Domain analysis of the actin-binding and actin-remodeling activities of drebrin.
Drebrin is an actin-binding protein which is expressed at highly levels in neurons. When introduced into fibroblasts, it has been known to bind to F-actin and to cause remodeling of F-actin. Here, we performed a domain analysis of the actin-binding and actin-remodeling activities of drebrin. Various fragments of drebrin cDNA were fused with green fluorescent protein cDNA and introduced into Chinese hamster ovary cells. Association of the fusion protein with F-actin and remodeling of the F-actin were examined. We found that the central 85-amino-acid sequence (residues 233-317) was sufficient for the binding to and remodeling of F-actin. The binding activity of this fragment was relatively low compared with that of full-length drebrin, but all the types of abnormalities of F-actin that are observed with full-length drebrin were also observed with this fragment. When this sequence was further fragmented, the actin-binding activity was greatly reduced and the actin-remodeling activity disappeared. The actin-binding activity of the central region of drebrin was confirmed by a cosedimentation assay of chymotryptic fragments of drebrin with purified actin. These data indicate that the actin-binding domain and actin-remodeling domain are identical and that this domain is located at the central region of drebrin.
Hayashi
K
K
Department of Neurobiology and Behavior, Department of Pharmacology, Gunma University School of Medicine, 3-39-22 Showamachi, Maebashi, 371, Japan. hayashi@sb.gunma-u.ac.jp
Ishikawa
R
R
Kawai-Hirai
R
R
Takagi
T
T
Taketomi
A
A
Shirao
T
T
D000199:Actins / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002918:Chymotrypsin; D006224:Cricetinae; D018076:DNA, Complementary; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional / Q000502:physiology; D009479:Neuropeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013314:Stress, Mechanical
D000199:Actins; D018076:DNA, Complementary; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C053386:drebrins; D049452:Green Fluorescent Proteins; D002918:Chymotrypsin