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Title: Pancreatic duodenum homeobox 1 (PDX-1) phosphorylation in pancreatic β-cells
Author: An, Rong
ISNI:       0000 0004 2687 1445
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2009
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Pancreatic Duodenum Homeobox (PDX-1) is a homeodomain transcription factor and a key regulator of β-cell development and differentiation. In mice and humans, mutations of the Pdx1 gene in β-cells have been linked to MODY and type 2 diabetes. Therefore, the regulation of PDX-1 transcriptional activity is very important. Phosphorylation is known to be a pivotal regulatory mechanism for several transcription factors. Therefore in this study, I focused on the regulation of phosphorylation on PDX-1 in intact β-cells and investigated the potential signalling pathways involved in its regulation. Firstly I showed that PDX-1 is phosphorylated under physiological conditions in pancreatic β-cell line. Additionally, recombinant PDX-1 is efficiently phosphorylated in vitro at Thr- 152 in the homeodomain by the nutrient-regulated protein kinase, PASK. To explore the physiological relevance of this site I made dephospho- (T152A) and phosphomimetic (T152E) mutants of PDX-1. In INSrαβ cells, a less differentiated cell line derived from INS-1 cells, wild-type PDX-1 and PDX-1T152A were partially redistributed from the cytosol to the nucleus in response to elevated glucose concentrations (20 vs 3 mM). By contrast, PDX-1T152D and PDX-1T152E displayed enhanced nuclear immunoreactivity at low glucose concentrations, and underwent nuclear export as glucose concentrations were raised. PDX- 1 binding to an oligonucleotide based on the consensus A3 binding site of the preproinsulin gene promoter was completely abolished by mutation of Thr-152 to an acidic amino acid. Furthermore, the PDX-1 phosphomimetic mutants failed to suppress preproglucagon gene expression in INSrαβ cells, an action fully preserved in the T152A mutant. However, metabolic labelling of INS-1(832/13) cells with 32Pi, as well as mass spectroscopic analysis, and the use of a phospho Thr-152-specific antibody, failed to reveal any clear evidence for phosphorylation at this site under a variety of in vivo conditions, including in cells overexpressing PASK. Thus, Thr-152 is not a major site of phosphorylation of PDX-1 in intact β-cells. However, a novel phosphorylation site, Ser-269, at the C-terminus of PDX-1 was identified by mass spectrometry. To explore the potential upstream regulatory kinase and the signalling pathway involved, I generated dephospho-(S269A) and phosphomimetic (S269E) mutant PDX-1 and a phospho-Ser-269-specific antibody. Candidate kinases were also identified using recombinant wild-type and mutant PDX-1 in in vitro phosphorylation experiments. In this thesis I demonstrate that PDX-1 is phosphorylated under physiological conditions in β-cell lines. I have shown that Thr152 is not a major phosphorylation site of PDX-1 in intact cells and we also defined the novel in vivo phosphorylation site of PDX-1. Moreover, we reported that PDX-1 is phosphorylated in vitro by a number of kinases including Aurora, Rsk1, PKCa, JNK. The work presented in the thesis provides evidence that PDX-1 phosphorylation is important in its role as a transcription regulator in mature β-cells; moreover, it suggests several physiological signalling networks to investigate.
Supervisor: Rutter, Guy ; Da Silva Xavier, Gabriela Sponsor: Overseas Research Student Award
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available