title: Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4.
abstract: The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, self-renewal and tumorigenesis in stem cells. However, although their transcriptional targets have been extensively studied, little is known about how these factors are regulated at the posttranslational level. In this study, we established an in vitro system to identify phosphorylation patterns of the OSKM factors by AKT kinase. OCT4, SOX2, KLF4 and c-MYC were expressed in Sf9 insect cells employing the baculoviral expression system. OCT4, SOX2 and KLF4 were localized in the nucleus of insect cells, allowing their easy purification to near homogeneity upon nuclear fractionation. All transcription factors were isolated as biologically active DNA-binding proteins. Using in vitro phosphorylation and mass spectrometry-based phosphoproteome analyses several novel and known AKT phosphorylation sites could be identified in OCT4, SOX2 and KLF4.
authors.affiliation: a Interfaculty Institute for Biochemistry ; University of Tübingen ; Tübingen , Germany.
authors.forename: Peter N
authors.initials: PN
authors.lastname: Malak
authors.affiliation: a Interfaculty Institute for Biochemistry ; University of Tübingen ; Tübingen , Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dannenmann
authors.affiliation: a Interfaculty Institute for Biochemistry ; University of Tübingen ; Tübingen , Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Hirth
authors.affiliation: a Interfaculty Institute for Biochemistry ; University of Tübingen ; Tübingen , Germany.
authors.forename: Oliver C
authors.initials: OC
authors.lastname: Rothfuss
authors.affiliation: a Interfaculty Institute for Biochemistry ; University of Tübingen ; Tübingen , Germany.|b German Cancer Consortium (DKTK) and German Cancer Research Center ; Heidelberg , Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Schulze-Osthoff
mesh_terms: D000818:Animals; D001665:Binding Sites; D005786:Gene Expression Regulation; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry; D050814:Octamer Transcription Factor-3 / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry*; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry* / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000737:chemistry* / Q000235:genetics; D061987:Sf9 Cells
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D051057:Proto-Oncogene Proteins c-akt
keywords: AKT; KLF4; OCT4; SOX2; c-MYC; phosphorylation; pluripotency; stem cells