title: PTTG's C-terminal PXXP motifs modulate critical cellular processes in vitro.
abstract: Human pituitary tumor-transforming gene (PTTG), known also as securin, is a multifunctional protein implicated in the control of mitosis and the pathogenesis of thyroid, colon, oesophageal and other tumour types. Critical to PTTG function is a C-terminal double PXXP motif, forming a putative SH3-interacting domain and housing the gene's sole reported phosphorylation site. The exact role of phosphorylation and PXXP structure in the modulation of PTTG action in vitro remains poorly understood. We therefore examined the mitotic, transformation, proliferation and transactivation function of the C-terminal PXXP motifs of human PTTG. Live-cell imaging studies using an EGFP-PTTG construct indicated that PTTG's regulation of mitosis is retained regardless of phosphorylation status. Colony-formation assays demonstrated that phosphorylation of PTTG may act as a potent inhibitor of cell transformation. In proliferation assays, NIH-3T3 cells stable transfected and overexpressing mutations preventing PTTG phosphorylation (Phos-) showed significantly increased [3H]thymidine incorporation compared with WT, whereas mutants mimicking constitutive phosphorylation of PTTG (Phos+) exhibited reduced cell proliferation. We demonstrated that PTTG transactivation of FGF-2 in primary thyroid and PTTG-null cell lines was not affected by PTTG phosphorylation but was prevented by a mutant disrupting the PXXP motifs (SH3-). Taken together, our data suggest that PXXP structure and phosphorylation are likely to exert independent and critical influences upon PTTG's diverse actions in vitro.
authors.affiliation: Division of Medical Sciences, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK. k.boelaert@bham.ac.uk
authors.forename: K
authors.initials: K
authors.lastname: Boelaert
authors.forename: R
authors.initials: R
authors.lastname: Yu
authors.forename: L A
authors.initials: LA
authors.lastname: Tannahill
authors.forename: A L
authors.initials: AL
authors.lastname: Stratford
authors.forename: F L
authors.initials: FL
authors.lastname: Khanim
authors.forename: M C
authors.initials: MC
authors.lastname: Eggo
authors.forename: J S
authors.initials: JS
authors.lastname: Moore
authors.forename: L S
authors.initials: LS
authors.lastname: Young
authors.forename: N J L
authors.initials: NJ
authors.lastname: Gittoes
authors.forename: J A
authors.initials: JA
authors.lastname: Franklyn
authors.forename: S
authors.initials: S
authors.lastname: Melmed
authors.forename: C J
authors.initials: CJ
authors.lastname: McCabe
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D064249:Securin
chemical_list: D009363:Neoplasm Proteins; D064249:Securin; C578002:pituitary tumor-transforming protein 1, human; D016222:Fibroblast Growth Factor 2