Isotype-specific degradation of Rac activated by the cytotoxic necrotizing factor 1
Isotype-specific degradation of Rac activated by the cytotoxic necrotizing factor 1
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DOI：
10.1074/jbc.m404346200
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发表时间：
2004-08-20
影响因子：
4.8
通讯作者：
Schmidt, G
中科院分区：
文献类型：
--
作者：
Pop, M;Aktories, K;Schmidt, G
来自大肠杆菌的细胞毒性坏死因子 1 (CNF1) 通过谷氨酰胺 61/63 的脱酰胺作用激活 Rho 家族成员。由于该氨基酸对于 GTP 水解至关重要，因此谷氨酰胺 61/63 的脱酰胺作用会产生组成型活性 Rho 蛋白。最近，研究表明，在 CNF1 处理的细胞中，CNF1 激活的 Rac 水平通过蛋白水解降解而迅速降低。在这里，我们研究了 CNF1 诱导的 Rac 降解的要求。通过过表达 His 标记的激活 Rac 突变体，我们表明组成型激活对于 Rac 的降解是必要的。然而，永久激活不足以降解，因为通过博德特氏菌皮肤坏死毒素在谷氨酰胺 61 处的转酰胺基作用而组成型激活的 Rac 不会被降解。与 GTP 酶激活蛋白 (Rac(N92D) 和 Rac(Y64H)) 和鸟苷核苷酸解离抑制剂 (Rac(H103E)) 相互作用缺陷的 His 标记 Rac 突变体的过表达在 CNF1 激活后会被降解，而无法与 CRIB 结构域效应子或大量 SH3 相互作用的 Rac(Y40C) 则不会被降解。 退化了。异戊二烯化和假定的有丝分裂破坏盒的存在对于 CNF 诱导的降解至关重要。与 Rac1 相比，Rac2 和 Rac3 在 CNF1 组成型激活后不会降解。使用定点诱变，我们将多碱基区域和氨基酸 90、107、147 和 151 定义为负责同种型特异性降解。
The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates members of the Rho family by deamidation of glutamine 61/63. Because this amino acid is crucial for GTP hydrolysis, deamidation of glutamine 61/63 results in constitutively active Rho proteins. Recently, it was shown that the level of CNF1-activated Rac is rapidly diminished in CNF1-treated cells by proteolytic degradation. Here, we studied the requirements for CNF1-induced Rac degradation. By overexpressing His-tagged activated Rac mutants we show that constitutive activation is necessary for degradation of Rac. However, permanent activation is not sufficient for degradation, because Rac that is constitutively activated by transamidation at glutamine 61 by the Bordetella dermonecrotic toxin is not degraded. Overexpression of His-tagged Rac mutants deficient in interaction with GTPase-activating protein (Rac(N92D) and Rac(Y64H)) and guanosine nucleotide dissociation inhibitor (Rac(H103E)) were degraded after activation by CNF1, whereas Rac(Y40C), which is not able to interact with CRIB domain effectors or plenty of SH3, was not degraded. Isoprenylation and the presence of a putative mitotic destruction box are essential for CNF-induced degradation. In contrast to Rac1, Rac2, and Rac3 were not degraded following constitutive activation by CNF1. Using site-directed mutagenesis, we defined the polybasic region and amino acids 90, 107, 147, and 151 as responsible for isotype-specific degradation.