The first reported mutations in the pituitary tumor-transforming gene binding factor

PBF is a ubiquitous glycoprotein which is over-expressed particularly in endocrine and endocrine-related cancers. Previously classified as a proto-oncogene, 11 substitution-missense mutations of PBF have now been reported in tumours from patients with ovarian, prostate and colorectal cancers via the COSMIC database, suggesting PBF may in fact be an oncogene. We have therefore examined the biological implications of all 11 mutations. Substitution mutations, which occurred across the 180 amino acid structure of PBF, with a potential hot-spot at residue 106, were re-created in tagged (HA and FLAG) and untagged constructs. Modelling of all mutants using I-TASSER identified potentially severe alterations in 3D structure for mutants W59F, R87C, S103L, G106R, G106W and R140W. SIFT predictions identified that all mutations except G106V would result in significant changes in protein function. Western blotting revealed that mutations C51R and G106R inhibited PBF dimerisation and glycosylation in vitro. Anisomycin half life studies in SW1736 thyroid and MCF7 breast cancer cells revealed that R87C and S103L mutations resulted in unchanged protein stability compared to WT (half life ~24 h), whereas C51R, W59F, G106R, G106V, G106W and R146W mutants were less stable, ranging from approximately 6 to 9 h. Interestingly, different tumour mutations at residue 106 revealed very different biochemical properties of the protein, with G106R showing significantly reduced glycosylation, dimerisation and half life, whereas G106V and G106W demonstrated WT levels of dimerization, and with G106V having 50% greater stability than G106W. This is the first description of mutations in PBF. Based on our initial biochemical characterisation of 3D protein structure, glycosylation, dimerisation and stability, we are now determining the functional consequences of these initial mutations on the known actions of PBF in endocrine neoplasia, to address the question of whether PBF mutations are oncogenic in vivo.