A new focal adhesion protein that interacts with integrin-linked kinase and regulates cell adhesion and spreading

J Cell Biol. 2001 Apr 30;153(3):585-98. doi: 10.1083/jcb.153.3.585.

Abstract

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 NH(2)-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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actinin*
  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • CHO Cells
  • Calcium-Binding Proteins
  • Calponins
  • Carrier Proteins / genetics
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism*
  • Cell Adhesion*
  • Cell Compartmentation
  • Cell Size*
  • Cricetinae
  • Cytoskeleton / chemistry
  • DNA-Binding Proteins / metabolism
  • Focal Adhesions / chemistry
  • Focal Adhesions / metabolism*
  • Humans
  • LIM Domain Proteins
  • Membrane Proteins
  • Microfilament Proteins
  • Molecular Sequence Data
  • Protein Binding
  • Protein Serine-Threonine Kinases / metabolism*
  • Sequence Homology, Amino Acid
  • Two-Hybrid System Techniques

Substances

  • Adaptor Proteins, Signal Transducing
  • Calcium-Binding Proteins
  • Carrier Proteins
  • DNA-Binding Proteins
  • LIM Domain Proteins
  • LIMS1 protein, human
  • Membrane Proteins
  • Microfilament Proteins
  • PARVA protein, human
  • PARVB protein, human
  • Actinin
  • integrin-linked kinase
  • Protein Serine-Threonine Kinases