Nieto-Pelegrin Elvira, Meiler Eugenia, Martín-Villa José Manuel, Benito-León María, Martinez-Quiles Narcisa
Department of Microbiology, School of Pharmacy, Complutense University, Madrid, Spain.
Division of Immunology, School of Medicine, Complutense University, Madrid, Spain.
PLoS Pathog. 2014 Mar 27;10(3):e1004022. doi: 10.1371/journal.ppat.1004022. eCollection 2014 Mar.
Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.
肠道致病性大肠杆菌（EPEC）感染会导致腹泻，这与发展中国家的高婴儿死亡率相关。EPEC粘附于上皮细胞并诱导肌动蛋白基座的形成。肌动蛋白聚合基本上是通过由Tir细菌效应蛋白介导的信号传导驱动的，该蛋白插入被感染细胞的质膜中。Tir结合Nck衔接蛋白，后者进而招募并激活N-WASP，它是威斯科特-奥尔德里奇综合征蛋白家族中普遍存在的成员。N-WASP激活Arp2/3复合物以促进肌动蛋白聚合。除了Tir-Nck-N-WASP途径的成分外，其他蛋白质也被招募到基座，但它们的功能尚不清楚。在这里，我们研究了Crk衔接蛋白的两种可变剪接异构体（CrkI/II）和旁系同源蛋白CrkL在EPEC形成基座过程中的功能。我们发现Crk异构体作为基座形成的冗余抑制剂。CrkII和CrkL的SH2结构域与Tir的磷酸化酪氨酸474结合，并与Nck竞争结合Tir，阻止其被招募到基座，从而抑制肌动蛋白聚合。EPEC感染诱导CrkII和CrkL中主要调节酪氨酸的磷酸化，可能阻止这些蛋白质的SH2结构域与Tir相互作用。磷酸化的CrkII和CrkL蛋白特异性定位于与EPEC接触的质膜。我们的研究揭示了Crk衔接蛋白在基座处的新作用，为这些癌蛋白如何调节肌动蛋白聚合开辟了新的视角。
