DETERMINATION OF FUNCTIONAL DOMAINS OF THE HUMAN TRANSCRIPTION FACTORPAX8 RESPONSIBLE FOR ITS NUCLEAR-LOCALIZATION AND TRANSACTIVATING POTENTIAL

The conserved structure of the transcription factors of the Pax gene f amily may reflect functional conservation. We have demonstrated that t he human Pax8 transcription factor is organized in several functional domains and contains two regions responsible for its nuclear localizat ion, in addition to an activating region at the carboxy terminus of th e protein and an inhibitory region encoded by the exon 9 present only in a splice variant PAX8a, Regions of PAX8 determining the nuclear loc alization of the PAX8/lacZ fusions contain short amino acid sequences similar to several described nuclear localization sites (NLS). These N LS were identified in the paired domain and between the octapeptide an d the residual homeodomain, respectively. The activating domain is enc oded by the exons 10 and 11 and its function is modulated by the adjac ent domains encoded by the exons 9 and 12, The domain encoded by exon 9 significantly inhibits the function of the activating domain. Pax8 i s expressed in thyroid cells and its product binds promoters of the th yroglobulin and thyroperoxidase genes through its paired domain, Thyro id cell growth and differentiation depend on thyrotropin which, by sti mulating cAMP synthesis, activates the cAMP-dependent protein kinase A (PKA). We have investigated a link between thyrotropin stimulation an d gene activation by Pax8. Stimulation of cAMP synthesis augments Pax8 -specific transcription in thyroid cells, indicating that PKA is invol ved in Pax8 activation. Cotransfection of GAL4/PAX8 fusions and the ca talytic subunit of PKA in A126, a PKA-deficient derivative of the PC12 pheochromocytoma cell line, synergistically activates the GAL4-specif ic reporter, suggesting the activating domain of PAX8 is dependent upo n the catalytic subunit of the PKA. We propose that this dependence is due to a hypothetical adaptor which forms a target for PKA and intera cts with the activating domain of PAX8. We show that PAX8 isolated fro m the thyroid cell line FTRL5 is a phosphoprotein in which phosphoryla tion is not dependant on cAMP pathway activation, Our results suggest that Pax8 is part of the cAMP signaling pathway and mediates thyrotrop in dependent gene activation in thyroid cells, Investigation of the PA X8 expression in a panel of Wilms tumors shows a striking correlation between the expression of PAX8 and another transcription factor. WT1, indicating that these two genes may interact in vivo.