Fragmin60 Encodes an Actin-binding Protein with a C2 Domain and Controls Actin Thr-203 Phosphorylation in PhysarumPlasmodia 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.

The Nucleo-cytoplasmic Actin-binding Protein CapG Lacks a Nuclear Export Sequence Present in Structurally Related Proteins

“…Capping of Actin Filaments-The experiments were performed essentially as described earlier (31,32). Briefly, preassembled unlabeled actin filaments (final concentration, 1 M) were used as "nuclei."…”

Identification of a Functional Switch for Actin Severing by Cytoskeletal Proteins

“…Gene knock-out experiments have implicated the actin severing properties of villin and gelsolin in their motogenic effect (5,6). However, all these proteins require very high calcium concentrations for actin severing (between 1 and 200 M) (7)(8)(9)(10)(11)(12)(13). Activation of gelsolin by Ca 2ϩ may be modulated by other factors as well (7,9,14).…”

Regulation of levels of actin threonine phosphorylation during life cycle of Physarum polycephalum

“…Physarum contains at least two other fragminlike molecules: amoebal fragmin (fragmin A; [Uyeda et al, 1988;T'Jampens et al, 1999]) and fragmin 60 [Furuhashi et al, 1992;Sklyarova et al, 2002]. The Physarum fragmin gene family is encoded by separate developmentally regulated genes; frgA is amoeba-specific [T'Jampens et al, 1999], whereas frgP and frg60 are plasmodium-specific [T'Jampens et al, 1997;Sklyarova et al, 2002]. The expression profile of afk is similar to those of frgP and frg60 [Sklyarova et al, 2002].…”

“…The Physarum fragmin gene family is encoded by separate developmentally regulated genes; frgA is amoeba-specific [T'Jampens et al, 1999], whereas frgP and frg60 are plasmodium-specific [T'Jampens et al, 1997;Sklyarova et al, 2002]. The expression profile of afk is similar to those of frgP and frg60 [Sklyarova et al, 2002]. Northern blot analysis showed that afk is not transcribed in amoeba.…”

“…Fragmin P is not present in amoebae, and Northern blot analysis has shown that the transcription of frgP is initiated during the long cell cycle that links the amoebal and plasmodial stages. Physarum contains at least two other fragminlike molecules: amoebal fragmin (fragmin A; [Uyeda et al, 1988;T'Jampens et al, 1999]) and fragmin 60 [Furuhashi et al, 1992;Sklyarova et al, 2002]. The Physarum fragmin gene family is encoded by separate developmentally regulated genes; frgA is amoeba-specific [T'Jampens et al, 1999], whereas frgP and frg60 are plasmodium-specific [T'Jampens et al, 1997;Sklyarova et al, 2002].…”