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A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction.

Yamaji S, Suzuki A, Sugiyama Y, Koide Y, Yoshida M, Kanamori H, Mohri H, Ohno S, Ishigatsubo Y - J. Cell Biol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.

ABSTRACT
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 beta1 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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ILK phosphorylates affixin in vitro. (A) Kinase activity of wild-type ILK and its point mutants overexpressed in COS-7 cells. Flag-tagged ILK or its mutants were overexpressed and immunoprecipitated from COS-7 cell lysates with monoclonal anti-Flag antibody. Kinase activities of the resultant immunocomplexes were examined using MBP as a substrate. Top, autoradiography showing 32P incorporation into MBP; bottom, Western blot analysis of the precipitated Flag-tagged ILK or its mutants using polyclonal anti-Flag antibody. (B) Full-length and COOH-terminal half of ss-affixin are phosphorylated by ILK in vitro. Recombinant full-length and COOH-terminal half of ss-affixin (RP2) were used as substrates to estimate the kinase activity of the ILK immunocomplexes prepared as described in A.
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Figure 5: ILK phosphorylates affixin in vitro. (A) Kinase activity of wild-type ILK and its point mutants overexpressed in COS-7 cells. Flag-tagged ILK or its mutants were overexpressed and immunoprecipitated from COS-7 cell lysates with monoclonal anti-Flag antibody. Kinase activities of the resultant immunocomplexes were examined using MBP as a substrate. Top, autoradiography showing 32P incorporation into MBP; bottom, Western blot analysis of the precipitated Flag-tagged ILK or its mutants using polyclonal anti-Flag antibody. (B) Full-length and COOH-terminal half of ss-affixin are phosphorylated by ILK in vitro. Recombinant full-length and COOH-terminal half of ss-affixin (RP2) were used as substrates to estimate the kinase activity of the ILK immunocomplexes prepared as described in A.

Mentions: Since the kinase domain of ILK is sufficient to associate with affixin, we next tested whether affixin can be phosphorylated by ILK in vitro. In Fig. 5 A, Flag-tagged wild-type or kinase-deficient ILK was overexpressed in COS-7 cells, and the kinase activity in the anti-Flag antibody immunoprecipitates was assayed using MBP as a substrate. The immunoprecipitates from cells expressing wild-type ILK showed enhanced kinase activity, whereas those from ILK (K220A)- and ILK(K220M)-expressing cells showed the background level of the activity. Surprisingly, the immunoprecipitate of ILK(E359K) reproducibly showed kinase activity comparable to wild-type ILK, suggesting that this mutant is not kinase deficient, although we do not know the reason for the discrepancy with the previous data (Novak et al. 1998). When we used recombinant affixin purified from E. coli as a substrate, the wild-type ILK immunoprecipitates but not the K220M immunoprecipitates phosphorylated affixin to the same extent as MBP, suggesting that affixin is a good substrate for ILK in vitro (Fig. 5 B). Consistent with the results on the narrowed binding site of the ILK–affixin interaction, the deletion mutant of affixin, RP2, was also phosphorylated by the ILK immunoprecipitates to a similar extent (Fig. 5 B). We also examined the possibility that affixin affects the kinase activity of ILK, since as shown in Fig. 4 D the activation loop of the kinase domain of ILK is involved in the interaction with affixin. However, the addition of recombinant affixin to the ILK immunoprecipitates did not affect 32P incorporation into MBP (data not shown), suggesting that affixin can be a substrate but not a regulator of ILK kinase activity.


A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction.

Yamaji S, Suzuki A, Sugiyama Y, Koide Y, Yoshida M, Kanamori H, Mohri H, Ohno S, Ishigatsubo Y - J. Cell Biol. (2001)

ILK phosphorylates affixin in vitro. (A) Kinase activity of wild-type ILK and its point mutants overexpressed in COS-7 cells. Flag-tagged ILK or its mutants were overexpressed and immunoprecipitated from COS-7 cell lysates with monoclonal anti-Flag antibody. Kinase activities of the resultant immunocomplexes were examined using MBP as a substrate. Top, autoradiography showing 32P incorporation into MBP; bottom, Western blot analysis of the precipitated Flag-tagged ILK or its mutants using polyclonal anti-Flag antibody. (B) Full-length and COOH-terminal half of ss-affixin are phosphorylated by ILK in vitro. Recombinant full-length and COOH-terminal half of ss-affixin (RP2) were used as substrates to estimate the kinase activity of the ILK immunocomplexes prepared as described in A.
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Related In: Results  -  Collection

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Figure 5: ILK phosphorylates affixin in vitro. (A) Kinase activity of wild-type ILK and its point mutants overexpressed in COS-7 cells. Flag-tagged ILK or its mutants were overexpressed and immunoprecipitated from COS-7 cell lysates with monoclonal anti-Flag antibody. Kinase activities of the resultant immunocomplexes were examined using MBP as a substrate. Top, autoradiography showing 32P incorporation into MBP; bottom, Western blot analysis of the precipitated Flag-tagged ILK or its mutants using polyclonal anti-Flag antibody. (B) Full-length and COOH-terminal half of ss-affixin are phosphorylated by ILK in vitro. Recombinant full-length and COOH-terminal half of ss-affixin (RP2) were used as substrates to estimate the kinase activity of the ILK immunocomplexes prepared as described in A.
Mentions: Since the kinase domain of ILK is sufficient to associate with affixin, we next tested whether affixin can be phosphorylated by ILK in vitro. In Fig. 5 A, Flag-tagged wild-type or kinase-deficient ILK was overexpressed in COS-7 cells, and the kinase activity in the anti-Flag antibody immunoprecipitates was assayed using MBP as a substrate. The immunoprecipitates from cells expressing wild-type ILK showed enhanced kinase activity, whereas those from ILK (K220A)- and ILK(K220M)-expressing cells showed the background level of the activity. Surprisingly, the immunoprecipitate of ILK(E359K) reproducibly showed kinase activity comparable to wild-type ILK, suggesting that this mutant is not kinase deficient, although we do not know the reason for the discrepancy with the previous data (Novak et al. 1998). When we used recombinant affixin purified from E. coli as a substrate, the wild-type ILK immunoprecipitates but not the K220M immunoprecipitates phosphorylated affixin to the same extent as MBP, suggesting that affixin is a good substrate for ILK in vitro (Fig. 5 B). Consistent with the results on the narrowed binding site of the ILK–affixin interaction, the deletion mutant of affixin, RP2, was also phosphorylated by the ILK immunoprecipitates to a similar extent (Fig. 5 B). We also examined the possibility that affixin affects the kinase activity of ILK, since as shown in Fig. 4 D the activation loop of the kinase domain of ILK is involved in the interaction with affixin. However, the addition of recombinant affixin to the ILK immunoprecipitates did not affect 32P incorporation into MBP (data not shown), suggesting that affixin can be a substrate but not a regulator of ILK kinase activity.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.

ABSTRACT
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 beta1 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.

Show MeSH
Related in: MedlinePlus