Predicted NES in PBF appears to be functional in vitro

Pituitary tumor transforming gene (PTTG) binding factor (PBF) is a proto-oncogene which is frequently upregulated in endocrine cancers. PBF has previously been determined to contain several putative signal sequences within its 180 amino acids. Previous studies have shown the nuclear localisation signal (NLS) to be functional and prediction software now suggests the presence of a putative leucine-rich nuclear export signal (NES) between residues 17 and 27. PBF is known to shuttle in and out of the nucleus, although as a small protein of ~25 kDa, this may be a passive process. Here, we hypothesised that the putative NES is functional in vitro and mediates active nuclear exit. Predicted NES residues were conserved in the PBF sequences of six out of seven analysed species. The most resounding NES prediction was present in human PBF, with an average NES score of 0.69 over the 11 consensus NES residues of sequence LXXXLXXLXL, where ‘L’ is a hydrophobic residue (often leucine) and ‘X’ is any other amino acid. Exportin-1 (CRM1), the central protein involved in nuclear export, was chemically inhibited and knocked down via siRNA in COS-7 and K1 cells, and the location of PBF assessed using immunofluorescence. When CRM1 was knocked down, cytoplasmic PBF levels were reduced as, more unexpectedly, were nucleolar levels. Fibrillarin, used as a marker of cellular nucleoli, co-localised with PBF in the presence of scrambled siRNA. However, when CRM1 was specifically knocked down, this co-localisation was no longer present, and there was a marked reduction in PBF staining corresponding with nucleolar location. Consistent findings were apparent with the CRM1 inhibitor, Leptomycin B. Thus, although the precise nuclear, nucleolar and cytoplasmic functions of PBF remain to be delineated, these data suggest that PBF has a conserved and functional nuclear export sequence, revealing that PBF exit from the nucleus is governed by an active process.