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

Abstract

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 α5β1 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.

ACKNOWLEDGMENTS

We thank Shoukat Dedhar for providing the human ILK cDNA and anti-human ILK antibody, Oliver Hobert (Massachusetts General Hospital) for sharing unpublished results, Keith Burridge (University of North Carolina at Chapel Hill) for valuable discussions, Mary Beckerle (University of Utah) for providing human zyxin cDNA, John Couchman and Anne Woods (University of Alabama at Birmingham) for providing rat kidney mesangial cells, and the Hybridoma Core Facility of the University of Alabama at Birmingham for technical assistance in the production of the mouse monoclonal anti-PINCH antibodies.

This work was supported in part by National Institutes of Health grant DK54639, research project grant 98-220-01-CSM from the American Cancer Society, and research grants from the American Heart Association, the American Lung Association, the Francis Families Foundation, and the V Foundation for Cancer Research (to C.W.). F.L. was supported by The Cell Adhesion and Matrix Research Center of the University of Alabama at Birmingham. C.W. is a V Foundation Scholar and a Parker B. Francis Fellow in Pulmonary Research of the Francis Families Foundation.

Y.T. and F.L. contributed equally to this work.