Old Dominion University ODU Digital Commons Chemistry & Biochemistry Faculty Publications Chemistry & Biochemistry 1995 Activation of Transfer RNA-Guanine Ribosyltransferase by Protein Kinase C Rana C. Morris Old Dominion University Bonnie J. Brooks Old Dominion University Panayota Eriotou Old Dominion University Deborah F. Kelly Old Dominion University Sandeep Sagar Old Dominion University See next page for additional authors Follow this and additional works at: https://digitalcommons.odu.edu/chemistry_fac_pubs Part of the Biochemistry Commons, Cell Biology Commons, and the Molecular Biology Commons Repository Citation Morris, Rana C.; Brooks, Bonnie J.; Eriotou, Panayota; Kelly, Deborah F.; Sagar, Sandeep; Hart, K. Lenore; and Elliot, Mark S., "Activation of Transfer RNA-Guanine Ribosyltransferase by Protein Kinase C" (1995). Chemistry & Biochemistry Faculty Publications. 163. https://digitalcommons.odu.edu/chemistry_fac_pubs/163 Original Publication Citation Morris, R. C., Brooks, B. J., Eriotou, P., Kelly, D. F., Sagar, S., Hart, K. L., & Elliott, M. S. (1995). Activation of transfer RNA-guanine ribosyltransferase by protein kinase C. Nucleic Acids Research, 23(13), 2492-2498. doi:10.1093/nar/23.13.2492 This Article is brought to you for free and open access by the Chemistry & Biochemistry at ODU Digital Commons. It has been accepted for inclusion in Chemistry & Biochemistry Faculty Publications by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. Authors Rana C. Morris, Bonnie J. Brooks, Panayota Eriotou, Deborah F. Kelly, Sandeep Sagar, K. Lenore Hart, and Mark S. Elliot This article is available at ODU Digital Commons: https://digitalcommons.odu.edu/chemistry_fac_pubs/163 2492-2498 Nucleic Acids Research, 1995, Vol. 23, No. 13 Q-D) 1995 Oxford University Press Activation of transfer RNA-guanine ribosyftransferase by protein kinase C Rana C. Morris, Bonnie J. Brooks, Panayota Eriotou, Deborah F. Kelly, Sandeep Sagar, K. Lenore Hart and Mark S. Elliott* Old Dominion University, Department of Chemistry and Biochemistry, Norfolk, VA 23529, USA Received March 16, 1995; Revised and Accepted May 23, 1995 ABSTRACT nucleosides have undefined functions in the molecular physiology of the cell. Yet, various individual members of this group of Transfer RNA-guanine ribosyltransferase (TGRase) modifications have been suggested to play important roles in the irreversibly incorporates queuine into the first position function of tRNA. One specific example is the queuosine in the anticodon of four tRNA isoacceptors. Rat brain modification of tRNA. The pre-formed base, queuine, is incorpo- protein kinase C (PKC) was shown to stimulate rat liver rated into tRNA by an irreversible post-transcriptional exchange of TGRase activity. TGRase preparations derived from rat queuine-for-guanine in the first position of the anticodon of four liver have been observed to decrease in activity over tRNA isoacceptors (aspartyl, asparaginyl, tyrosyl and histidyl- time in storage at -20 or -700C. Contamination of the tRNAs) (1-4). This unique base exchange reaction is catalyzed by samples by phosphatases was indicated by a p-nitro- the enzyme tRNA-guanine ribosyltransferase (TGRase) (EC phenylphosphate conversion test. The addition of 2.4.2.29) (3,4). micromolar concentrations of the phosphatase inhibi- A of the function of queuine in tors sodium pyrophosphate and sodium fluoride into great deal research regarding TGRase isolation buffers resulted in a greater return of tRNA has been undertaken. The overall picture that emerges TGRase activity than without these inhibitors. Inactive positions queuosine-modified tRNA as a controlling agent in the TGRase preparations were reactivated to their original differentiation, development and stress management of several activity with the addition of PKC. In assays combining cell types. Alterations in the levels of queuosine-modified tRNA both TGRase and PKC enzymes, inhibitors of protein have been observed during differentiation and development for kinase C (sphingosine, staurosporine, H-7 and D.discoideum (5), plants (6) and Drosophila (7-9), and during calphostin C) all blocked the reactivation of TGRase, development and aging in the rat (10). Queuosine-modified whereas activators of protein kinase C (calcium, tRNA in E.coli appearto protect the organism from stress invoked diacylglycerol and phosphatidyl serine) increased the by suboptimal growth conditions (11). Queuosine-modified activity of TGRase. None of the PKC modulators tRNA also appears to be involved with lactate dehydrogenase affected TGRase activity directly. Alkaline phospha- (12,13) and cytochrome b559 (14) expression in mammalian tase, when added to assays, decreased the acfivity of systems, and therefore