V-1, a protein expressed transiently during murine cerebellar development, regulates actin polymerization via interaction with capping protein.
V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the alpha and beta subunits of the capping protein (CP, alternatively called CapZ or beta-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1.CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 x 10(-7) m and a molecular stoichiometry of approximately 1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development.
Taoka
Masato
M
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan. mango@comp.metro-u.ac.jp
Ichimura
Tohru
T
Wakamiya-Tsuruta
Akiko
A
Kubota
Yoshiaki
Y
Araki
Takeshi
T
Obinata
Takashi
T
Isobe
Toshiaki
T
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000818:Animals; D051347:CapZ Actin Capping Protein; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002531:Cerebellum / Q000254:growth & development*; D051345:Destrin; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007668:Kidney; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D051339:Actin Depolymerizing Factors; D000199:Actins; C495842:CAPZA1 protein, human; D051347:CapZ Actin Capping Protein; C495843:Capza1 protein, mouse; C495847:Capza2 protein, mouse; D002352:Carrier Proteins; D051345:Destrin; D036341:Intercellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C065700:myotrophin