Department of Physiology, The University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163, USA.
Cell Signal. 2012 Apr;24(4):931-42. doi: 10.1016/j.cellsig.2011.12.016. Epub 2011 Dec 29.
Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subsequently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, migration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of the EGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, we demonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine regulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted with integrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreading and migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activation of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activation in live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increased Src kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially interacts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuated by spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physically and functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to the trans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activation of FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanistically how polyamines modulate Src function at the molecular level.
细胞内多胺水平受鸟氨酸脱羧酶 (ODC) 的活性高度调节，ODC 催化多胺生物合成的第一个限速反应，产生腐胺，随后腐胺转化为亚精胺和精胺。我们已经表明，多胺调节肠上皮细胞的增殖、迁移和凋亡。多胺在 EGFR 和 Src 水平上调节关键信号事件。然而，多胺的确切作用机制尚不清楚。在本研究中，我们证明 ODC 在细胞铺展过程中定位于片状伪足和黏附斑。精胺通过调节 EGFR 与 Src 的相互作用来调节 EGF 诱导的迁移。EGFR 与整合素 β3、Src 和粘着斑激酶 (FAK) 相互作用。在铺展和迁移过程中，活性Src (pY418-Src) 与 FAK 定位在一起。精胺阻止 EGF 诱导的 EGFR 与整合素 β3、Src 和 FAK 的结合。Src 和 FAK 的激活是 HEK293 细胞中 EGF 诱导迁移所必需的。使用基于 FRET 的 Src 报告器在活 HEK293 细胞中进行 EGFR 介导的 Src 激活显示，多胺耗竭显着增加 Src 激酶活性。体外结合研究表明，精胺直接与 Src 结合，并优先与 Src 的 SH2 结构域相互作用。精胺严重削弱了 Src 和 EGFR 之间的物理相互作用。因此，精胺作为一种分子开关，在物理和功能上调节 EGFR-Src 偶联。EGFR 激活后，整合素 β3、FAK 和 Src 被募集到 EGFR，导致 EGFR 和 Src 的转激活以及 Src 介导的 FAK 磷酸化。FAK 的激活诱导 Rho-GTPases，随后发生迁移。这是第一项在分子水平上定义多胺如何调节 Src 功能的机制研究。
