Tumour protein D52 is hypothesised to be involved in regulated secretion in the pancreatic acinar cell, indicated by rapid phosphorylation in response to secretagogue stimulation. The phosphorylation status of TPD52 in response to glucose stimulation in the pancreatic β cell was analysed by an <i>in vitro</i> method involving the incorporation of <sup>32</sup>P-ATP into the TPD52 protein. Limitations of the system prevented the full confirmation of the rapid phosphorylation of TPD52 in response to glucose stimulation, however preliminary data suggested this was likely to occur. Bio-informatic phosphorylation site prediction was used and we hypothesised that CaMKII was the key enzyme phosphorylating TPD52 in the β cell at the serine phosphorylation site within the motif SPTFKsFEEKV. Proteomic analysis of the phosphorylated TPD52 isoform confirmed the novel identification of the motif phosphorylated by CaMKII. To determine the effect of TPD52 on regulated secretion we reduced TPD52 RNA levels using vector based siRNA. Two stable knockdown cell lines were created showing a 50 % and 80 % reduction in RNA levels. No difference was observed in glucose stimulated insulin secretion between TPD52 knockdown and control cells. Patch clamp experiments also showed no significant difference in capacitance changes following depolarisation between knockdown and control clones. Quantification of insulin granule number and size from TEM pictures confirmed a high level of inter-clonal variation. Focus shifted to explore the previously published protein interaction between TPD52 and MAL2 as both proteins have been identified within the β cell. The original interaction between MAL2 and TPD52 was confirmed; however, when natural isoforms with shorter N-terminal regions were substituted for TPD52 and MAL2 the interaction was diminished slightly. This suggests that the N-terminal regions are not integral to the interaction between the proteins, but are perhaps required for stability of the complex.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:499641 |
| Date | January 2009 |
| Creators | Manning, Yashka |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=25933 |
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