title: Phosphorylation of the N-terminal domain regulates subcellular localization and DNA binding properties of the peptidyl-prolyl cis/trans isomerase hPar14.
abstract: Human parvulin 14 (hPar14) is a folding helper enzyme belonging to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases). This enzyme is thought to play a role in cell-cycle and chromatin remodeling. Although hPar14 was nuclearly localized and bound to double-stranded DNA, the molecular basis of the subcellular localization and the functional regulation remained unknown. Here we show that subcellular localization and DNA-binding ability of hPar14 is regulated by posttranslational modification of its N-terminal domain. As proved by MALDI-TOF mass spectrometry and MS/MS fragmentation, hPar14 is phosphorylated at Ser19 in vitro and in vivo. In human HeLa cells the protein is most likely modified by casein kinase 2 (CK2). Phosphorylation of hPar14 is inhibited both in vitro and in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific inhibitor of CK2 activity. Mutation of Ser19 to Ala abolishes phosphorylation and alters the subcellular localization of hPar14 from predominantly nuclear to significantly cytoplasmic. Immunostaining shows that a Glu19 mutant of hPar14, which mimics the phosphorylated state of Ser19, is localized around the nuclear envelope, but does not penetrate into the nucleoplasm. In contrast to wild-type hPar14, the in vitro DNA-binding affinity of the Glu19 mutant is strongly reduced, suggesting that only the dephosphorylated protein is the active DNA-binding form of hPar14 in the nucleus.
authors.affiliation: Max Planck Research Unit, Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle, Germany.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Reimer
authors.forename: Matthias
authors.initials: M
authors.lastname: Weiwad
authors.forename: Angelika
authors.initials: A
authors.lastname: Schierhorn
authors.forename: Peter-Karl
authors.initials: PK
authors.lastname: Ruecknagel
authors.forename: Jens-Ulrich
authors.initials: JU
authors.lastname: Rahfeld
authors.forename: Peter
authors.initials: P
authors.lastname: Bayer
authors.forename: Gunter
authors.initials: G
authors.lastname: Fischer
mesh_terms: D015153:Blotting, Western; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000737:chemistry; D006367:HeLa Cells; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D009154:Mutation; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection
chemical_list: D002843:Chromatin; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012965:Sodium Chloride; D012694:Serine; D004004:Dichlororibofuranosylbenzimidazole; D006493:Heparin; D004247:DNA; C000606846:PIN1 protein, human; C000606848:PIN4 protein, human; D019696:Peptidylprolyl Isomerase