaffinity analogue.
Binding of eIF-4E to the 5' m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the molecular basis for this recognition, photoaffinity labeling with [gamma-32P]8-N3GTP was used in binding site studies of human recombinant cap binding protein eIF-4E. Competitive inhibition of this cap analogue by m7GTP and capped mRNA indicated probe specificity for interaction at the protein binding site. Saturation of the binding site with [gamma-32P]8-N3GTP further demonstrated the selectivity of photoinsertion. Aluminum (III)-chelate chromatography and reverse-phase HPLC were used to isolate the binding site peptide resulting from digestion of photolabeled eIF-4E with modified trypsin. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113-Arg 122.Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [gamma-32P]8-N3GTP. A detailed understanding of eIF-4E.m7G mRNA cap interactions may lead the way to regulating this essential protein-RNA interaction for specific mRNA in vivo.
Affinity Labels↗
Expression of functional eIF-4Ehuman: purification, detailed characterization, and its use in isolating eIF-4E binding proteins.
Protein-mRNA cap interactions represent a critical point for regulating gene expression in vivo. For example, a rapid stimulation of gene expression at the mRNA level is mediated by insulin regulating the availability of functional cap-binding protein (eIF-4E). In addition, several viruses modify cap binding proteins to regulate host vs viral gene expression. However, little is known about the molecular details of eIF-4E interactions with m7GTP mRNA caps, with regulatory proteins (e.g., eIF-4E binding proteins), and with proteins within the eIF-4F complex. To study these protein-mRNA and protein-protein interactions in mammalian systems we have constructed a T7 polymerase-driven expression vector containing the coding sequence for human eIF-4E. Recombinant eIF-4Ehuman was purified in a functional state by m7GTP affinity chromatography and Mono Q FPLC. This recombinant protein has biological and physical characteristics that are similar or identical to native eIF-4E. Fluorescence titration studies determined the equilibrium constant for recombinant eIF-4E/m7GTP binding to be 10.1 +/- 0.3 x 10(5) M(-1). To isolate eIF-4E binding proteins, recombinant eIF-4E was linked to agarose beads and incubated with cell lysates. Several proteins were isolated, including a 220-kDa protein that was confirmed to be the p220 subunit of eIF-4F by its proteolysis during incubation with lysates of poliovirus-infected cells. We conclude that recombinant eIF-4E produced in Escherichia coli provides a useful tool for studying eIF-4E/protein and eIF-4E/mRNA cap interactions and their role in regulating mammalian gene expression.
Chromatography, Affinity↗
Erythropoietin stimulates phosphorylation of eIF-4E and identification of a 37-kD phosphoprotein that binds mRNA caps in erythroblasts.
To explore the mechanism of erythropoietin action on differentiation of erythroblasts, we have examined its effect on regulating phosphorylation of the 25-kD mRNA cap binding protein (eIF-4E). Erythroblasts from the spleens of mice infected with the anemia strain of Friend virus (FVA cells) were studied. Erythropoietin stimulated phosphorylation of eIF-4E in FVA cells within 30 minutes, and this effect was maximal at 60 minutes. Phosphoamino acid analysis and tryptic phosphopeptide map analysis of eIF-4E isolated from both control and erythropoietin-treated cells identified a predominant phosphopeptide containing phosphoserine. However, when cells were incubated with 1 muM okadaic acid, eIF-4E was phosphorylated on both serine and threonine residues and three additional tryptic phosphopeptides were detected. We also identified a 37-kD phosphoprotein (pp37) that bound specifically to the m7GTP cap structure and coimmunoprecipitated with eIF-kD protein was phosphorylated on both serine and threonine residues. These results indicate that phosphorylation of eIF-4E is a target in erythropoietin-initiated signal transduction events and that this phosphorylation precedes observable effects of erythropoietin on macromolecular biosynthesis. Although of pp37 remains to be studied, it may represent a developmentally regulated mRNA cap binding protein.
Anemia↗
Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis.
