Phosphorylation of the N-terminal domain regulates subcellular localization and DNA binding properties of the peptidyl-prolyl cis/trans isomerase hPar14.

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.