Steinbacher, S. et al. The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain. EMBO J. 18, 2923-2929

Abteilung Strukturforschung, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.
The EMBO Journal (Impact Factor: 10.43). 07/1999; 18(11):2923-9. DOI: 10.1093/emboj/18.11.2923
Source: PubMed


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 actin-fragmin 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 A resolution. The three-dimensional fold reveals a catalytic module of approximately 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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    • "Such specificity is achieved by protrusion of AceK substrate recognition helix into the active site of ICDH dimer [28]. Another example of APK substrate specificity comes from structures solved for actin–fragmin kinase [26], which suggests evolution of an elaborate complementary surface that specifically binds to the actin–fragmin dimer. This specific binding of substrates by APKs is in contrast to EPKs and ELKs, which generally display substrate diversity [30,31]. "
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    ABSTRACT: Protein phosphorylation in eukaryotes is carried out by a large and diverse family of protein kinases, which display remarkable diversity and complexity in their modes of regulation. The complex modes of regulation have evolved as a consequence of natural selection operating on protein kinase sequences for billions of years. Here we describe how quantitative comparisons of protein kinase sequences from diverse organisms, in particular prokaryotes, have contributed to our understanding of the structural organization and evolution of allosteric regulation in the protein kinase domain. An emerging view from these studies is that regulatory diversity and complexity in the protein kinase domain evolved in a 'modular' fashion through elaboration of an ancient core component, which existed before the emergence of eukaryotes. The core component provided the conformational flexibility required for ATP binding and phosphoryl transfer in prokaryotic kinases, but evolved into a highly regulatable domain in eukaryotes through the addition of exaggerated structural features that facilitated tight allosteric control. Family and group-specific features are built upon the core component in eukaryotes to provide additional layers of control. We propose that 'modularity' and 'conformational flexibility' are key evolvable traits of the protein kinase domain that contributed to its extensive regulatory diversity and complexity.
    Philosophical Transactions of The Royal Society B Biological Sciences 09/2012; 367(1602):2529-39. DOI:10.1098/rstb.2012.0015 · 7.06 Impact Factor
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    • "For survey of similarities within the thioredoxin-like clan, the CLANS algorithm [34] was run on a set of sequences including (a) all the Pfam “seeds” from the 17 families of the “protein kinase domain” clan (CL0016), (b) the 143 representative SELO domains (see Results) along with 10 representative mchC proteins, (c) other proteins with expected similarity to the PKL clan, including all the Pfam “seeds” from the Pfam families: Alpha_kinase (PF02816) [36], PI3_PI4_kinase (PF00454) [130], Act-Frag_cataly kinase, PF09192, [131], PPDK_N (PF01326) [132], PIP5K (PF01504) [133]. For these families, structural similarity to the PKL kinases is known [8]. "
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    ABSTRACT: Selenoproteins serve important functions in many organisms, usually providing essential oxidoreductase enzymatic activity, often for defense against toxic xenobiotic substances. Most eukaryotic genomes possess a small number of these proteins, usually not more than 20. Selenoproteins belong to various structural classes, often related to oxidoreductase function, yet a few of them are completely uncharacterised. Here, the structural and functional prediction for the uncharacterised selenoprotein O (SELO) is presented. Using bioinformatics tools, we predict that SELO protein adopts a three-dimensional fold similar to protein kinases. Furthermore, we argue that despite the lack of conservation of the “classic” catalytic aspartate residue of the archetypical His-Arg-Asp motif, SELO kinases might have retained catalytic phosphotransferase activity, albeit with an atypical active site. Lastly, the role of the selenocysteine residue is considered and the possibility of an oxidoreductase-regulated kinase function for SELO is discussed. The novel kinase prediction is discussed in the context of functional data on SELO orthologues in model organisms, FMP40 a.k.a.YPL222W (yeast), and ydiU (bacteria). Expression data from bacteria and yeast suggest a role in oxidative stress response. Analysis of genomic neighbourhoods of SELO homologues in the three domains of life points toward a role in regulation of ABC transport, in oxidative stress response, or in basic metabolism regulation. Among bacteria possessing SELO homologues, there is a significant over-representation of aquatic organisms, also of aerobic ones. The selenocysteine residue in SELO proteins occurs only in few members of this protein family, including proteins from Metazoa, and few small eukaryotes (Ostreococcus, stramenopiles). It is also demonstrated that enterobacterial mchC proteins involved in maturation of bactericidal antibiotics, microcins, form a distant subfamily of the SELO proteins. The new protein structural domain, with a putative kinase function assigned, expands the known kinome and deserves experimental determination of its biological role within the cell-signaling network.
    PLoS ONE 02/2012; 7(2):e32138. DOI:10.1371/journal.pone.0032138 · 3.23 Impact Factor
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    ABSTRACT: Amoebae and plasmodia constitute the two vegetative growth phases of the Myxomycete Physarum. In vitro and in vivo phosphorylation of actin in plasmodia is tightly controlled by fragmin P, a plasmodium-specific actin-binding protein that enables actin phosphorylation by the actin-fragmin kinase. We investigated whether amoebal actin is phosphorylated by this kinase, in spite of the lack of fragmin P. Strong actin phosphorylation was detected only following addition of recombinant actin-fragmin kinase to cell-free extracts of amoebae, suggesting that amoebae contain a protein with properties similar to plasmodial fragmin. We purified the complex between actin and this protein to homogeneity. Using an antibody that specifically recognizes phosphorylated actin, we demonstrate that Thr203 in actin can be phosphorylated in this complex. A full-length amoebal fragmin cDNA was cloned and the deduced amino acid sequence shows 65% identity with plasmodial fragmin. However, the fragmins are encoded by different genes. Northern blots using RNA from a developing Physarum strain demonstrate that this fragmin isoform (fragmin A) is not expressed in plasmodia. In situ localization showed that fragmin A is present mainly underneath the plasma membrane. Our results indicate that Physarum amoebae express a fragmin P-like isoform which shares the property of binding actin and converting the latter into a substrate for the actin-fragmin kinase.
    European Journal of Biochemistry 11/1999; 265(1):240-50. DOI:10.1046/j.1432-1327.1999.00721.x · 3.58 Impact Factor
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