A Fragmin-Like Protein from Plasmodium of Physarum polycephalum That Severs F-Actin and Caps the Barbed End of F-Actin in a Ca2+-Sensitive Way1

Furuhashi, Kiyoshi; Hatano, Sadashi

doi:10.1093/oxfordjournals.jbchem.a122850

Cited by 22 publications

(15 citation statements)

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“…The catalytic loop insertion and the surrounding helices H6, H7 and H8 therefore probably form a relatively flat substrate recognition domain that diverges significantly from the elaborately structured substrate recognition domain in eukaryotic protein kinases (Figures 1 and 2). Since only the structure of gelsolin segment‐1 in complex with actin is known (McLaughlin et al ., 1993), where segment‐1 binds to a cleft between subdomains 1 and 3, the role of fragmin, fragmin60 (Furuhashi et al ., 1989, 1992) or Dictyostelium severin in substrate recognition by cAFK remains unclear. However, this structure shows that the phosphorylated loop around Thr203 is considerably flexible in comparison to the actin–DNase I complex, and this may explain why neighbouring threonine residues in actin can also be phosphorylated (Furuhashi et al ., 1992; Gettemans et al ., 1992).…”

Section: Resultsmentioning

confidence: 99%

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain

Steinbacher

Hof

Eichinger³

et al. 1999

The EMBO Journal

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Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actinfragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 Å resolution. The threedimensional fold reveals a catalytic module of~160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity.

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Section: Resultsmentioning

confidence: 99%

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain

Steinbacher

Hof

Eichinger³

et al. 1999

The EMBO Journal

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“…The villin core retains the Ca 2þ -dependent actin-severing, -capping, and -nucleating functions of villin, whereas the headpiece endows villin with the ability to form microfilament bundles (Friederich et al, 1990;Hartwig and Kwiatkowski, 1991). Proteins comprising three gelsolin-like domains, like severin from Dictyostelium (Yamamoto et al, 1982), fragmin from Physarum (Furuhashi and Hatano, 1989), and CapG from vertebrates, are F-actin capping and/or severing factors. These are encoded by distinct genes.…”

Section: Introductionmentioning

confidence: 99%

ACTIN BINDING PROTEIN29 fromLiliumPollen Plays an Important Role in Dynamic Actin Remodeling

et al. 2007

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Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca 2þ -and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.

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“…A second substrate of the AFK in vitro is a dimer between actin and a 60-kDa protein which is immunologically related to fragmin, called fragmin 60 (Furuhashi and Hatano, 1989 ;Furuhashi et al, 1992). The anti-fragmin antibodies used in this study cross react weakly with a 60-kDa protein after western blotting of a Physurum cytosolic extract (data not shown) and we tentatively identify this polypeptide as fragmin 60.…”

Section: Discussionmentioning

confidence: 99%

In vivo Phosphorylation of Actin in Physarum Polycephalum

Corte

Gettemans

Waelkens

et al. 1996

European Journal of Biochemistry

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Actin‐fragmin is a heterodimeric protein complex from Physarum polycephalum microplasmodia that is phosphorylated in vitro at residues Thr203 and Thr202 of the actin subunit by the endogenous actin‐fragmin kinase. Following phosphorylation, the F‐actin capping activity of the complex becomes Ca2+‐dependent, suggesting a fundamental regulatory role in controlling F‐actin growth [Gettemans, J., De Ville, Y., Waelkens E. and Vandekerckhove, J. (1995)J. Biol. Chem. 270, 2644–26511. In this study we analysed actin phosphorylation in vivo. We demonstrate that the actin‐fragmin complex constitutes the only substrate of the actin‐fragmin kinase in plasmodia. Monomeric actin is not phosphorylated. Immunoprecipitation of actin‐fragmin reveals that approximately 405% of the actin sub‐unit of the complex is phosphorylated in vivo. However, using purified substrate and kinase, the complex can be quantitatively phosphorylated as judged by two‐dimensional gel electrophoresis. Through comparative phosphopeptide fingerprinting, we show that the phosphorylation sites in vivo are identical to those identified in vitro. We additionally characterized a complex of actin and the NH2‐terminal half of fragmin (residues 1–168) that is also phosphorylated by the same kinase. In contrast to actin‐fragmin, phosphorylation of the complex between actin and residues 1–168 of fragmin is independent of Ca2+ because the second Ca2+‐dependent regulatory actin‐binding domain is missing. By artificially varying the actin‐fragmin concentration or the actin‐fragmin kinase activity present in microplasmodia cytosolic extracts, we attempted to detect alternative protein substrates for the actin‐fragmin kinase. The fact that none could be identified suggests that the control and properties of actin‐fragmin phosphorylation observed in vitro may stand as a model for F‐actin growth control in Physarum cells.

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A Fragmin-Like Protein from Plasmodium of Physarum polycephalum That Severs F-Actin and Caps the Barbed End of F-Actin in a Ca2+-Sensitive Way1

Cited by 22 publications

References 0 publications

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain

ACTIN BINDING PROTEIN29 fromLiliumPollen Plays an Important Role in Dynamic Actin Remodeling

In vivo Phosphorylation of Actin in Physarum Polycephalum

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