We report the molecular cloning and characterization of a 49-kDa form of casein kinase I from rat testis. A cDNA clone encoding the enzyme, designated casein kinase I delta, contained an open reading frame of 1284 nucleotides that predicts a polypeptide of 428 amino acids with a M(r) of 49,121. The predicted amino acid sequence shares 76% identity with casein kinase I alpha, a 37-kDa form recently cloned from bovine brain (Rowles, J., Slaughter, C., Moomaw, C., Hsu, J., and Cobb, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9548-9552), and 65% identity with HRR25, a 57-kDa form of casein kinase I from yeast shown to be involved in DNA repair (Hoekstra, M. F., Liskay, R. M., Ou, A. C., DeMaggio, A. J., Burbee, D. G., and Heffron, F. (1991) Science 253, 1031-1034). Northern analysis of rat or rabbit RNA revealed three hybridizing species of 3.5-4.1, 2.2, and 1.9 kilobase pairs (kb). The largest message was detected in all tissues examined, whereas the 1.9- and 2.2-kb species were found predominantly in testis. A probe corresponding to the 3'-untranslated region of the casein kinase I delta cDNA hybridized only to the 1.9-kb transcript. Expression of the casein kinase I delta cDNA in Escherichia coli resulted in active enzyme that phosphorylated casein, phosvitin, and the peptide substrate DDDDVASLPGLRRR. Enzyme activity was associated with a predominant polypeptide of 55-kDa, although COOH-terminal degradation products of 50 and 42 kDa were also present in partially purified enzyme. Recombinant casein kinase I delta was inhibited by the specific casein kinase I inhibitor, CKI-7, half-maximally at 12 microM. Heparin inhibited recombinant casein kinase I delta when phosvitin was the substrate, with half-maximal inhibition at 11.5 micrograms/ml. However, if the peptide substrate was used, heparin activated recombinant casein kinase I delta 4-5-fold, with half-maximal activation at 9.5 micrograms/ml. A truncated form of casein kinase I delta, lacking the COOH-terminal 111 amino acids, was no longer activated by heparin. Casein kinase I delta therefore represents a separate member of the casein kinase I family distinguished by its larger size and unique kinetic behavior with respect to heparin.
Amino Acid Sequence↗
Novel phosphorylation sites of eukaryotic initiation factor-4F and evidence that phosphorylation stabilizes interactions of the p25 and p220 subunits.
Only serine phosphorylation of eukaryotic initiation factor-4E (eIF-4E) has been previously reported in intact cells. We found that treatment of HepG2 cells with okadaic acid resulted in as much as 20% of eukaryotic initiation factor (eIF)-4E phosphorylation occurring on threonine residues and that tryptic phosphopeptide maps showed several previously unrecognized phosphopeptides. Analysis of p220 from control and okadaic acid-treated cells demonstrated serine and threonine phosphorylation under both conditions. However, a unique pattern of phosphopeptides in okadaic acid-treated cells was observed. The most notable finding was that hyperphosphorylation of eIF-4E and p220 increased binding of p220 but not eIF-4E to the m7GTP cap structure. We suggest that phosphorylation of eIF-4E is more complicated than previously recognized and that hyperphosphorylation of eIF-4E and p220 recruits more p220 into the protein complex that associates with mRNA caps. A better understanding of these protein-protein and protein-mRNA interactions may aid the design of anti-sense directed chemistries that disrupt such interactions for a specific target mRNA (Baker, B.F., Miraglia, L., and Hagedorn, C. H. (1992) J. Biol. Chem. 267, 11495-11499).
Cells, Cultured↗
Modulation of eucaryotic initiation factor-4E binding to 5'-capped oligoribonucleotides by modified anti-sense oligonucleotides.
The 5' cap structure of eucaryotic mRNA plays a pivotal role in mRNA metabolism. This report demonstrates that anti-sense oligonucleotides equipped with 3'-overhanging nucleotides modulate the amount of recombinant human eucaryotic initiation factor-4E that binds to a 5'-capped oligoribonucleotide. The degree of inhibition or enhancement of protein binding is dependent upon the number and sequence of overhanging nucleotides. A 45% inhibition of complexation was observed by the addition of one 3'-overhanging guanosine residue. Addition of a second residue (+2/GN) resulted in a higher degree of inhibition, 77-88%. In contrast, addition of one adenosine residue enhanced the formation of the eucaryotic initiation factor-4E-m7GpppRNA complex by 213%. Modulation of protein interactions with the 5'-cap structure is likely to effect several biological events, including pre-mRNA processing, transport of the mRNA from the nucleus to the cytoplasm and translation of the target mRNA. This targeting strategy in anti-sense chemistry may have practical applications in experimental biology and medicine.
Base Sequence↗
Phosphoprotein phosphatase 2A dephosphorylates eIF-4E and does not alter binding to the mRNA cap.
