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

• Published in: The Journal of biological chemistry Vol. 279; no. 7; p. 5501
• Main Authors: Tsygankova, Oxana M, Feshchenko, Elena, Klein, Peter S, Meinkoth, Judy L
• Format: Journal Article
• Language: English
• Published: United States 13-02-2004
• Subjects: Animals; Blotting, Western; Bromodeoxyuridine - pharmacology; Cell Division; Cell Line; Cyclic AMP - metabolism; Cysteine Endopeptidases - metabolism; DNA Mutational Analysis; Dose-Response Relationship, Drug; Glycogen Synthase Kinase 3 - metabolism; Glycogen Synthase Kinase 3 beta; GTPase-Activating Proteins - metabolism; Humans; Multienzyme Complexes - antagonists & inhibitors; Multienzyme Complexes - metabolism; Mutagenesis, Site-Directed; Mutation; Phosphorylation; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; rap1 GTP-Binding Proteins - metabolism; Rats; Rats, Wistar; Repressor Proteins - metabolism; Ribosomal Protein S6 Kinases, 70-kDa - metabolism; Thyroid Gland - metabolism; Thyrotropin - metabolism; Time Factors; Transfection; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins
• ISSN: 0021-9258

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 3beta (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.