title: Pdx1 is post-translationally modified in vivo and serine 61 is the principal site of phosphorylation.
abstract: Maintaining sufficient levels of Pdx1 activity is a prerequisite for proper regulation of blood glucose homeostasis and beta cell function. Mice that are haploinsufficient for Pdx1 display impaired glucose tolerance and lack the ability to increase beta cell mass in response to decreased insulin signaling. Several studies have shown that post-translational modifications are regulating Pdx1 activity through intracellular localization and binding to co-factors. Understanding the signaling cues converging on Pdx1 and modulating its activity is therefore an attractive approach in diabetes treatment. We employed a novel technique called Nanofluidic Proteomic Immunoassay to characterize the post-translational profile of Pdx1. Following isoelectric focusing in nano-capillaries, this technology relies on a pan specific antibody for detection and it therefore allows the relative abundance of differently charged protein species to be examined simultaneously. In all eukaryotic cells tested we find that the Pdx1 protein separates into four distinct peaks whereas Pdx1 protein from bacteria only produces one peak. Of the four peaks in eukaryotic cells we correlate one of them to a phosphorylation Using alanine scanning and mass spectrometry we map this phosphorylation to serine 61 in both Min6 cells and in exogenous Pdx1 over-expressed in HEK293 cells. A single phosphorylation is also present in cultured islets but it remains unaffected by changes in glucose levels. It is present during embryogenesis but is not required for pancreas development.
authors.affiliation: Department of Beta-cell Regeneration, Hagedorn Research Institute, Gentofte, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Frogne
authors.forename: Kathrine Beck
authors.initials: KB
authors.lastname: Sylvestersen
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubicek
authors.forename: Michael Lund
authors.initials: ML
authors.lastname: Nielsen
authors.forename: Jacob
authors.initials: J
authors.lastname: Hecksher-Sørensen
mesh_terms: D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D002645:Chickens / Q000254:growth & development / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018149:Glucose Intolerance; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D012694:Serine / Q000235:genetics*; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001786:Blood Glucose; D018398:Homeodomain Proteins; D007328:Insulin; C491328:Nkx6-1 protein, mouse; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D012694:Serine