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PHOSPHO-REGULATION OF ACA8, A PLASMA MEMBRANE CA2+-ATPASE OF ARABIDOPSIS THALIANA
Get PDFACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca2+-ATPase of Arabidopsis thaliana. Phospho-proteomic studies identified several phosphopeptides corresponding to portions of its regulatory N-terminus. Each of the Ser found to be phosphorylated in those studies (S19, S22, S27, S29, S57, and S99) has been mutated to Asp, to mimic phosphorylation of the ACA8 N-terminus, and to Ala to prevent phosphorylation. Mutants have been expressed in Saccharomyces cerevisiae and characterized: as shown by the low activation by CaM, mutants S19D, S57D, S22D and S27D are deregulated. Moreover, the low response to CaM of ACA8 mutants S22A, S27A, and S29A points the relevance of these serine residues per se in determining the amplitude of the response of ACA8 to CaM. To analyse the effect of S to D mutation on the kinetic of CaM binding, His-tagged N-termini of wild-type and mutant ACA8 (6His-1M-I116) were expressed in Escherichia coli, affinity-purified and used in surface plasmon resonance experiments. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (KD 10-fold higher than wild-type ACA8) and S99D (KD about half that of wild-type ACA8). Since S19 is in a consensus motive for calcium-dependent protein kinases (CDPKs) the ACA8 N-terminus has been subjected to in vitro phosphorylation assays with two isoforms of A. thaliana CDPKs: CDPK1, that phosphorylates ACA2 (an endoplasmic reticulum localised isoform of A. thaliana ACA) and CDPK16, a plasma membrane localised isoform of CDPK. Results show that both kinases are able to phosphorylate ACA8 N-terminus, but CDPK16 with higher extent. Phosphorylation of mutant 6His-1M-I116 peptides mapped CDPK16 phosphorylation site at S19 and at S22. Furthermore, we identified by two-hybrid screening two isoforms of CBL-interacting protein kinases (CIPKs) as putative interactors of ACA8 N-terminus region: CIPK9 and CIPK14. BiFC analysis in Nicotiana benthamiana confirmed the two-hybrid results, showing that interaction between ACA8 full length and CIPK9 or CIPK14 occurs in planta at the plasma membrane. Moreover, phosphorylation assay demonstrate that both kinases phosphorylate ACA8 N-terminus in vitro. Implications of these results are discussed
Single point mutations in the small cytoplasmic loop of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana, generate partially deregulated pumps
No full textACA8 is a type 2B Ca2+-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu(252) to Asn(345) sequence. Mutation to Ala of any of six tested acidic residues (Glu(252), Asp(273), Asp(291), Asp(303), Glu(302), or Asp(332)) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wildtype ACA8. Moreover, its phenotype is stronger than that of D291NACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca2+-ATPase plays a role in the attainment of the auto-inhibited state
Mutation of phosphorylable serine residues in the N-terminus of the plasma membrane Ca-ATPase ACA8
No full textPhosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana
No full textACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca2+-ATPase of Arabidopsis thaliana. Several phosphopeptides corresponding to portions of the regulatory N-terminus of ACA8 have been identified in phospho-proteomic studies. To mimic phosphorylation of the ACA8 N-terminus, each of the serines found to be phosphorylated in those studies (Ser19, Ser22, Ser27, Ser29, Ser57, and Ser99) has been mutated to aspartate. Mutants have been expressed in Saccharomyces cerevisiae and characterized: mutants S19D and S57D-and to a lesser extent also mutants S22D and S27D-are deregulated, as shown by their low activation by CaM and by tryptic cleavage of the N-terminus. The His-tagged N-termini of wild-type and mutant ACA8 (6His-M-1-I-116) were expressed in Escherichia coli, affinity-purified, and used to analyse the kinetics of CaM binding by surface plasmon resonance. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (K-D similar to 10-fold higher than wild-type ACA8) and S99D (K-D about half that of wild-type ACA8). The ACA8 N-terminus is phosphorylated in vitro by two isoforms of A. thaliana calcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-M-1-I-116 peptides shows that CPK16 is able to phosphorylate the ACA8 N-terminus at Ser19 and at Ser22. The possible physiological implications of the subtle modulation of ACA8 activity by phosphorylation of its N-terminus are discussed
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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