title: Rapid nucleotide exchange renders Asp-11 mutant actins resistant to depolymerizing activity of cofilin, leading to dominant toxicity in vivo.
abstract: Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8562, Japan.
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Umeki
authors.forename: Jun
authors.initials: J
authors.lastname: Nakajima
authors.forename: Taro Q P
authors.initials: TQ
authors.lastname: Noguchi
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Tokuraku
authors.forename: Akira
authors.initials: A
authors.lastname: Nagasaki
authors.forename: Kohji
authors.initials: K
authors.lastname: Ito
authors.forename: Keiko
authors.initials: K
authors.lastname: Hirose
authors.forename: Taro Q P
authors.initials: TQ
authors.lastname: Uyeda
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D051339:Actin Depolymerizing Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D058105:Polymerization; D011485:Protein Binding; D055550:Protein Stability; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D009711:Nucleotides; D015800:Protozoan Proteins; D001224:Aspartic Acid; D000255:Adenosine Triphosphate