Jacob, Sara Mae; (1993) An investigation into the post-translational modifications of Annexin VI. Masters thesis (M.Phil), UCL (University College London).

Text An_investigation_into_the_post.pdf Download (8MB)

Abstract

The annexins are a major, novel class of calcium/phospholipid-binding proteins, the functions of which are unknown. To date, there are thirteen members of this family cloned from species diverse as humans to Dictyostelium. The canonical annexin structure comprises four internal repeats- each of approximately 70 amino acids, with the exception of annexin VI, a 68 kDa protein comprised of eight repeats. Annexins I and II are known to be in vivo substrates for cellular protein kinases. To date, of all the other annexins, only annexin VI has been shown to be phosphorylated in whole cells. Since phosphorylation is known to play a major role in signal transduction and cell transformation, the aim of this project was to characterize the phosphorylation of annexin VI. In studies using murine Swiss 3T3 fibroblasts, it became apparent that annexin VI is phosphorylated in a growth-dependent manner on both serine and threonine. However, an unexpected result was that the majority of phosphorus (when cells were 32Pi-labelled over a 24h period) was incorporated into two novel phospho-entities (which I call phospho-X and phospho-Z) that were not known phosphoamino-acids. Many proteins are post-translationally modified, and in many instances the identification of these modifications can provide insight into the proteins' function. There are several post-translational modifications in which phosphorus forms part of the structure, including ADP-ribosylation and the glycosylphosphatidylinositol (GPI) anchor. However, these possibilities were eliminated on the basis of metabolic labelling experiments. Mass spectroscopy suggested that phospho-X was a low molecular weight entity. Due to either the chemical nature of the post-translational modification or the peptide to which it was attached, conventional site-mapping techniques were ineffective in identifying the modified residues in annexin VI. However, limited chemical cleavage with N-chlorosuccinimide clearly indicated that annexin VI is modified at more than one site, one lying close to the N-terminus and the other in the C-terminal half of the protein. To facilitate the site-mapping studies, annexin VI was expressed in the heterologous cell-line A431 by stable transfection. Annexin VI in the transfected cells was shown to incorporate phospho-X and phospho-Z. In conclusion, these data describe both a potentially novel post-translational modification of annexin VI and a model system for its further study.

Download activity - last month

Export as JSONXMLCSV

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item