A Novel Binding Factor Facilitates Nuclear Translocation and Transcriptional Activation Function of the Pituitary Tumor-transforming Gene Product

Abstract

Pituitary tumor-transforming gene (PTTG) is a recently characterized oncogene whose expression product contains a transcriptional activation domain at the C terminus. To understand the mechanisms involved in PTTG biological functions, we used yeast two-hybrid screening to identify proteins that interact with PTTG. This study reports the isolation and characterization of a novel PTTG-binding factor (PBF). PBF contains an open reading frame of 179 amino acids with a predicted molecular mass of 22 kDa. In Northern blot analyses, PBF mRNA was ubiquitously expressed in human tissues. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that PBF interacts specifically with PTTG under both in vitro and in vivo conditions. The PTTG binding domain in PBF was located within the C-terminal 30-amino acid region that contain a nuclear localization signal. Immunofluorescence and subcellular fractionation studies showed that PTTG is predominantly expressed in the cytoplasm with partial nuclear localization, whereas PBF is localized both in the cytoplasm and the nucleus. The interaction between PBF and PTTG facilitated PTTG translocation from the cytoplasm to the nucleus. Furthermore, PBF is required for transcriptional activation of basic fibroblast growth factor by PTTG. In summary, we have characterized a novel PTTG-binding protein that facilitates PTTG nuclear translocation and potentiates its transcriptional activation function. Pituitary tumor-transforming gene (PTTG)1 was isolated by its differential mRNA expression in rat pituitary tumor cells (1). Overexpression of PTTG induces cell transformation and generates tumors in nude mice (1). Several human PTTG homologues have since been cloned (2-5). High level expression of PTTG mRNA in multiple types of tumors as well as in carcinoma cell lines (2-7) suggests that PTTG may be involved in tumorigenesis of many tissues in addition to the pituitary. In normal adult tissues, PTTG mRNA expression is restricted to a small number of tissues, including testis, thymus, and placenta (1-3).The mechanisms involved in PTTG biological function are largely unknown. To elucidate the biological function of PTTG, we have used a yeast two-hybrid system to identify proteins that associate with PTTG (8). This report shows that the ribosomal protein S10 and a novel human homologue of the bacterial heat-shock protein, DnaJ (HSJ2), interact specifically with PTTG under both in vitro and in vivo conditions (8). Association of PTTG with these proteins indicates that PTTG may link to the ribosome and is involved in the regulation of translation (8). In addition, during the rat spermatogenic cycle, PTTG mRNA is expressed stage specifically in only spermatocytes and spermatids, suggesting that PTTG may play a role in rat spermatogenesis (8). The C-terminal portion of human PTTG was shown to function as a transcriptional activator when fused to a heterologous DNA binding domain (2). In NIH3T3 cells that overexpress PTTG, increased expression of bFGF mRNA and protein was obs...

Expression and function of the novel proto-oncogene PBF in thyroid cancer: a new target for augmenting radioiodine uptake

“…PBF also possesses an extracellular N-terminal cysteine-rich region, similar to that found in the membrane-associated plexins, semaphorins and integrins, and hence referred to as a PSI domain (Bork et al 1999). In contrast to evidence supporting the characterisation of PBF as a membrane protein, the presence of a bipartite nuclear localisation signal (NLS) near the C-terminus suggested that PBF may also be a nuclear protein (Chien & Pei 2000).…”

“…PTTG, tagged with green fluorescent protein (GFP-PTTG), was predominantly observed in the cytoplasm, with partial nuclear localisation, whereas co-transfection of HA-PBF resulted in increased nuclear translocation of GFP-PTTG. This effect was abrogated when the mutated NLS was used, indicating that PBF with an intact NLS is required for PTTG translocation into the nucleus (Chien & Pei 2000).…”

“…Specific binding between PTTG and PBF was demonstrated both in vitro and in vivo through pulldown and co-immunoprecipitation assays in COS-7 cells. Through deletion analysis, the PTTG binding domain in PBF was identified within the C-terminal 30 amino acids and the corresponding domain in PTTG was found to be between amino acids 123 and 154 (Chien & Pei 2000).…”

“…PBF was discovered through a yeast two-hybrid screen to identify proteins that interact with the human securin PTTG (Chien & Pei 2000). Specific binding between PTTG and PBF was demonstrated both in vitro and in vivo through pulldown and co-immunoprecipitation assays in COS-7 cells.…”

“…PBF, also known as PTTG1-interacting protein (PTTG1IP), encodes a protein of 180 amino acids with a predicted molecular mass of 22 kDa (Chien & Pei 2000). Located on chromosome 21q22.3, PBF had previously been cloned and termed C21orf3 (also known as C21orf1).…”