Pdx1 Is Post-Translationally Modified In vivo and Serine 61 Is the Principal Site of Phosphorylation Thomas Frogne1, Kathrine Beck Sylvestersen2, Stefan Kubicek3, Michael Lund Nielsen2, Jacob Hecksher- Sørensen1* 1 Department of Beta-cell Regeneration, Hagedorn Research Institute, Gentofte, Denmark, 2 Center for Protein Research, University of Copenhagen, Copenhagen, Denmark, 3 Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria 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. Citation: Frogne T, Sylvestersen KB, Kubicek S, Nielsen ML, Hecksher-Sørensen J (2012) Pdx1 Is Post-Translationally Modified In vivo and Serine 61 Is the Principal Site of Phosphorylation. PLoS ONE 7(4): e35233. doi:10.1371/journal.pone.0035233 Editor: Massimo Federici, University of Tor Vergata, Italy Received June 14, 2011; Accepted March 14, 2012; Published April 11, 2012 Copyright: 2012 Frogne et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was made possible by funding from the JDRF www.jdrf.org; grant #26-2008-639 (to JH-S) and EU FP7 Marie Curie Fellowship www. mariecurie.org PIOF-GA-2008-21135 (SK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The Hagedorn Research Institute is affiliated to Novo Nordisk. JH-S owns equity shares in Novo Nordisk A/S. JH-S is 100% employed by Novo Nordisk. TF is 100% funded by a grant from JDRF. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: jhes@hagedorn.dk Introduction Pancreatic and duodenal homeobox 1 (Pdx1) also known as Ipf1 and Stf1 is a master regulator of pancreas development [1,2,3]. Pdx1 was first cloned and described in Xenopus, as Xlhbox8, where it was shown to be expressed in the developing endoderm and pancreas [4]. In mouse embryos Pdx1 is expressed in the endoderm from e8.5 where it defines the regions that will form the dorsal and ventral pancreas [1,2,5]. The evidence that Pdx1 is instrumental for pancreas development comes from both mouse and human where depletion of a functional Pdx1 protein results in pancreas agenesis [1,2,6]. Conversely, over expression of Pdx1 in endoder- mal cells outside the presumptive pancreas can activate events reminiscent of pancreas development. In chicken embryos forced expression of Pdx1 in the developing endoderm partially induces pancreas development. Thus, ectopic Pdx1 quenches the expres- sion of non-pancreatic genes such as Sox2 and Cdx in regions outside the presumptive pancreas [7] while it induces pancreatic markers like Nkx6.1 [8]. In Xenopus, over expression of a modified Pdx1, carrying the VP16 transcriptional activation domain can cause conversion of liver to pancreas [9]. Postnatally Pdx1 is expressed in the mature b-cell where it serves as an important regulator of glucose homeostasis [10,11]. In humans, mutations in the Pdx1 gene have been associated with type 2 diabetes and maturity onset diabetes of the young 4 (MODY4) [12,13]. This role is conserved in evolution and impaired glucose tolerance has been observed in several animal models where Pdx1 protein levels have been depleted or reduced [10,14,15,16,17,18]. Furthermore, the diabetic pheno- type observed following Pdx1 inactivation is reversible and blood glucose levels can be normalized if Pdx1 expression is reactivated [19]. In the sand rat (Psammonys obesus) which normally lacks Pdx1 in the b-cells, reintroduction of the protein greatly improves glucose stimulated insulin transcription [15]. Several studies have shown that Pdx1 directly influences glucose homeostasis by binding and regulating the promoters of genes such as insulin (ins), glut2 (Slc2a2) and glucokinase (Gck) [3,20,21,22]. However, in addition to the immediate effects of Pdx1 on glucose homeostasis studies in mice haploinsufficient for Pdx1 have revealed a long term requirement for correct Pdx1 dosage. In the mature b-cell the loss of one Pdx1 allele affects both glucose stimulated insulin release and b-cell survival [11]. Furthermore, the compensatory increase in b-cell mass associated with impaired insulin signaling relies on Pdx1 dosage. Mice that are double heterozygous for mutations in the insulin receptor (Ir) and the insulin receptor substrate (Irs) display impaired insulin signaling and respond by increasing their the b-cell mass. This expansion is abolished in mice that are haploinsufficient PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35233