Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
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Two Regions Responsible for the Actin Binding of p57, a Mammalian Coronin Family Actin-Binding Protein
Teruaki OkuSaotomo ItohMasamitsu OkanoAkiko SuzukiKensuke SuzukiShizuo NakajinTsutomu TsujiWilliam Michael NauseefSatoshi Toyoshima
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2003 Volume 26 Issue 4 Pages 409-416

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Abstract

The actin-binding protein p57, a member of the coronin protein family, is expressed in a variety of immune cells. It has five WD repeats and a coiled-coil motif containing a leucine zipper, both of which are known to mediate protein–protein interactions. In order to identify the precise actin-binding regions in p57, and to assess the contribution of these structural motifs, we prepared various truncated p57 as fusion proteins with glutathione S-transferase (GST) and examined their actin-binding activity. A co-sedimentation assay demonstrated that p571—371 (C-terminal truncated p57) had the ability to bind F-actin, but p57372—461 (a fragment containing the coiled-coil motif) did not. A segment consisting of the N-terminal 34 amino acids of p57 (p571—34) was found to bind to F-actin in the co-sedimentation assay. Furthermore, fluorescence microscopic observation showed that p571—34 was co-localized with F-actin in COS-1 cells after the transfection with the p571—34 construct. Deletion of 10KFRHVF15, a sequence conserved among coronin-related proteins, from p571—34 abolished its actin-binding activity, suggesting that this sequence with basic and hydrophobic amino acids is crucial for p57 to bind to F-actin. However, the N-terminal deletion mutant p5763—461 retained the binding ability to F-actin. This result suggests the presence of a second actin-binding region. Further deletion analysis revealed that p57111—204, which includes the second and third WD repeats, also exhibited weak actin-binding activity in the co-sedimentation assay. Taken together, these data strongly suggest that at least two regions within Met-1 to Asp-34 and Ile-111 to Glu-204 of p57 are responsible for its binding to the actin cytoskeleton.

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© 2003 The Pharmaceutical Society of Japan
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