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© The Rockefeller University Press,
0021-9525/1998//1227 $5.00
The Journal of Cell Biology, Volume 143, Number 5,
, 1998 1227-1238
Article |
Nexilin: A Novel Actin Filament-binding Protein Localized at Cell–Matrix Adherens Junction
Takai Biotimer Project, ERATO, Japan Science and Technology Corporation (c/o JCR Pharmaceuticals Co., Ltd.), Kobe 651-2241, Japan
We isolated two novel actin filament (F-actin)–binding proteins from rat brain and rat 3Y1 fibroblast. They were splicing variants, and we named brain big one b-nexilin and fibroblast small one s-nexilin. b-Nexilin purified from rat brain was a protein of 656 amino acids (aa) with a calculated molecular weight of 78,392, whereas s-nexilin, encoded by the cDNA isolated from rat 3Y1 cells by the reverse transcriptase-PCR method, was a protein of 606 aa with a calculated molecular weight of 71,942. b-Nexilin had two F-actin– binding domains (ABDs) at the NH2-terminal and middle regions, whereas s-nexilin had one ABD at the middle region because 64 aa residues were deleted and 14 aa residues were inserted in the first NH2-terminal ABD of b-nexilin, and thereby the first ABD lost its activity. b- and s-nexilins bound along the sides of F-actin, but only b-nexilin showed F-actin cross-linking activity. b-Nexilin was mainly expressed in brain and testis, whereas s-nexilin was mainly expressed in testis, spleen, and fibroblasts, such as rat 3Y1 and mouse Swiss 3T3 cells, but neither b- nor s-nexilin was detected in liver, kidney, or cultured epithelial cells. An immunofluorescence microscopic study revealed that s-nexilin was colocalized with vinculin, talin, and paxillin at cell– matrix adherens junction (AJ) and focal contacts, but not at cell–cell AJ, in 3Y1 cells. Overexpressed b- and s-nexilins were localized at focal contacts but not at cell–cell AJ. These results indicate that nexilin is a novel F-actin–binding protein localized at cell–matrix AJ.
Key Words: actin-binding protein • focal contact • stress fiber • adherens junction • integrin
Abbreviations used in this paper: aa, amino acid(s); ABD, F-actin–binding domain(s); AJ, adherens junction(s); F-actin, actin filament; G-actin, actin monomer; GST, glutathione S-transferase; His6, six histidine residues; S1, subfragment 1.
The work performed at Osaka University Medical School was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, and Culture, Japan (1997), by grants-in-aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from the Ministry of Health and Welfare, Japan (1997), and by grants from the Human Frontier Science Program (1997).
Address all correspondence to Yoshimi Takai, Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565-0871, Osaka, Japan. Tel.: 81-6-879-3410. Fax: 81-6-879-3419. E-mail: ytakai{at}molbio.med.osaka-u.ac.jp
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