The Pax gene family encodes transcription factors that are essential in organogenesis and in the differentiation of various organs in higher eukaryotes. Pax proteins have a DNA binding domain at the N-terminus, and a transcriptional activation domain at the C-terminus. How these domains interact with the transcriptional machinery of the cell is still unclear. In the present paper, we describe the identification by means of immunological screening of the WW domain binding protein WBP-2 as a biochemical interactor of Pax8 (a WW domain is a protein-interaction domain containing two conserved tryptophan residues). Pax8 is required for the morphogenesis of the thyroid gland and for the maintenance of the thyroid differentiated cellular phenotype. WBP-2 was identified originally as a WW domain binding protein, and its function is still unknown. WBP-2 binds to Pax8 in vitro in pulldown assays, and in vivo in tissue culture cells in co-immunoprecipitation assays. Interestingly, Pax8 does not contain a WW domain. Our results point to the identification of a new protein-interacting domain that is present in the C-terminal portion of Pax8 and that is required for protein–protein interaction with WBP-2. Our results demonstrate that WBP-2 is not a transcriptional co-activator of Pax8, but rather behaves as an adaptor molecule, as suggested in other studies.
Abbreviations used: CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; LUC, luciferase; NIS, sodium/iodide symporter; RT-PCR, reverse transcription–PCR; Tg, thyroglobulin; TPO, thyroperoxidase; TTF, thyroid-specific transcription factor; TRITC, tetramethylrhodamine β-isothiocyanate; WBP, WW domain binding protein; tWBP-2, thyroid-specific transcript of WBP-2; WW domain, protein-interaction domain containing two conserved tryptophan residues.