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Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction.

ABSTRACT: The linking of integrin to cytoskeleton is a critical event for an effective cell migration. Previously, we have reported that a novel integrin-linked kinase (ILK)-binding protein, affixin, is closely involved in the linkage between integrin and cytoskeleton in combination with ILK. In the present work, we demonstrated that the second calponin homology domain of affixin directly interacts with alpha-actinin in an ILK kinase activity-dependent manner, suggesting that integrin-ILK signaling evoked by substrate adhesion induces affixin-alpha-actinin interaction. The overexpression of a peptide corresponding to the alpha-actinin-binding site of affixin as well as the knockdown of endogenous affixin by small interference RNA resulted in the blockade of cell spreading. Time-lapse observation revealed that in both experiments cells were round with small peripheral blebs and failed to develop lamellipodia, suggesting that the ILK-affixin complex serves as an integrin-anchoring site for alpha-actinin and thereby mediates integrin signaling to alpha-actinin, which has been shown to play a critical role in actin polymerization at focal adhesions.

SUBMITTER: Yamaji S

PROVIDER: S-EPMC2172344 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction.

Yamaji Satoshi S Suzuki Atsushi A Kanamori Heiwa H Mishima Wataru W Yoshimi Ryusuke R Takasaki Hirotaka H Takabayashi Maki M Fujimaki Katsumichi K Fujisawa Shin S Ohno Shigeo S Ishigatsubo Yoshiaki Y

The Journal of cell biology 20040501 4

The linking of integrin to cytoskeleton is a critical event for an effective cell migration. Previously, we have reported that a novel integrin-linked kinase (ILK)-binding protein, affixin, is closely involved in the linkage between integrin and cytoskeleton in combination with ILK. In the present work, we demonstrated that the second calponin homology domain of affixin directly interacts with alpha-actinin in an ILK kinase activity-dependent manner, suggesting that integrin-ILK signaling evoked ...[more]

PMID: 15159419

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Project description:BackgroundAnnexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or null mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.ResultsTo identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.ConclusionsIn overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

Dataset Information

Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction.

Publications

Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction.

Similar Datasets

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