Abstract
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 Ca2+/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 p60src and 80 kDa cortactin, an actin-binding protein and known p60src 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 (Y464, Y471) or cortactin (Y421, Y466, and Y482) by p60src 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.
Original language | English (US) |
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Pages (from-to) | 511-519 |
Number of pages | 9 |
Journal | Biochemical and Biophysical Research Communications |
Volume | 298 |
Issue number | 4 |
DOIs | |
State | Published - 2002 |
Externally published | Yes |
Keywords
- Arp 2/3 complex
- Cortactin
- Cytoskeleton
- Endothelial cell MLCK
- p60
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology