title: Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells.
abstract: Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.
authors.affiliation: Division of Infectious Diseases, The Saban Research Institute, Children's Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027, USA.
authors.forename: Rajyalakshmi S
authors.initials: RS
authors.lastname: Rudrabhatla
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Selvaraj
authors.forename: Nemani V
authors.initials: NV
authors.lastname: Prasadarao
mesh_terms: D001921:Brain / Q000098:blood supply / Q000166:cytology / Q000382:microbiology*; D002478:Cells, Cultured; D015536:Down-Regulation; D042783:Endothelial Cells / Q000382:microbiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000382:microbiology*; D004926:Escherichia coli / Q000472:pathogenicity*; D006801:Humans; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
chemical_list: D018994:Myosin Light Chains; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein