ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser 298 Augments Hepatic Gluconeogenesis Bandish Kapadia 1☯ , Navin Viswakarma 1¤☯ , Kishore V. L. Parsa 1 , Vasundhara Kain 1 , Soma Behera 1 , Sashidhara Kaimal Suraj 2 , Phanithi Prakash Babu 2 , Anand Kar 3 , Sunanda Panda 3 , Yi-jun Zhu 4 , Yuzhi Jia 4 , Bayar Thimmapaya 5 , Janardan K. Reddy 4* , Parimal Misra 1* 1 Department of Biology, Dr Reddy’s Institute of Life Sciences, An Associate Institute of University of Hyderabad, Hyderabad, Andhra Pradesh, India, 2 Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, India, 3 Department of Life Sciences, Devi Ahilya University, Indore, Madhya Pradesh, India, 4 Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America, 5 Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America Abstract PRIP- Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB- binding protein (CBP)/ E1A binding protein pγ00 (Epγ00) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERKβ phosphorylates PIMT at Ser β98 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMT Sβ98D ) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMT Sβ98D but not PIMT Sβ98A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U01β6, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T 4 hormone to rats activated ERK1/β resulting in enhanced PIMT ser β98 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERKβ-mediated phosphorylation of PIMT at Ser β98 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. Citation: Kapadia B, Viswakarma N, Parsa KVL, Kain V, Behera S, et al. (β01γ) ERKβ-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser β98 Augments Hepatic Gluconeogenesis. PLoS ONE 8(1β): e8γ787. doi:10.1γ71/journal.pone.008γ787 Editor: Hak Hotta, Kobe University, Japan Received July β9, β01γ; Accepted November 8, β01γ; Published December 17, β01γ Copyright: © β01γ Kapadia 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 supported by DBT (Department of Biotechnology, India) funded Grant (No. BT/PR1γ5β7/BRB/0/765/β010) awarded to PM and KVLP, and the National Institutes of Health Grant DK08γ16γ and DK097β49 to J.K.R. and Rβ1A1094β96 to B. T. BK and VK are financially supported by Senior Research Fellowship from CSIR and post-doctoral fellowship from DBT, India respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: parimalm@drils.org (PM); jkreddy@northwestern.edu (JKR) ☯ These authors contributed equally to this work. ¤ Current address: Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, Illinois, United States of America Introduction PIMT/NCoA6IP (PRIP Interacting protein with Methyl Transferase domain) was first isolated as a transcriptional coactivator PRIP/NCoA6 interacting protein (NCoA6IP) in a yeast two hybrid screen [1]. PIMT is expressed in several tissues including liver, kidney and skeletal muscle [1]. PIMT is expressed ubiquitously and contains an RNA binding motif, a putative methyltransferase domain and an S-adenosyl methionine (SAM) binding domain, suggesting that it may function as an RNA methyltransferase[1]. Later it was demonstrated that PIMT hypermethylates small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) [β]. DTL, the Drosophila homolog of PIMT/NCoA6IP has an essential role in PLOS ONE | www.plosone.org 1 December β01γ | Volume 8 | Issue 1β | e8γ787