Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of β1 integrin, seems to be a key component of FAs, but its exact role in cell–substrate interaction remains to be 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 tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin–ILK signaling required for the establishment of cell–substrate adhesion.
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11 June 2001
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June 11 2001
A Novel Integrin-Linked Kinase–Binding Protein, Affixin, Is Involved in the Early Stage of Cell–Substrate Interaction
Satoshi Yamaji,
Satoshi Yamaji
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Atsushi Suzuki,
Atsushi Suzuki
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Yuki Sugiyama,
Yuki Sugiyama
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Yu-ichi Koide,
Yu-ichi Koide
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Michihiko Yoshida,
Michihiko Yoshida
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Heiwa Kanamori,
Heiwa Kanamori
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Hiroshi Mohri,
Hiroshi Mohri
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Shigeo Ohno,
Shigeo Ohno
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Yoshiaki Ishigatsubo
Yoshiaki Ishigatsubo
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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Satoshi Yamaji
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
Atsushi Suzuki
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
Yuki Sugiyama
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
Yu-ichi Koide
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
Michihiko Yoshida
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
Heiwa Kanamori
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
Hiroshi Mohri
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
Shigeo Ohno
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
Yoshiaki Ishigatsubo
aThe First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
bDepartment of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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.
Online ISSN: 1540-8140
Print ISSN: 0021-9525
© 2001 The Rockefeller University Press
2001
The Rockefeller University Press
J Cell Biol (2001) 153 (6): 1251–1264.
Citation
Satoshi Yamaji, Atsushi Suzuki, Yuki Sugiyama, Yu-ichi Koide, Michihiko Yoshida, Heiwa Kanamori, Hiroshi Mohri, Shigeo Ohno, Yoshiaki Ishigatsubo; A Novel Integrin-Linked Kinase–Binding Protein, Affixin, Is Involved in the Early Stage of Cell–Substrate Interaction. J Cell Biol 11 June 2001; 153 (6): 1251–1264. doi: https://doi.org/10.1083/jcb.153.6.1251
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