Epac is required for TSH mitogenic action: role of its DEP domain

DANIEL HOCHBAUM; KYOUNGJA HONG; FERNANDO RIBEIRO-NETO; DANIEL L. ALTSCHULER

Congreso; The Endocrine Society's 89th Annual Meeting; 2007

cAMP stimulates cell proliferation in several experimental models. However, the effector pathways mediating cAMP action are still ill-defined. The thyroid gland represents a prototypical compartment where thyroid-stimulating hormone (TSH), via cAMP, stimulates a mitogenic response. Both PKA-dependent and PKA-independents events are required for TSH?s full mitogenic activity. Intracellular increase of cAMP rapidly leads to PKA-dependent phosphorylation and PKA-independent activation of the small-G-protein Rap1b. Its expression in cells where cAMP is mitogenic is associated with an increase in cAMP-mediated G1/S phase entry, and expression of active Rap1 in mouse transgenic thyroids induces a TSH-dependent tumorigenic phenotype. Thus, Rap1b is a transducer of the cAMP mitogenic signal. Epac (exchange protein activated by cAMP) is a cAMP-dependent activator of Rap1 whose action is completely independent of PKA activity. In order to address whether Epac-dependent Rap1 activation could represent the PKA-independent step in TSH action we have decided to develop a dominant negative approach. Expression of Epac?s N-terminal regulatory domain (N-Epac), containing the DEP and cAMP binding domains but devoid of any catalytic activity was able to block agonist-mediated Rap activation as well as TSH-dependent cell proliferation. N-Epac?s inhibitory action was fully dependent on the presence of the DEP domain. N-Epac?s dominant negative inhibitory action can be rescued by co-transfection of a constitutively active Rap1 protein. Expression of a DEP-deficient Epac construct is incapable of mediating agonist-dependent Rap activation. Moreover, the inhibitory action of R279E Epac, a mutation that abolishes cAMP binding, depended entirely on the presence of its DEP domain. Therefore, these results indicate that Epac-mediated Rap activation is strictly required for TSH-mitogenic action in a way that is fully dependent on its DEP domain. In an attempt to assess the role of DEP in Epac?s action, binding partners were identified and their potential role/s in Epac subcellular localization and function will be presented.