Pancreatic duodenum homeobox 1 (PDX-1) phosphorylation in pancreatic β-cells

Abstract

Pancreatic Duodenum Homeobox (PDX-1) is a homeodomain transcription factor and akey regulator of ?-cell development and differentiation. In mice and humans, mutations ofthe Pdx1 gene in ?-cells have been linked to MODY and type 2 diabetes. Therefore, theregulation of PDX-1 transcriptional activity is very important.Phosphorylation is known to be a pivotal regulatory mechanism for several transcriptionfactors. Therefore in this study, I focused on the regulation of phosphorylation on PDX-1 inintact ?-cells and investigated the potential signalling pathways involved in its regulation.Firstly I showed that PDX-1 is phosphorylated under physiological conditions in pancreatic?-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 thephysiological relevance of this site I made dephospho- (T152A) and phosphomimetic(T152E) mutants of PDX-1. In INSr?? cells, a less differentiated cell line derived fromINS-1 cells, wild-type PDX-1 and PDX-1T152A were partially redistributed from the cytosolto 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 glucoseconcentrations, and underwent nuclear export as glucose concentrations were raised. PDX-1 binding to an oligonucleotide based on the consensus A3 binding site of the preproinsulingene promoter was completely abolished by mutation of Thr-152 to an acidic amino acid.Furthermore, the PDX-1 phosphomimetic mutants failed to suppress preproglucagon geneexpression in INSr?? 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 forphosphorylation at this site under a variety of in vivo conditions, including in cells overexpressingPASK. Thus, Thr-152 is not a major site of phosphorylation of PDX-1 in intact?-cells. However, a novel phosphorylation site, Ser-269, at the C-terminus of PDX-1 wasidentified by mass spectrometry. To explore the potential upstream regulatory kinase andthe signalling pathway involved, I generated dephospho-(S269A) and phosphomimetic(S269E) mutant PDX-1 and a phospho-Ser-269-specific antibody. Candidate kinases werealso identified using recombinant wild-type and mutant PDX-1 in in vitro phosphorylationexperiments.In this thesis I demonstrate that PDX-1 is phosphorylated under physiological conditions in?-cell lines. I have shown that Thr152 is not a major phosphorylation site of PDX-1 inintact 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 includingAurora, Rsk1, PKCa, JNK. The work presented in the thesis provides evidence that PDX-1phosphorylation is important in its role as a transcription regulator in mature ?-cells;moreover, it suggests several physiological signalling networks to investigate.