Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation.
Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation.
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DOI：
10.2142/biophysico.bppb-v18.001
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发表时间：
2021
影响因子：
1.5
通讯作者：
Sako Y
中科院分区：
文献类型：
--
作者：
Yoshizawa R;Umeki N;Yamamoto A;Murata M;Sako Y
RTK - RAS - MAPK系统是细胞命运决定的主要信号通路。在多种受体酪氨酸激酶（RTK）类型中，已知细胞外信号调节激酶（ERK，即丝裂原活化蛋白激酶，MAPK）的瞬时激活会刺激细胞增殖，而其持续激活则诱导细胞分化。然而，在这两种情况下，RAS的激活都是瞬时的，这表明在正常细胞中，对其活性进行严格的时间调控至关重要。质膜胞质侧的RAS通过生长因子刺激后发生磷酸化的RTK招募GRB2 / SOS复合物，进而由SOS激活。衔接蛋白GRB2既能直接识别磷酸化的RTK，也能通过另一种衔接蛋白SHC间接识别。我们在此利用活细胞中的单分子成像和荧光相关光谱技术，研究了SHC途径下GRB2招募的调控机制。我们用分化因子（神经调节蛋白）刺激MCF7细胞，并观察了包括GRB2和SHC在内的细胞信号分子的易位、复合物形成及磷酸化情况。我们的研究结果表明，SHC对GRB2 / SOS - RAS途径存在双相调控：在刺激早期（<10分钟），SHC通过增加质膜上GRB2 / SOS复合物的结合位点来提高RAS活性的幅度。然而在后期，SHC抑制RAS激活，并通过在细胞质中形成复合物，将GRB2分子从膜上隔离。后一种机制与MEK和/或ERK对RAS的其他负反馈调节机制协同作用，共同完成RAS的瞬时激活动力学过程。
RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS.