Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.
Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin "pedestals." One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspF(U) (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY(458)) sequence in the Tir C-terminal cytoplasmic domain. Tir, EspF(U), and N-WASP form a complex, but neither EspF(U) nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspF(U) identified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspF(U), and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal NPY(458) sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.
Vingadassalom
Didier
D
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Kazlauskas
Arunas
A
Skehan
Brian
B
Cheng
Hui-Chun
HC
Magoun
Loranne
L
Robbins
Douglas
D
Rosen
Michael K
MK
Saksela
Kalle
K
Leong
John M
JM
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001287:Attachment Sites, Microbiological; D001422:Bacterial Adhesion; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D019453:Escherichia coli O157 / Q000166:cytology* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055232:Proline-Rich Protein Domains; D011485:Protein Binding; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018909:src Homology Domains
D000199:Actins; C523100:BAIAP2L1 protein, human; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C489109:EspFU protein, E coli; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C109287:Tir protein, E coli