Serine-71 phosphorylation of Rac1 modulates downstream signaling.
Serine-71 phosphorylation of Rac1 modulates downstream signaling.
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DOI：
10.1371/journal.pone.0044358
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Gerhard R
中科院分区：
文献类型：
--
作者：
Schwarz J;Proff J;Hävemeier A;Ladwein M;Rottner K;Barlag B;Pich A;Tatge H;Just I;Gerhard R
Rho GTPases Rac1 和 Cdc42 通过向不同的信号通路发出信号来调节多种细胞功能。据信，GTP酶激活位置处特定效应子的存在决定了下游信号传导的途径。我们之前报道过 EGF 诱导的 Rac1/Cdc42 Ser-71 磷酸化。通过使用 Rac1 和 Cdc42 的磷酸模拟 S71E 突变体，我们研究了 Ser-71 磷酸化对与选定效应蛋白结合的影响。当 Ser-71 被交换为谷氨酸作为磷模拟取代时，Rac1 和 Cdc42 的组成型活性 (Q61L) 变体分别与其特定相互作用伙伴 Sra-1 和 N-WASP 以及其共同效应蛋白 PAK 的结合被取消。然而，与常见效应蛋白 IQGAP1/2/3 或 MRCK α 的相互作用几乎不受影响。这种矛盾的行为在功能测定中很明显。与 Rac1 Q61L 相比，磷模拟 Rac1 Q61L/S71E 不能诱导膜皱褶增加。相反，Rac1 Q61L/S71E 允许丝状伪足形成，这与占主导地位的 Sra-1/Wave 信号通路的废除一致。此外，与Rac1转染的细胞相反，Rac1 S71E未能激活PAK1/2。另一方面，Rac1 Q61L/S71E 在激活 NF-κB 方面与 Rac1 Q61L 一样有效，说明磷酸化 Rac1 的正信号转导。总之，这些数据表明 Rac1 和 Cdc42 在丝氨酸 71 处的磷酸化代表了一种可逆机制，可以改变 GTPase/效应器偶联的特异性，并优先解决选定的下游途径。
The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.
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