title: NANOG is multiply phosphorylated and directly modified by ERK2 and CDK1 in vitro.
abstract: NANOG is a divergent homeobox protein and a core component of the transcriptional circuitry that sustains pluripotency and self-renewal. Although NANOG has been extensively studied on the transcriptional level, little is known regarding its posttranslational regulation, likely due to its low abundance and challenging physical properties. Here, we identify eleven phosphorylation sites on endogenous human NANOG, nine of which mapped to single amino acids. To screen for the signaling molecules that impart these modifications, we developed the multiplexed assay for kinase specificity (MAKS). MAKS simultaneously tests activity for up to ten kinases while directly identifying the substrate and exact site of phosphorylation. Using MAKS, we discovered site-specific phosphorylation by ERK2 and CDK1/CyclinA2, providing a putative link between key signaling pathways and NANOG.
authors.affiliation: Stem Cells and Regenerative Medicine, Morgridge Institute for Research, Madison, WI 53715, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Brumbaugh
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Russell
authors.affiliation: Stem Cells and Regenerative Medicine, Morgridge Institute for Research, Madison, WI 53715, USA.
authors.forename: Pengzhi
authors.initials: P
authors.lastname: Yu
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA ; Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Westphall
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA ; Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.affiliation: Stem Cells and Regenerative Medicine, Morgridge Institute for Research, Madison, WI 53715, USA ; Department of Cell & Regenerative Biology, University of Wisconsin-Madison, Madison, WI 53706, USA ; Department of Molecular, Cellular & Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
mesh_terms: D000595:Amino Acid Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D056751:Cyclin A2 / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D000071317:Nanog Homeobox Protein; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D056751:Cyclin A2; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D010748:Phosphopeptides; D016203:CDC2 Protein Kinase; D019950:Mitogen-Activated Protein Kinase 1