A Novel Integrin-Linked Kinase-Binding Protein, Affixin, Is Involved in the Early Stage of Cell-Substrate Interaction

doi:10.1083/jcb.153.6.1251

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Published online 11 June 2001. doi:10.1083/jcb.153.6.1251

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© The Rockefeller University Press, 0021-9525/2001//1251 $5.00
The Journal of Cell Biology, Volume 153, Number 6, , 2001 1251-1264

Original Article

A Novel Integrin-Linked Kinase–Binding Protein, Affixin, Is Involved in the Early Stage of Cell–Substrate Interaction

Satoshi Yamaji^a, Atsushi Suzuki^b, Yuki Sugiyama^b, Yu-ichi Koide^b, Michihiko Yoshida^a, Heiwa Kanamori^a, Hiroshi Mohri^a, Shigeo Ohno^b, and Yoshiaki Ishigatsubo^a^,b

^a The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
^b Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
The First Department of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan.81-45-786-344481-45-787-2630

ishigatsu{at}med.yokohama-cu.ac.jp

Focal adhesions (FAs) are essential structures for cell adhesion,migration, and morphogenesis. Integrin-linked kinase (ILK),which is capable of interacting with the cytoplasmic domainof β1 integrin, seems to be a key component of FAs, butits exact role in cell–substrate interaction remains tobe clarified. Here, we identified a novel ILK-binding protein,affixin, that consists of two tandem calponin homology domains.In CHOcells, affixin and ILK colocalize at FAs and at the tipof the leading edge, whereas in skeletal muscle cells they colocalizeat the sarcolemma where cells attach to the basal lamina, showinga striped pattern corresponding to cytoplasmic Z-band striation.When CHO cells are replated on fibronectin, affixin and ILKbut not FA kinase and vinculin concentrate at the cell surfacein blebs during the early stages of cell spreading, which willgrow into membrane ruffles on lamellipodia. Overexpression ofthe COOH-terminal region of affixin, which is phosphorylatedby ILK in vitro, blocks cell spreading at the initial stage,presumably by interfering with the formation of FAs and stressfibers. The coexpression of ILK enhances this effect. Theseresults provide evidence suggesting that affixin is involvedin integrin–ILK signaling required for the establishmentof cell–substrate adhesion.

Key Words: affixin • cell spreading • focal adhesion • integrin-linked kinase • integrin

Abbreviations used in this paper: CH, calponin homology; ECM,extracellular matrix; FA, focal adhesion; FAK, FA kinase; GST,glutathione S-transferase; ILK, integrin-linked kinase; MBP,myelin basic protein; SF, stress fiber.

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