Sato Tatsuhiro, Nakashima Akio, Guo Lea, Tamanoi Fuyuhiko
Department of Microbiology, Immunology & Molecular Genetics, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095, USA.
J Biol Chem. 2009 May 8;284(19):12783-91. doi: 10.1074/jbc.M809207200. Epub 2009 Mar 19.
Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.
Rheb G蛋白通过激活mTORC1在TSC/Rheb/mTOR信号通路中发挥关键作用。利用从缺乏营养的哺乳动物细胞中用抗raptor抗体免疫沉淀的mTORC1，Rheb对mTORC1的激活在体外能够如实地重现。添加重组Rheb可显著提高这种mTORC1制剂对4E-BP1的体外低激酶活性。另一方面，添加Rheb不会激活用抗rictor抗体从哺乳动物细胞中免疫沉淀的mTORC2。mTORC1的激活对Rheb具有特异性，因为其他G蛋白如KRas、RalA/B和Cdc42不会激活mTORC1。Rheb1和Rheb2均可激活mTORC1。此外，这种激活依赖于结合的GTP的存在。我们还发现Rheb的效应结构域是mTORC1激活所必需的。FKBP38是最近提出的Rheb作用的介质，在体外似乎不参与Rheb依赖的mTORC1激活，因为缺乏FKBP38的mTORC1制剂仍可被Rheb激活。添加Rheb会导致底物蛋白4E-BP1与mTORC1的结合显著增加。PRAS40是一种含有TOR信号（TOS）基序的蛋白，它与4EBP1与mTORC1的结合竞争，可抑制Rheb诱导的mTORC1激活。缺乏raptor的mTORC1制剂不会被Rheb激活。Rheb不会诱导mTOR的自磷酸化。这些结果表明，Rheb诱导4E-BP1与mTORC1结合的改变，从而调节mTORC1的激活。
