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Ras Binding Triggers Ubiquitination of the Ras Exchange Factor Ras-GRF2

机译：Ras绑定触发Ras交换因子Ras-GRF2的泛素化。

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Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.
机译：Ras是由上游鸟嘌呤核苷酸交换因子激活的小GTP酶，其中之一是Ras-GRF2。 GRF2是一种广泛表达的蛋白质，具有多个可识别的序列基序，包括Ras交换子基序（REM），包含破坏盒的PEST区（DB）和Cdc25结构域。 Cdc25域具有鸟嘌呤核苷酸交换因子活性，并与Ras相互作用。本文中，我们检查了GRF2中的DB基序是否通过遍在蛋白途径导致蛋白水解。基于Son-of-sevenless（Sos）蛋白的REM和Cdc25区的已解析结构，REM可以在Ras结合过程中稳定Cdc25域。 GRF2的DB基序位于REM和Cdc25结构域之间，引人猜测它可能在Ras结合后暴露于泛素化机制。 GRF2激活后，GRF2蛋白水平急剧下降，显性负性Ras诱导GRF2降解，这表明破坏GRF2不需要Ras下游信号传导，并且与Ras的结合对于降解很重要。 GRF2在体内被泛素化，可以使用质谱法检测到。在蛋白酶体抑制剂存在下，Ras-GRF2以高分子量结合物的形式积累，表明GRF2被26S蛋白酶体破坏。删除数据库可减少GRF2的泛素化。缺少Cdc25结构域的GRF2没有被泛素化，这表明不能结合Ras的蛋白质不能被正确地靶向破坏。 Cdc25结构域内消除Ras结合的点突变也消除了泛素化，这表明与Ras结合对于GRF2的泛素化是必需的。我们得出结论，由GTPase结合诱导的构象变化暴露了DB，从而靶向破坏GRF2。

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