Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane

• Published in: Journal of cellular physiology 2006-09, Vol.208 (3), p.620-628
• Main Authors: Stroeken, Peter J.M., Alvarez, Belén, Rheenen, Jacco van, Wijnands, Yvonne M., Geerts, Dirk, Jalink, Kees, Roos, Ed
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Cell Membrane - enzymology; Cercopithecus aethiops; COS Cells; DNA - genetics; Fluorescence Resonance Energy Transfer; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Mice; Phosphotyrosine - metabolism; Polymerase Chain Reaction; Protein-Serine-Threonine Kinases - metabolism; rho-Associated Kinases; Transfection
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.20699

The integrin cytoplasmic domain‐associated protein‐1 (ICAP‐1) binds via its C‐terminal PTB (phosphotyrosine‐binding) domain to the cytoplasmic tails of β1 but not other integrins. Using the yeast two‐hybrid assay, we found that ICAP‐1 binds the ROCK‐I kinase, an effector of the RhoA GTPase. By coimmunoprecipitation we show that ICAP‐1 and ROCK form complexes in cells and that ICAP‐1 contains two binding sites for ROCK. In cells transfected with both ICAP‐1 and ROCK, the proteins colocalized at the cell membrane predominantly in lamellipodia and membrane ruffles, but also in retraction fibers. ROCK was not found at these sites when ICAP‐1 was not co‐transfected, indicating that ICAP‐1 translocated ROCK. In lamellipodia ICAP‐1 and ROCK colocalized with endogenous β1 integrins and this colocalization was also observed with the isolated ICAP‐1 PTB domain. The plasma membrane localization of ROCK did not depend on β1 integrin ligation or ROCK kinase activity, and in truncated ROCK proteins it required the presence of the ICAP‐1‐binding domain. To show that the interaction was direct, we measured fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) fused to ICAP‐1 and yellow fluorescent protein (YFP) fused to ROCK. FRET was observed in lamellipodia in cells that were induced to spread. These results indicate that ICAP‐1‐mediated binding of ROCK to β1 integrin serves to localize the ROCK‐I kinase to both the leading edge and the trailing edge where ROCK affects cell migration. J. Cell. Physiol. 208: 620–628, 2006. © 2006 Wiley‐Liss, Inc.