title: Fragmin60 encodes an actin-binding protein with a C2 domain and controls actin Thr-203 phosphorylation in Physarum plasmodia and sclerotia.
abstract: We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.
authors.affiliation: Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Rommelaere Institute, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Sklyarova
authors.forename: Veerle
authors.initials: V
authors.lastname: De Corte
authors.forename: Kris
authors.initials: K
authors.lastname: Meerschaert
authors.forename: Liesbeth
authors.initials: L
authors.lastname: Devriendt
authors.forename: Berlinda
authors.initials: B
authors.lastname: Vanloo
authors.forename: Juliet
authors.initials: J
authors.lastname: Bailey
authors.forename: Lynnette J
authors.initials: LJ
authors.lastname: Cook
authors.forename: Mark
authors.initials: M
authors.lastname: Goethals
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Damme
authors.forename: Magda
authors.initials: M
authors.lastname: Puype
authors.forename: Joel
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Jan
authors.initials: J
authors.lastname: Gettemans
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D017985:Dalteparin / Q000737:chemistry* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005786:Gene Expression Regulation; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010804:Physarum / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
chemical_list: D000199:Actins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; D013912:Threonine; D005982:Glutathione Transferase; D017985:Dalteparin; D002118:Calcium