title: Thyroid-stimulating hormone/cAMP and glycogen synthase kinase 3beta elicit opposing effects on Rap1GAP stability.
abstract: 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.
authors.affiliation: Department of Pharmacology, Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Oxana M
authors.initials: OM
authors.lastname: Tsygankova
authors.forename: Elena
authors.initials: E
authors.lastname: Feshchenko
authors.forename: Peter S
authors.initials: PS
authors.lastname: Klein
authors.forename: Judy L
authors.initials: JL
authors.lastname: Meinkoth
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002455:Cell Division; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D020690:GTPase-Activating Proteins / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D013961:Thyroid Gland / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C116934:E6-target protein 1; D020690:GTPase-Activating Proteins; D009097:Multienzyme Complexes; D012097:Repressor Proteins; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D013972:Thyrotropin; D000242:Cyclic AMP; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D038362:Glycogen Synthase Kinase 3; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D020827:rap1 GTP-Binding Proteins; D001973:Bromodeoxyuridine