Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity
Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity
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DOI：
10.1128/mcb.24.18.7965-7975.2004
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发表时间：
2004-09-01
影响因子：
5.3
通讯作者：
Guan, KL
中科院分区：
文献类型：
--
作者：
Li, Y;Inoki, K;Guan, KL
结节性硬化症(TSC)是一种由TSC1或TSC2肿瘤抑制基因突变引起的遗传病。最近的研究表明，TSC2具有针对小G蛋白Rheb的GAP(GTPase激活蛋白)活性，并抑制其刺激mTOR信号通路的能力。Rheb和TSC2组成一对独特的GTP酶和GAP，因为Rheb有很高的基础GTP水平，而TSC2没有在RAS-GAP中发现的催化精氨酸手指。为了研究TSC2和Rheb在mTOR信号转导中的作用，我们分析了TSC2刺激的Rheb GTPase活性。我们发现，Arg15是RAS中相当于Gly12的残基，对于Rheb作为TSC2 GAP的底物是重要的。此外，我们还鉴定了TSC2 GAP活性所必需的天冬酰胺残基。我们证明了TSC2的GAP和RHEB的一种新的催化机理，即TSC2使用催化的“天冬酰胺拇指”来代替RAS-GAP中的精氨酸手指。此外，我们还发现Rheb的法尼化和膜定位不是Rheb刺激S6激酶(S6K)磷酸化所必需的。对TSC2的TSC1结合缺陷突变体的分析表明，TSC1对TSC2的GAP活性并不是必需的，但可能作为TSC1/TSC2复合体的调节成分发挥作用。我们的数据进一步证明，GAP活性对于TSC2抑制S6K磷酸化的细胞功能是必不可少的。
Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated that TSC2 displays GAP (GTPase-activating protein) activity specifically towards the small G protein Rheb and inhibits its ability to stimulate the mTOR signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, because Rheb has high basal GTP levels and TSC2 does not have the catalytic arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to function as a substrate for TSC2 GAP. In addition, we identified asparagine residues essential for TSC2 GAP activity. We demonstrated a novel catalytic mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that farnesylation and membrane localization of Rheb is not essential for Rheb to stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but may function as a regulatory component in the TSC1/TSC2 complex. Our data further demonstrate that GAP activity is essential for the cellular function of TSC2 to inhibit S6K phosphorylation.