Immunolocalization of human p14(ARF) to the granular component of the interphase nucleolus

The human p14(ARF) protein is encoded by an alternative transcript from the INK4a/ARF locus on chromosome 9p21, a locus frequently afflicted in human tumors. By use of two novel specific antisera against p14(ARF) we show that the protein is localized mainly in nucleoli but also in the nucleoplasm. T ransfection of full-length and deletion mutant GFP-p14(ARF) fusion proteins confirmed this subcellular localization and assigned the nucleolar localiz ation signal to the exon 2-encoded C-terminal region. In order to determine p14(ARF) expression in human tumor cells, we examined p14(ARF) in 32 tumor cell lines by immunofluorescence staining. Nucleolar p14(ARF) was detected in 10 lines, all of which lacked functional p53. Double immunostaining wit h p14(ARF) and B23/nucleophosmin or fibrillarin antibodies using 3D microsc opy revealed that p14(ARF) is located mainly in the granular component of t he nucleolus. p14(ARF) was also found in distinct granular aggregates scatt ered throughout the nucleoplasm. RNase digestion or selective inhibition of rRNA transcription by low doses of actinomycin D caused nucleoplasmic tran slocation of p14(ARF). This indicates that the nucleolar localization of p1 4(ARF) is dependent on ongoing transcriptional activity in intact functiona l nucleoli. (C) 2000 Academic Press.