We have shown that the small GTPase Rap1b, a protein known to antagonize the mitogenic and transforming activity of Ras, is endowed with both mitogenic and tumorigenic properties. Rap1b can be activated by cAMP, an intracellular message known to either stimulate or inhibit cell proliferation. The oncogenic property of Rap1b was revealed in a model system in which cAMP stimulates cell proliferation and was linked to Rap's ability to promote S phase entry. We have now tested the significance of the mitogenic action of Rap1b in a physiologically relevant model, the differentiated thyroid follicular cells, a system that requires thyroid-stimulating hormone (TSH), acting via cAMP, to mediate a full mitogenic response. Here we report that cAMP-dependent hormonal stimulation of DNA synthesis requires Rap1b in a manner dependent on its phosphorylation by protein kinase A.

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G 1 /S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMPdependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.