Farnesylation-defective Rheb Increases Axonal Length Independently of mTORC1 Activity in Embryonic Primary Neurons
Farnesylation-defective Rheb Increases Axonal Length Independently of mTORC1 Activity in Embryonic Primary Neurons
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DOI：
10.5607/en.2019.28.2.172
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发表时间：
2019-04-01
影响因子：
2.4
通讯作者：
Huh, Sung-Oh
中科院分区：
文献类型：
--
作者：
Choi, Seunghyuk;Sadra, Ali;Huh, Sung-Oh
Rheb（在大脑中富集的Ras同源物）是一种小的GTPase蛋白，通过调节mTORC1（哺乳动物-rapamycin- target-of- complex- 1）活性，在新皮层发育的细胞信号传导中起重要作用。已知mTORC1控制各种生物过程，包括在溶酶体膜室形成复合物的轴突生长。因此，通过Rheb半胱氨酸残基（C180）的法尼化，Rheb锚定在溶酶体膜上，是促进mTOR活性所必需的。为了测试Rheb法尼化的意义，我们在大鼠海马神经元和小鼠胚胎神经元中过度表达了Rheb法尼化突变形式Rheb C180S。有趣的是，我们发现Rheb C180S在两种测试系统中都保持了与野生型Rheb相似的轴突伸长促进作用。另一方面，Rheb C180S没有表现出野生型Rheb的多轴突促进作用。mTORC1的下游靶点phospho-4EBP1的水平在Rheb C180S转染的神经元中惊人地增加，尽管磷酸化mTOR的水平与对照载体转染相比显着降低。特异性mTORC1抑制剂雷帕霉素也不能完全消除转染细胞中Rheb C180S的轴突伸长特征。我们的数据表明，非膜腔室中的Rheb可以通过4EBP1的磷酸化和mtorc1独立通路促进轴突伸长。
Rheb (Ras homolog enriched in the brain) is a small GTPase protein that plays an important role in cell signaling for development of the neocortex through modulation of mTORC1 (mammalian-target-of-rapamycin-complex- 1) activity. mTORC1 is known to control various biological processes including axonal growth in forming complexes at the lysosomal membrane compartment. As such, anchoring of Rheb on the lysosomal membrane via the farnesylation of Rheb at its cysteine residue (C180) is required for its promotion of mTOR activity To test the significance of Rheb farnesylation, we overexpressed a farnesylation mutant form of Rheb, Rheb C180S, in primary rat hippocampal neurons and also in mouse embryonic neurons using in utero electroporation. Interestingly, we found that Rheb C180S maintained promotional effect of axonal elongation similar to the wild-type Rheb in both test systems. On the other hand, Rheb C180S failed to exhibit the multiple axon-promoting effect which is found in wild-type Rheb. The levels of phospho-4EBP1, a downstream target of mTORC1, were surprisingly increased in Rheb C180S transfected neurons, despite the levels of phosphorvlated mTOR being significantly decreased compared to control vector transfectants. A specific mTORC1 inhibitor, rapamycin, also could not completely abolish axon elongation characteristics of Rheb C180S in transfected cells. Our data suggests that Rheb in a non-membrane compartment can promote the axonal elongation via phosphorylation of 4EBP1 and through an mTORC1-independent pathway.