Biomedical subjects

C H Hagedorn

Publications and source records attributed to C H Hagedorn.

At least 19 recordsLinked to original sources

PubMed1992

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↗

PubMed1992

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↗

PubMed1992

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↗

PubMed1992

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↗

PubMed1991

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↗

PubMed1991

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↗

PubMed1991

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↗

PubMed1991

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↗

PubMed1990

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↗

PubMed1988

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↗

PubMed1988

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↗

PubMed1988

Identification of a protein kinase activity in rabbit reticulocytes that phosphorylates the mRNA cap binding protein.

The 25 kDa mRNA cap binding protein can be purified in a partially phosphorylated state and the extent of its phosphorylation appears to be regulated during heat shock and mitosis in mammalian cells. We demonstrated that a nonabundant serine protein kinase activity exists in rabbit reticulocytes that phosphorylates the 25 kDa cap binding protein in both the free (eIF-4E) and complexed (eIF-4F) state. This kinase was not inhibited by the cAMP-dependent protein kinase inhibitory peptide IAAGRTGRRNAIHDILVAA, did not phosphorylate S6 ribosomal protein, did not phosphorylate p220 of eIF-4F as protein kinase C does and no other substrates for this kinase were apparent in reticulocyte ribosomal salt wash. The molecular identity of this kinase, the specific site(s) of eIF-4E that it phosphorylates and its in vivo regulatory role remain to be studied.

Animals↗

PubMed1988

Selective inhibition of proteins synthesized from different mRNA species in reticulocyte lysates containing L-pyrroline-5-carboxylic acid.

L-Pyrroline-5-carboxylic acid is a naturally occurring nonprotein amino acid present in human plasma that changes concentrations with diet. L-pyrroline-5-carboxylic acid inhibited net synthesis of globin in untreated reticulocyte lysates in a dose dependent manner. This inhibition was greater than that observed with equimolar GSSG or NADP+ and was prevented by a NADPH generating system. L-pyrroline-5-carboxylic acid also inhibited net synthesis of proteins from brome mosaic and alfalfa mosaic virus mRNAs to different extents. However, no effect on the translation of the naturally uncapped encephalomyocarditis virus mRNA was observed. In general, mRNAs that are considered strongly competitive, such as alfalfa mosaic virus 2 and 4, were more resistant to this inhibitory process. These results indicate that pyrroline-5-carboxylic acid can initiate a differential effect on proteins synthesized from different mRNA species by an as yet unidentified mechanism.

Animals↗

PubMed1988

Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides). The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vmax with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but less than 2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont.

Animals↗

PubMed1986

Demonstration of a NADPH-linked delta 1-pyrroline-5-carboxylate-proline shuttle in a cell-free rat liver system.

These studies indicate that the interconversions of delta 1-pyrroline-5-carboxylate and proline can function as a shuttle that generates extra-mitochondrial NADP+ and transfers hydride ions into mitochondria in a cell-free rat liver system. A phosphate-free buffer with high concentrations of triethanolamine and 2-mercaptoethanol prevented the cold inactivation of pyrroline-5-carboxylate reductase (EC 1.5.1.2) in liver extracts. This enzyme had an apparent KmNADPH that was 2% of the apparent KmNADH X VmaxNADPH was approx. 50% of VmaxNADH. Unlabeled proline was converted to [5-3H]proline in incubations containing liver soluble fraction, mitochondria and a [4S-3H]NADPH generating system. This demonstrated one turn of the proposed shuttle in a homologous liver system. [5-3H]Proline production increased linearly over 60 min and decreased by 87% or more when specific components were eliminated. Rotenone was required for maximal activity, suggesting that inhibition of delta 1-pyrroline-5-carboxylate efflux would be required for significant shuttle activity in vivo. Both the relative concentrations of NADPH and NADH in liver cytosol and the kinetic characteristics of liver pyrroline-5-carboxylate reductase predict that the described shuttle should be overwhelmingly linked to NADPH rather than NADH. A NADPH-linked delta 1-pyrroline-5-carboxylate-proline shuttle may occur in hepatocytes and function at specific times to regulate pathways limited by cytosolic [NADP+].

Animals↗

PubMed1986

Catalytic transfer of hydride ions from NADPH to oxygen by the interconversions of proline and delta 1-pyrroline-5-carboxylate.

Studies in reconstituted systems indicate that the interconversions of proline and delta 1-pyrroline-5-carboxylate can constitute a shuttle which transfers reducing equivalents into mitochondria as proline and oxidizing potential out as delta 1-pyrroline-5-carboxylate. The studies reported here determine if the transfer of reducing equivalents can be stoichiometrically greater than the utilization of shuttle intermediates. First, 3HOH production from [5-3H]proline was used to quantitate proline oxidation under conditions where delta 1-pyrroline-5-carboxylate could recycle back to proline in a system containing mitochondrial particles and pyrroline-5-carboxylate reductase. In parallel incubations the metabolic fate of [U-14C]proline was determined. 3HOH production continued to increase while no net change occurred in 14C-labeled proline and delta 1-pyrroline-5-carboxylate indicating catalytic activity in this system. In another system that contained intact mitochondria and pyrroline-5-carboxylate reductase, proline oxidation was quantitated by both [U-14C]proline recoveries and 3HOH production from [5-3H]proline. Proline oxidation was the same by both methods in incubations lacking NADPH and pyrroline-5-carboxylate reductase. However, it was significantly underestimated by [U-14C]proline recoveries as compared to 3HOH production in incubations containing all shuttle components. These results indicate catalytic functioning of the proposed shuttle in a reconstituted system containing intact mitochondria. This shuttle may function at specific times to catalytically generate cytosolic NADP+ and in turn regulate enzymes limited by [NADP+]. We suggest that the proposed shuttle may function to increase ribose-5-phosphate synthesis by the oxidative limb of the pentose phosphate pathway and inturn increase PP-ribose-P and purine synthesis during the initiation of cell growth.

Adenosine Triphosphate↗

PubMed1984

Pyrroline-5-carboxylate in human plasma.

Pyrroline-5-carboxylate is the intermediate in the interconversions of proline, ornithine, and glutamate. Recent studies have suggested that pyrroline-5-carboxylate has regulatory properties in a wide range of tissues. Physiologic studies in humans have been limited by the unavailability of a sufficiently sensitive assay for this compound. Until now pyrroline-5-carboxylate has not been detectable in plasma of normal humans or even in plasma of patients with type II hyperprolinemia. We now report a method for measuring pyrroline-5-carboxylate made possible by a preparation of purified Escherichia coli pyrroline-5-carboxylate reductase. This method is more sensitive than currently available methods by two or three logs and is applicable for all biologic fluids. We have quantitated pyrroline-5-carboxylate in normal plasma, urine, and saliva. In addition, we have found pyrroline-5-carboxylate levels 10x to 20x normal in two patients with type II hyperprolinemia. The ability to measure physiologic concentrations of pyrroline-5-carboxylate now enables further characterization of its role as a regulatory molecule.

Chromatography, Ion Exchange↗

PubMed1983

Transfer of reducing equivalents into mitochondria by the interconversions of proline and delta 1-pyrroline-5-carboxylate.

Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers 3H from [1-3H]glucose to NADP+ to delta 1-pyrroline-5-carboxylate and yields [3H]proline. The formation of [3H]proline depends on the presence of NADP, delta 1-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and delta 1-pyrroline-5-carboxylate reductase. The production of [3H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to delta 1-pyrroline-5-carboxylate by mitochondrial proline oxidase. delta 1-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by delta 1-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of delta 1-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.

Animals↗