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© The Rockefeller University Press,
0021-9525/2001//585 $5.00
The Journal of Cell Biology, Volume 153, Number 3,
, 2001 585-598
Original Article |
A New Focal Adhesion Protein That Interacts with Integrin-Linked Kinase and Regulates Cell Adhesion and Spreading
carywu{at}imap.pitt.edu
Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH2-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti–CH-ILKBP antibodies, and green fluorescent protein–CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP–ILK–PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.
Key Words: focal adhesion • integrin-linked kinase • calponin homology • integrins • cell adhesion
© 2001 The Rockefeller University Press
The online version of this article contains supplemental material.Y. Tu and Y. Huang contributed equally to this work.
Abbreviations used in this paper: ANK, ankyrin; CH, calponin homology; CH-ILKBP, CH domain-containing ILK-binding protein; ECM, extracellular matrix; ERK, extracellular signal–regulated kinase; FA, focal adhesion; FAK, FA kinase; GFP, green fluorescent protein; GST, glutathione S-transferase; ILK, integrin-linked kinase; MBP, maltose-binding protein.
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