The phosphorylation and dephosphorylation of the 25 kDa mRNA cap binding protein eukaryotic initiation factor-4E (eIF-4E) is regulated during different physiologic and pathophysiologic states that include cell growth and the late phase of adenovirus infection. We have found that okadaic acid is much more effective in increasing the phosphorylated fraction of eIF-4E than phorbol 12-myristate 13-acetate in Hep G2 cells. Phosphoprotein phosphatase 2A dephosphorylated eIF-4E isolated from both phorbol 12-myristate 13-acetate- or okadaic acid-treated cells, whereas alkaline and acid phosphatase were relatively ineffective. The ability of purified [35S]eIF-4E isolated from okadaic acid-treated cells to bind mRNA caps was compared to phosphoprotein phosphatase 2A-treated [35S]eIF-4E and found to be no different. This suggests that alternative explanations for the previously observed effects of eIF-4E phosphorylation on protein synthesis must be considered. In addition, our results indicate that the in vivo phosphorylation of eIF-4E is not catalyzed solely by protein kinase C.
Ethers, Cyclic↗
Yeast casein kinase I homologues: an essential gene pair.
We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues.
Amino Acid Sequence↗
Protein kinase C phosphorylates both serine and threonine residues of the mRNA cap binding protein eIF-4E.
Several lines of evidence indicate that phosphorylation of the 25 kDa mRNA cap binding protein (eIF-4E) stimulates the efficiency of translational initiation. While the protein kinases which catalyze this reaction in intact cells have not been completely identified, evidence suggests that protein kinase C phosphorylates serine residues of eIF-4E in intact cells. In this study we demonstrate that protein kinase C also phosphorylates threonine residues of recombinant human eIF-4E in vitro. Phosphorylation of threonine and serine was observed over a range of eIF-4E and salt concentrations. However, relatively low levels of phosphorylation were seen even under optimal conditions. Similar results were observed with native eIF-4E purified from human erythrocytes. These findings demonstrate that protein kinase C can phosphorylate both serine and threonine residues of eIF-4E in vitro, but suggest that protein kinase C may not be the primary enzyme that phosphorylates eIF-4E in vivo.
Amino Acid Sequence↗
Use of a monoclonal antibody (APE-1) directed against the conserved GTPEYLAPE motif present in protein kinases as an affinity purification reagent.
cDNA sequence studies have predicted the existence of protein kinases that have not been characterized at the protein level. This laboratory has previously shown that polyclonal rabbit antibodies directed against a highly conserved sequence motif, GTPEYLAPE, present in the catalytic domain of many protein kinases will react during immunoblots with several protein kinases containing the same or homologous motifs. In this report I describe the development of a mouse monoclonal antibody, designated APE-1, that reacts with the GTPEYLAPE motif. In addition, the use of this monoclonal antibody in the affinity purification of kinase activities from rabbit reticulocyte lysate is described. This approach may provide a general method for the purification of previously uncharacterized protein kinases that share regions which are homologous to the GTPEYLAPE motif. Refinements in this method may permit the rapid purification of nonabundant and/or rapidly inactivated protein kinases that have not been isolated using other approaches.
Amino Acid Sequence↗
Platelet-derived growth factor stimulates phosphorylation of the 25 kDa mRNA cap binding protein (eIF-4E) in human lung fibroblasts.
Platelet-derived growth factor exerts rapid effects on protein synthesis and polysome formation in cultured cells. We report that platelet-derived growth factor stimulates a rapid phosphorylation of eIF-4E in WI-38 human lung fibroblasts. The effect was dependent on both time and PDGF concentrations. Phosphoserine was the sole phosphoamino acid identified and tryptic phosphopeptide maps showed a single phosphopeptide under both control and PDGF conditions. Phosphorylation of eIF-4E may be of the events required for initiating entry into G1 and commitment into S phase of the cell cycle.
Amino Acids↗
Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F.
Eukaryotic initiation factor 4F, a multi-protein mRNA cap binding complex, was isolated by m7GTP-Sepharose affinity chromatography from human mammary epithelial cells (184A1N4) incubated with [32P] orthophosphate. Treatment of cells with epidermal growth factor resulted in enhanced phosphorylation of both p28 (eIF-4E) and p220 subunits. The identities of the p28 and p220 subunits were confirmed by immunoprecipitation. The phosphorylation was both rapid and sustained in duration; p28 attained maximal levels (2-3-fold) within 30 min of treatment and remained elevated for at least 2 h, while p220 reached one-half maximal levels by 30 min, and maximal levels (3-4-fold) by 2 h of treatment. Two phosphorylated isoforms of p28 and multiple phosphorylated forms of p220 were detected by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of 6 N HCl hydrolyzates of p28 and p220 isolated from epidermal growth factor-treated and control cells indicated that serine is the predominant phosphorylated amino acid in both instances. In no case was phosphotyrosine observed. Pretreatment of cells with 1 microM okadaic acid resulted in the hyperphosphorylation of both p28 and p220 subunits. These results suggest that mitogenic growth factors and cellular serine/threonine phosphatases (pp1 and/or pp2A) serve essential roles in regulating phosphorylation levels of eukaryotic initiation factor 4F and support the concept that translational control is a component of the signal transduction mechanisms involved in growth regulation.
