Activation of transfer RNA-guanine ribosyltransferase by protein kinase C

Rana C. Morris, Bonnie J. Brooks, Panayota Eriotou, Deborah F. Kelly, Sandeep Sagar, K. Lenore Hart, Mark S. Elliott

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Abstract

Transfer RNA-guanine ribosyltransferase (TGRase) irreversibly incorporates queuine into the first position in the anticodon of four tRNA isoacceptors. Rat brain protein kinase C (PKC) was shown to stimulate rat liver TGRase activity. TGRase preparations derived from rat liver have been observed to decrease in activity over time in storage at -20 or -70°C. Contamination of the samples by phosphatases was indicated by a p-nitrophenylphosphate conversion test. The addition of mlcromolar concentrations of the phosphatase inhibitors sodium pyrophosphate and sodium fluoride into TGRase isolation buffers resulted in a greater return of TGRase activity than without these inhibitors. Inactive TGRase preparations were reactivated to their original activity with the addition of PKC. In assays combining both TGRase and PKC enzymes, inhibitors of protein kinase C (sphingosine, staurosporlne, H-7 and calphostin C) all blocked the reactivation of TGRase, whereas activators of protein kinase C (calcium, dlacylglycerol and phosphatidyl serine) increased the activity of TGRase. None of the PKC modulators affected TGRase activity directly. Alkaline phosphatase, when added to assays, decreased the activity of TGRase and also blocked the reactivation of TGRase with PKC. Denaturing PAGE and autoradiography was performed on TGRase isolates that had been labelled with 32P by PKC. The resulting strong 60 kDa band (containing the major site for phosphorylation) and weak 34.5 kDa band (containing the TGRase activity) are suggested to associate to make up a 104 kDa heterodime that comprises the TGRase enzyme. This was corroberated by native and denaturing sizeexclusion chromatography. These results suggest that PKC-dependent phosphorylation of TGRase is tied to efficient enzymatic function and therefore control of the queuine modification of tRNA.

Original languageEnglish (US)
Pages (from-to)2492-2498
Number of pages7
JournalNucleic acids research
Volume23
Issue number13
DOIs
StatePublished - Jul 11 1995

All Science Journal Classification (ASJC) codes

  • Genetics

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    Morris, R. C., Brooks, B. J., Eriotou, P., Kelly, D. F., Sagar, S., Lenore Hart, K., & Elliott, M. S. (1995). Activation of transfer RNA-guanine ribosyltransferase by protein kinase C. Nucleic acids research, 23(13), 2492-2498. https://doi.org/10.1093/nar/23.13.2492