title: Phosphorylation regulates human OCT4.
abstract: The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706-1532, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Brumbaugh
authors.forename: Zhonggang
authors.initials: Z
authors.lastname: Hou
authors.forename: Jason D
authors.initials: JD
authors.lastname: Russell
authors.forename: Sara E
authors.initials: SE
authors.lastname: Howden
authors.forename: Pengzhi
authors.initials: P
authors.lastname: Yu
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Ledvina
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D050814:Octamer Transcription Factor-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D050814:Octamer Transcription Factor-3; C472575:SALL4 protein, human; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors; D013912:Threonine; D012694:Serine; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1