Amino Acids↗
Casein kinase I phosphorylates the 25-kDa mRNA cap-binding protein.
The 25-kDa mRNA cap-binding protein (eIF-4E) exists in both phosphorylated and dephosphorylated forms in eukaryotic cells. Phosphorylated eIF-4E appears to be preferentially associated with 48 S initiation complexes and with the 220-kDa subunit of eIF-4F. In addition, dephosphorylation of eIF-4E has been observed during heat shock and mitosis which are accompanied by decreased protein synthesis. However, the control of eIF-4E phosphorylation and its regulatory role remain poorly understood. Using eIF-4E as a substrate we have identified and purified from rabbit reticulocytes a protein kinase that phosphorylates eIF-4E in vitro. This enzyme phosphorylated eIF-4E on both serine and threonine residues with an apparent Km of 3.7 microM. The molecular mass of the enzyme and specificity for substrates other than eIF-4E suggested that this enzyme was a species of casein kinase I. This was confirmed by comparing the phosphopeptide map of the purified reticulocyte enzyme with that of rabbit skeletal muscle casein kinase I and by comparing phosphopeptide maps of eIF-4E phosphorylated in vitro by each enzyme. We conclude that casein kinase I phosphorylates eIF-4E in vitro and suggest that eIF-4E may be phosphorylated by casein kinase I in intact cells under some physiologic conditions.
Animals↗
Development and characterization of polyclonal antibodies against a conserved sequence in the catalytic domain of protein kinases.
Using a synthetic oligopeptide (CGGGTPEYLAPEGGK) crosslinked to keyhole limpet hemocyanin we have raised polyclonal rabbit antibodies against a 9 residue homologous region found in the catalytic domain of most protein kinases. These antibodies reacted during Western immunoblotting with cAMP dependent protein kinase catalytic subunit, phosphorylase kinase gamma subunit and calcium calmodulin dependent protein kinase II which have homologous sequences of GTPEYLAPE, GTPSYLAPE and GTPGYLSPE, respectively. Five other protein kinases did not react with anti-GTPEYLAPE antibodies during Western immunoblotting. Affinity-purified antibodies were able to detect as little as 50 ng of cAMP dependent protein kinase and 200 ng of Ca2+/calmodulin dependent protein kinase II. Immunoblotting of A431 cell plasma membrane vesicles indicated the presence of an approximately 55 kDa protein that contains the conserved sequence and is likely to be a protein kinase. Antibodies directed against conserved sequences present in protein kinases, or possibly other enzymes, may be useful in identifying previously uncharacterized enzymes at the protein level.
Amino Acid Sequence↗
Induction of eIF-4E phosphorylation by the addition of L-pyrroline-5-carboxylic acid to rabbit reticulocyte lysate.
Addition of L-pyrroline-5-carboxylic acid to reticulocyte lysates inhibits protein synthesis and induced phosphoproteins of 25 and 14 kDa. The 25 kDa phosphoprotein had the same Mr and pI as phosphorylated eIF-4E. Incubation of lysates with L-pyrroline-5-carboxylic acid did not alter the crosslinking of eIF-4E to reovirus mRNA caps. These results suggest that modifications of the translational apparatus other than eIF-4E phosphorylation may mediate the inhibitory effect seen with L-pyrroline-5-carboxylic acid and/or that phosphorylation of eIF-4E may effect functions subsequent to its interaction with the mRNA cap such as protein-protein interactions with other cap-specific translation factors.
Animals↗
Phosphorylation of the p220 subunit of eIF-4F by cAMP dependent protein kinase and protein kinase C in vitro.
Changes in the extent of phosphorylation of the 25 kDa subunit of eIF-4F occur during several major biological events including mitosis and heat shock in mammalian cells and shortly after fertilization of sea urchin (Lytechinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by cGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and cGMP dependent protein kinases whereas p48 of eIF-4F was not. The effect of these phosphorylation events on eIF-4F function, its assembly or disassembly, susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigated.
Adenosine Triphosphate↗