Two regions responsible for the actin binding of p57, a mammalian coronin family actin-binding protein.
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 p57(1-371) (C-terminal truncated p57) had the ability to bind F-actin, but p57(372-461) (a fragment containing the coiled-coil motif) did not. A segment consisting of the N-terminal 34 amino acids of p57 (p57(1-34)) was found to bind to F-actin in the co-sedimentation assay. Furthermore, fluorescence microscopic observation showed that p57(1-34) was co-localized with F-actin in COS-1 cells after the transfection with the p57(1-34) construct. Deletion of (10)KFRHVF(15), a sequence conserved among coronin-related proteins, from p57(1-34) 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 p57(63-461) retained the binding ability to F-actin. This result suggests the presence of a second actin-binding region. Further deletion analysis revealed that p57(111-204), 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.
Oku
Teruaki
T
Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan.
Itoh
Saotomo
S
Okano
Masamitsu
M
Suzuki
Akiko
A
Suzuki
Kensuke
K
Nakajin
Shizuo
S
Tsuji
Tsutomu
T
Nauseef
William Michael
WM
Toyoshima
Satoshi
S
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D000199:Actins; D008840:Microfilament Proteins; D038941:Polypyrimidine Tract-Binding Protein; C072061:coronin proteins