Tsygankova Oxana M, Feshchenko Elena, Klein Peter S, Meinkoth Judy L
Department of Pharmacology, Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2004 Feb 13;279(7):5501-7. doi: 10.1074/jbc.M305824200. Epub 2003 Dec 2.
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.
除了调节Rap活性外，关于Rap GTP酶激活蛋白Rap1GAP的调节和功能知之甚少。结节性硬化蛋白和E6TP1蛋白水平通过泛素介导的蛋白水解受到严格调控。已经证明这些RapGAP与SPA-1一样具有肿瘤抑制作用。研究了Rap1GAP是否发挥类似作用。我们现在报告，在促甲状腺激素（TSH）依赖的甲状腺细胞中，Rap1GAP蛋白水平受到动态调节。在撤除TSH后，Rap1GAP的磷酸化净增加，随后被蛋白酶体介导降解。序列分析在Rap1GAP C末端结构域中鉴定出两个假定的破坏框。糖原合酶激酶3β（GSK3β）使从甲状腺细胞免疫沉淀的Rap1GAP磷酸化，并且GSK3β抑制剂可防止内源性Rap1GAP的磷酸化和降解。在人胚肾293细胞中共表达GSK3β和Rap1GAP可刺激蛋白酶体依赖性的Rap1GAP周转。突变分析确定了丝氨酸525在调节Rap1GAP稳定性中的作用。在甲状腺细胞中过表达Rap1GAP会损害TSH/cAMP刺激的p70S6激酶活性和细胞增殖。这些数据首次表明Rap1GAP蛋白水平受到严格调控，并且首次支持Rap1GAP作为肿瘤抑制因子的作用。
