Novel interaction of cortactin with endothelial cell myosin light chain kinase.
Inflammatory mediators such as thrombin evoke increases in vascular permeability through activation of endothelial contractile mechanisms which involve increased levels of MLC phosphorylation catalyzed by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK). We previously noted that the high molecular weight endothelial MLCK isoform (EC MLCK) is stably associated with a complex containing p60(src) and 80kDa cortactin, an actin-binding protein and known p60(src) target. In this study we have utilized in vitro binding assays to confirm specific interaction between EC MLCK and cortactin. Tyrosine phosphorylation of either EC MLCK (Y(464), Y(471)) or cortactin (Y(421), Y(466), and Y(482)) by p60(src) significantly increased this direct association. Site-specific antibody and peptide studies subsequently confirmed EC MLCK AA #972-979 and 1019-1025 as sites of cortactin interaction. EC MLCK-cortactin interaction in vitro failed to modulate MLCK enzymatic activity but appeared to inhibit EC MLCK binding to F-actin, while EC MLCK abolished cortactin-mediated augmentation of Arp2/3-stimulated actin polymerization. These data suggest that cortactin-EC MLCK interaction may be a novel determinant of endothelial cortical actin-based cytoskeletal rearrangement.
Dudek
Steven M
SM
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
Birukov
Konstantin G
KG
Zhan
Xi
X
Garcia
Joe G N
JG
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D051356:Cortactin; D004727:Endothelium / Q000166:cytology / Q000201:enzymology; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D000199:Actins; D051356:Cortactin; D008840:Microfilament Proteins; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase