Thyroid-stimulating hormone/cAMP and glycogen synthase kinase 3β elicit opposing effects on Rap1GAP stability

Beyond regulating Rap activity, little is known regarding the regulation and function of the Rap GTPase-activating protein Rap1GAP. Tuberin and E6TP1 protein levels are tightly regulated through ubiquitin-mediated proteolysis. A role for these RapGAPs, along with SPA- 1, as tumor suppressors has been demonstrated. Whether Rap1GAP performs a similar role was investigated. We now report that Rap1GAP protein levels are dynamically regulated in thyroid- stimulating hormone ( TSH)- dependent thyroid cells. Upon TSH withdrawal, Rap1GAP undergoes a net increase in phosphorylation followed by proteasome- mediated degradation. Sequence analysis identified two putative destruction boxes in the Rap1GAP C- terminal domain. Glycogen synthase kinase 3 beta ( GSK3beta) phosphorylated Rap1GAP immunoprecipitated from thyroid cells, and GSK3beta inhibitors prevented phosphorylation and degradation of endogenous Rap1GAP. Co- expression of GSK3beta and Rap1GAP in human embryonic kidney 293 cells stimulated proteasome- dependent Rap1GAP turnover. Mutational analysis established a role for serine 525 in the regulation of Rap1GAP stability. Overexpression of Rap1GAP in thyroid cells impaired TSH/ cAMP-stimulated p70S6 kinase activity and cell proliferation. These data are the first to show that Rap1GAP protein levels are tightly regulated and are the first to support a role for Rap1GAP as a tumor suppressor.