Crk adaptors negatively regulate actin polymerization in pedestals formed by enteropathogenic Escherichia coli (EPEC) by binding to Tir effector.

[wiskott-aldrich syndrome]

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 Arp 2 /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 SH 2 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 SH 2 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 .