The phosphorylation of eukaryotic ribosomal proteins

Rankine, Andrew David (1976) The phosphorylation of eukaryotic ribosomal proteins. PhD thesis, University of Glasgow.

Full text available as:
[img]
Preview
PDF
Download (36MB) | Preview

Abstract

The work described in this thesis extends studies on the phosphorylation of ribosomal proteins to Krebs Ascites and Baby Hamster Kidney cells. When these cells were incubated with [32P]-orthophosphate, radioactivity became associated with ribosomal proteins, and was shown to be in the form of phosphoproteins by both chemical and enzymic criteria. About 18 phosphoproteins were detected when protein extracted from 80s ribosomes was separated by polyacrylamide gel electrophoresis. However, many of these appeared to be non-ribosomal contaminants, for far fewer phosphoproteins were seen with purified ribosomes or ribosomal subunits. Five phosphoproteins were found on ribosomal subunits analysed by electrophoresis in gels containing sodium dodecyl sulphate, and three on gels containing urea at pH 4.5. This difference may have been due to some of the phosphoproteins being relatively acidic (and thus of low mobility at pH 4.5), or alternatively because of the poorer resolving power of the latter system of gel electrophoresis. The number of ribosomal phosphophrotein detected in this work is more than was originally reported for other tissues, but is still much less than has been found by workers who have phosphorylated ribosomes in vitro using protein kinases. The ribosomal subunit on which a pair of phosphoproteins of low molecular weight was located, differed for Ascites and Baby Hamster Kidney cells, the proteins were on the large subunit of Ascites cells, and on the small subunit of Baby Hamster Kidney cells. The most probable explanation of this result is that these proteins are at the interface between the ribosomal subunits. Such a location is consistent with the previously suggested role for these proteins in holding the subunits together as inactive monosomes. Two-dimensional gel electrophoresis was used to try to identify unequivocally the ribosomal phosphoproteins. Only one protein was definitely identified by this method; a phosphoprotein of the small subunit of both Ascites and Baby Hamster Kidney cells, which was found to be the protein designated S6 in the standard nomenclature. The radioactivity associated with S6 comigrated with an anodic 'tail' of the protein, which apparently represents a number of increasingly phosphorylated derivatives of S6. This observation meant that it was possible to estimate the extent of phosphorylation of S6 merely by staining the protein of the phosphorylated derivatives, even when these contained no radioactivity. Studies were performed to see whether the functional activity of ribosomes correlated in any way with the phosphorylation of ribosomal proteins (particularly S6). When protein synthesis was inhibited in Ascites cells, there was no change in either the specific radioactivity of the phosphate in the ribosomal protein, the number of phosphorylated proteins resolved by one-dimensional gel electrophoresis, or the visible extent of phosphorylation of S6 in protein analysed by two-dimensional gel electrophoresis. This result contrasts with studies on the phosphorylation of S6 in other tissues, and may be due to high levels of phosphoprotein phosphatase in Ascites cells. The phosphorylation of S6 was, however, very extensive in growing Baby Hamster Kidney cells, demonstrating that a high level of phosphorylation of S6 under physiological conditions does not require protein synthesis to be inhibited. In studies to investigate the relationship between cyclic AMP had no effect on the phosphorylation of S6 in Ascites cells, a result which again contrasts with those in other tissues. More significantly, the extent of phosphorylation of S6 in Baby Hamster Kidney cells under different growth conditions in vivo did not correlate with the cellular concentration of cyclic AMP, indicating that elevated levels of this nucleotide are not essential for the phosphorylation of S6 in vivo. There was greater phosphorylation of S6 in growing, rather than resting, Baby Hamster Kidney cells, a situation which was not altered when the resting cells were stimulated to grow for short periods b the addition of fresh medium. S6 was also found to be extensively phosphorylated in the livers of young mice, but not in those of adult mice. These results are discussed in relation to a model in which ribosomes are phosphorylated during synthesis and thereafter are normally slowly dephosphorylated. It is suggested that the phosphorylation of S6 may have a role in the assembly or extra-nuclear transport of ribosomes, or in the control of their lifespan in the cytoplasm.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: David Leader
Keywords: Molecular biology
Date of Award: 1976
Depositing User: Enlighten Team
Unique ID: glathesis:1976-73122
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: http://theses.gla.ac.uk/id/eprint/73122

Actions (login required)

View Item View Item

Downloads

Downloads per month over past year