Abstract
The putative HLA-DR associated protein I (PHAPI) is a nuclear protein with a molecular mass of 32 kDa, which consists of about 250 amino acids. The cDNAs encoding the wild-type and mutated PHAPI were ligated into a eukaryotic expression vector, which produces the GFP fusion protein, and were expressed in the mouse fibroblast BALB3T3 cell line. The wild-type PHAPI and a series of mutated PHAPIs which lacked each 20-amino acid segment within the leucine-rich N-terminal region were localized to nuclei, whereas a mutant protein which lacked the acidic C-terminal tail including the potential nuclear localization signal was diffusely spread throughout the whole cell. We have shown previously that PHAPI is phosphorylated in vitro by unknown protein kinases, at Ser-204 in human PHAPI. To investigate the phosphorylation of PHAPI in vivo, mutant PHAPI in which Ser-204 was changed to Ala was also expressed in the cells and was metabolically labeled with a radioactive orthophosphate. The mutant PHAPI was localized to nuclei in the same manner as wild-type PHAPI and was still phosphorylated at Ser residues although the phosphorylation level was slightly reduced compared to that of the wild-type PHAPI. These results suggest that PHAPI might be phosphorylated in vivo at several Ser residues including Ser-204, and that the phosphorylation of Ser-204 is not essential for the nuclear localization of PHAPI.