may be involved with management of TGRase and also blocked the reactivation of TGRase oxidative stress in eukaryotic cells. with PKC. Denaturing PAGE and autoradiography was Transfer RNA isolated from neoplastic tissues and transformed performed on TGRase isolates that had been labelled cell lines is queuosine-hypomodified to various degrees (2,15,16). with 32p by PKC. The resuflting strong 60 kDa band The degree of hypomodification has been related to the staging (containing the major site for phosphorylation) and toward malignancy in human lymphomas and leukemias (17), and weak 34.5 kDa band (containing the TGRase activity) in lung (18) and ovarian tumors (19). Queuosine-deficient murine are suggested to associate to make up a 104 kDa erythroleukemia cells demonstrated a significant increase in tRNA heterodimer that comprises the TGRase enzyme. This queuosine levels when forced to differentiate (20). The differenti- was corroberated by native and denaturing size- ation of these cells and the corresponding increase in queuosine exclusion chromatography. These results suggest that level were effectively blocked by chronic treatment with the tumor PKC-dependent phosphorylation of TGRase is tied to promoter TPA (20). These data suggest that undermodification of efficient enzymatic function and therefore control of tRNA with queuosine is related to growth enhancing or tumor the queuine modification of tRNA. promoting events. Chronic exposure of the phorbol ester tumor promoter PDD to INTRODUCTION cultured normal human fibroblasts induced a large and transient inhibition of queuosine modification levels in tRNA (21). The There are a large number ofmodified nucleosides found in transfer decrease in queuosine content of tRNA always occured immedi- RNA (tRNA) molecules. For the most part, these modified ately before an increase in the population density of the cultures * To whom correspondence should be addressed Nucleic Acids Research, 1995, Vol. 23, No. 13 2493 (21). Concurrent addition of exogenous queuine effectively Mono-Q buffer Ten mM Tris-Cl, pH 7.5, 1 mM MgCl2 and 0.5 blocked the PDD-induced increase in population density, and mM dithiothreitol. maintained the queuosine modification at near normal levels. In addition, it was demonstrated that PDD was unable to directly Reaction buffer. Ten mM Tris-Cl, pH 7.5, 35 mM KCl, 1 mM inhibit TGRase preparations in vitro (22). These data imply an MgCl2, 0.5 mM dithiothreitol. indirect mechanism of action for phorbol esters on TGRase, Denaturing sample buffer Ten mM Tris-Cl, pH 7.4, 0.5% SDS, queuosine modification of tRNA, and their relationship to cell 20% glycerol and 0.01% bromophenol blue. growth. Mammals are incapable ofsynthesizing their own queuine, and must obtain it from their diet or gut flora (23,24). Mammalian Isolation of TGRase from rat liver cells grown in culture obtain from serum queuine animal used to The purification of TGRase was performed by adaptation of supplement the growth media (3,25). This suggests that efficient previously published methods (29). Five adult Sprague-Dawley cellular uptake of across the dietary queuine cell membrane is a rats were sacrificed by cervical dislocation and their livers were prerequisite for the insertion of the base into tRNA by TGRase. rapidly excised and rinsed in ice-cold homogenization buffer. The It was demonstrated that cellular uptake of queuine is sensitive to tissue was homogenized in 100 ml buffer, filtered through two phorbol esters (22,26), and more recently that queuine is uptake layers of sterile gauze and centrifuged at 10 000 g for 30 min at modulated by protein kinase C (PKC) activity (27). Activators of 4°C. The resulting supernatant was centrifuged at 100 000 g for PKC-stimulated queuine uptake in cultured human fibroblasts, 1 h at 4°C. The supematant was decanted through sterile gauze, whereas inhibitors of PKC reduced queuine to uptake a measur- brought to a final volume of 300 ml in homogenization buffer and able base-line level (27). loaded by gravity feed onto a 13 x 5 cm DE-52 ion exchange Chronic exposure of cultured cells to phorbol esters has been pre-equilibrated with homogenization buffer. A 2 1 wash of shown to induce a down-regulation of PKC (28). We have homogenization buffer lacking the protease inhibitors and Triton observed a decrease in the of tRNA with queuosine-modification X-100 was drawn through the column by low pressure vacuum chronic exposure of PDD to human and a of fibroblasts, reversal using a water-aspirator. TGRase was eluted from the column with this phenomenon by concurrent addition of exogenous queuine 11 ofhomogenization buffer containing 0.2 M KCl. Protein in the (21). If PKC levels were diminished in cells chronically exposed 0.2