The phosphorylation of ribosomal proteins in baby hamster kidney fibroblasts

Kennedy, Iain M (1982) The phosphorylation of ribosomal proteins in baby hamster kidney fibroblasts. PhD thesis, University of Glasgow.

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Abstract

This thesis describes studies directed towards understanding the function of the phosphorylation of a particular ribosomal protein - S6 - in eukaryotic cells. Two experimental approaches to determine the extent of phosphorylation of ribosomal protein S6 were adopted. One involved labelling the cells with radioactive orthophosphate and quantitatively estimating the extent of phosphorylation of the ribosomes by measuring the radioactivity incorporated into the protein and the specific radioactivity of the presumed precursor, ATP, The other involved qualitative estimation of the phosphorylation by analysing the ribosomal proteins in a system of gel electrophoresis in which the phosphorylated derivatives of the protein are resolved from the unphosphorylated protein. This latter method had the advantage that it could be applied when it was not possible to label certain subcellular fractions of ribosomes with (32P)-orthophosphate. It had previously been found that the phosphorylation of ribosomal protein S6 was greater in pre-confluent hamster fibroblasts than in confluent fibroblasts. Part of this thesis describes attempts to determine whether this was due to there being a greater proportion of highly phosphorylated newly-synthesised ribosomes in the pre-confluent cells, as might have been expected if the function of the phosphorylation was in the nucleus. The extent of phosphorylation of ribosomal protein S6 in nucleoli was first examined electrophoretically after labelling the cells with (35S)-methionine, isolating the nucleoli and extracting their protein. However this approach was unsuccessful because of insufficient incorporation of radioactivity into the nucleolar ribosomal proteins. When nucleoli were incubated in vitro with (32P)-ATP, histones became labelled with showing that protein kinase activity was present. However no radioactivity could be detected in ribosomal protein S6, so that the extent (if any) of its phosphorylation could not be determined in this way. The extent of phosphorylation of ribosomal protein S6 was also investigated in 'native' ribosomal subunits, as they include ribosomal subunits which have newly entered the cytoplasm from the nucleus. Proteins from 'native' ribosomal subunits were again analysed by two-dimensional gel electrophoresis. The results here were complicated by the presence of non-ribosomal proteins, but seemed to indicate that ribosomal protein S6 was in a low state of phosphorylation in 'native' 40S subunits. This conclusion was finally confirmed when the extent of phosphozylation of ribosomal protein S6 was compared in newly-synthesised and older cytoplasmic ribosomes. These two populations of ribosomes were identified by differentially labelling the cells with (35S)-methionine, either for 20 minutes before harvesting (newly-synthesised ribosomes), or with a 3 hour pulse of (35S)-methionine followed by a 'chase' with non-radioactive methionine for 2 days before harvesting (2-day old ribosomes). Electrophoretic analysis clearly showed that both newly-synthesised and 2-day old ribosomes had ribosomal protein S6 in a low state of phosphorylation. These results demonstrated that newly- synthesised ribosomes were no more phosphorylated than older ribosomes, in contrast to the original hypothesis. This therefore suggested that the proposal that the function of the phosphorylation was in the nucleus was incorrect. The remainder of the work described in this thesis was concerned with the phosphorylation of ribosomal proteins in cells infected with, or transfoimed by viruses. It had been reported that in cells infected with several different viruses the (35P)-labelling of ribosomal protein S6 increased, although no change in the position of electrophoretic migration of the protein was detected. The work described here clearly demonstrates that in cells infected with pseudorabies virus there is an increase of between 350% and 750% in the extent of phosphorylation of ribosomal protein S6, as estimated by both of the methods mentioned above. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: David Leader
Keywords: Biochemistry
Date of Award: 1982
Depositing User: Enlighten Team
Unique ID: glathesis:1982-72480
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: http://theses.gla.ac.uk/id/eprint/72480

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