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Molecular and Cellular Biology, March 1999, p. 2425-2434, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The LIM-Only Protein PINCH Directly Interacts with Integrin-Linked Kinase and Is Recruited to Integrin-Rich Sites in Spreading Cells

Yizeng Tu, Fugang Li, Silvia Goicoechea, and Chuanyue Wu*

Department of Cell Biology and The Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019

Received 31 August 1998/Returned for modification 13 October 1998/Accepted 11 December 1998

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha 5beta 1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways.

* Corresponding author. Mailing address: 217 Volker Hall, Department of Cell Biology and The Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, 1670 University Blvd., Birmingham, AL 35294-0019. Phone: (205) 975-2253. Fax: (205) 934-7029. E-mail: cwu{at}cellbio.bhs.uab.edu.

Molecular and Cellular Biology, March 1999, p. 2425-2434, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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