ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding
ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding
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DOI：
10.4161/auto.7.7.15491
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发表时间：
2011-07-01
期刊：
影响因子：
13.3
通讯作者：
Tee, Andrew R.
中科院分区：
文献类型：
--
作者：
Dunlop, Elaine A.;Hunt, David K.;Tee, Andrew R.
蛋白质合成和自噬是两个截然相反的过程，它们控制细胞生长以响应营养供应。哺乳动物/机械靶的雷帕霉素复合体1(MTORC1)途径是控制蛋白质合成的主要调控因子，最近被证明通过磷酸化和失活自噬调节蛋白ULK1来抑制自噬。ULK1还抑制mTORC1底物S6K1的磷酸化，表明mTORC1和ULK1之间存在复杂的信号相互作用。在这里，我们证明了ULK1在体内和体外诱导Raptor的多位点磷酸化。使用磷酸特异性抗体，我们发现Ser855和Ser859被ULK1强烈磷酸化，Ser792也被中度磷酸化。有趣的是，ULK1的过表达也增加了猛禽Ser863和mTOR自动磷酸化位点Ser2481的磷酸化，这是一种mTORC1依赖的方式。尽管有证据表明，在ULK1过表达后mTORC1激酶活性增加，但mTORC1介导的S6K1和4E-BP1的磷酸化显著受到抑制。ULK1的表达对mTORC1各组分之间的蛋白质相互作用没有影响，但确实降低了Raptor与底物4E-BP1的结合能力。此外，ULK1的shRNA敲除导致mTORC1底物的磷酸化增加，而Ser859和Ser792处的Raptor的磷酸化降低。我们提出了一种新的机制，即ULK1通过阻碍底物与Raptor的对接而抑制mTORC1。这是一个新的负反馈环，当营养供应有限时，激活自噬来维持mTORC1抑制。
Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.