The glycerophosphoinositols (GPIs) are ubiquitous, bioactive metabolites that are produced by the phospholipase A2 IVa activity on the membrane phosphoinositides. Glycerophosphoinositol (GroPIns) and glycerophosphoinositol 4-phosphate (GroPIns4P) are the most active and well studied of the GPIs. When added exogenously, the GPls can enter cells and have multiple effects, such as modulation of actin cytoskeleton organisation in fibroblasts and reduction of the invasive potential of metastatic cells. To gain insights into the molecular mechanisms of the effects of the GPIs, I set out to identify their protein targets. Therefore, a proteomic approach based on high- throughput differential-LC-MS/MS analysis was used with the modified GPIs: with a biotin moiety bound to their glycerol backbone. The targets identified include proteins involved in cell signalling, cytoskeleton organisation, protein folding and metabolic processes. Among these, I focussed my attention on Src-homology phosphatase-l (Shpl), a well-known regulator of Src activation, as it might be related to the reported signalling pathway leading to GroPlns4P-mediated modulation of the actin cytoskeleton, which involves Src. Based on biochemical data, I provide evidence of a direct interaction between Shp 1 and both GroPIns4P .and GroPIns. During my project, Shpl was studied in the context of GroPIns4P-induced membrane ruffle formation in NIH 3T3 fibroblasts, where inhibition of enzymatic activity of Shp 1 completely abolished GroPIns4P-mediated reorganisation of the actin cytoskeleton. In addition, I have also shown that GroPIns4P treatment results in the dephosphorylation of the inhibitory tyrosine residue of Src, as a consequence of increased binding between Shp 1 and Src. A role for Shp 1 is also demonstrated in GroPIns-mediated inhibition of tumour cell invasion. In A375MM melanoma cells, GroPIns treatment results in inhibition of extracellular matrix degradation, and this activity is suppressed when the inactive mutant form of Shp 1 is expressed, while it is essentially unaffected by expression of the native enzyme. In agreement with these results, a lack of effect of GroPIns was observed also in Shpl knock-down cells. In conclusion, my PhD project has led to the definition of Shpl as the first direct GPI target that has been identified to date, and it reveals a positive role for Shpl in the mechanisms of action of both GroPIns4 P and GroPIns.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:578003 |
| Date | January 2012 |
| Creators | Varone, Alessia |
| Publisher | Open University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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