Modulation of myosin light-chain phosphorylation by p21-activated kinase 1 in Escherichia coli invasion of human brain microvascular endothelial cells.
Cytoskeletal dynamics, modulated by actin-myosin interactions, play an important role in Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC). Herein, we show that inhibitors of myosin function, butanedione monoxide and ML-7, significantly blocked the E. coli invasion of HBMEC. The invasive E. coli induces myosin light-chain (MLC) phosphorylation during the invasion process, which gets recruited to the site of actin condensation beneath the bacteria. We also show that invading E. coli downregulates the activity of p21-activated kinase 1 (PAK1), which is an upstream regulator of MLC kinase (MLCK). Overexpression of wild-type PAK1 and constitutively active PAK1 in HBMEC inhibits E. coli invasion significantly with a concomitant decrease in MLC phosphorylation. The inhibition of E. coli invasion by these PAK1 mutants is due to the absence of phospho-MLC at the actin condensation points. In contrast, the dominant-negative PAK1 shows no effect either on the invasion or on MLC phosphorylation or phospho-MLC recruitment to the actin focal points, suggesting that activated PAK1 inactivates MLCK. Taken together, these results suggest that E. coli invasion of HBMEC induces MLC phosphorylation by inhibiting the activity of PAK1 and the recruitment of phosphorylated MLC to the site of actin condensation beneath the bacteria for efficient internalization of E. coli into HBMEC.
Rudrabhatla
Rajyalakshmi S
RS
Division of Infectious Diseases, Childrens Hospital Los Angeles and Keck School of Medicine, University of Southern California, Los Angeles, California 90027, USA.
Sukumaran
Sunil K
SK
Bokoch
Gary M
GM
Prasadarao
Nemani V
NV
D000199:Actins / Q000378:metabolism; D001921:Brain / Q000098:blood supply / Q000382:microbiology*; D002478:Cells, Cultured; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000166:cytology / Q000382:microbiology*; D004926:Escherichia coli / Q000472:pathogenicity*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D054462:p21-Activated Kinases
D000199:Actins; D018994:Myosin Light Chains; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha