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Functie en regulatie van de reversibele methylatie van proteïne fosfatase 2A
Get PDFReversible phosphorylation of serine/threonine residues is one of the most frequent post-translational modifications of proteins and is an important regulatory mechanism in many cellular processes. The steady-state phosphorylation status of a protein is controlled by the opposing actions of protein kinases and phosphatases. Protein phosphatase 2A (PP2A) is a major family of multimeric serine/threonine phosphatases and belongs together with protein phosphatase 4 (PP4) and 6 (PP6) to the family of the PP2A-like phosphatases. PP2A is implicated in the regulation of a plethora of cellular processes such as apoptosis, transcription, translation, DNA replication, signal transduction, cell division and tumourigenesis. The participation of PP2A in these pleiotropic functions implies that its activity must be tightly controlled in vivo. The PP2A core enzyme consists of a catalytic subunit (C subunit/PP2AC) and a scaffolding A subunit (PR65/A subunit) through which one of several regulatory B subunits (PR55/B, PR61/B’ or PR72/B”) can bind. Proper functioning and regulation of PP2A is achieved by the association of the regulatory B subunits with the PP2A core enzyme, resulting in the formation of heterotrimeric PP2A holoenzymes with specific catalytic properties, subcellular locations and substrate specificities. Therefore, the assembly of the complex with the appropriate B-type subunit is the key to specificity and regulation of PP2A. In this process, methylation and phosphorylation of the conserved C-terminal PP2AC tail seem to play a crucial role. Methylation occurs on the carboxyl group of the carboxyterminal leucine 309 residue of PP2AC and is catalyzed by an S-adenosylmethionine-dependent leucine carboxyl methyltransferase (LCMT1), whereas demethylation is catalyzed by a specific phosphatase methylesterase (PME-1). However, neither the exact consequences nor the regulation of PP2AC methylation and phosphorylation are very clear.
In this work, I showed that the diverse regulatory B subunits require distinct modifications or structural features of the PP2AC C-terminal tail for heterotrimer formation and that PP2AC methylation is essential for cell viability. More specifically, carboxylmethylation of the C-terminal leucine 309 residue of PP2AC is absolutely necessary for formation of PP2A trimers with the PR55/B subunits (PP2AT55), whereas it is not absolutely required for PP2AT61,72 formation. PP2AT61a,b,e assembly is specifically inhibited by phosphorylation-mimicking mutations of tyrosine 307, whereas introduction of a negative charge at position threonine 304 inhibits only PP2AT55 formation. These results have important implications since they show that the selectivity for the B-class subunits can be based on the methylation and the phosphorylation state of PP2AC. This opens the possibility for dynamic subunit interchanges partially regulated by covalent modifications of the PP2AC C-terminal tail, thereby explaining differential trimeric assembly of PP2A.
Further, I demonstrated that PME-1, in contrast to LCMT1, is mainly nuclear correlating with the predominant occurrence of demethylated PP2AC in the nucleus. The nuclear targeting of PME-1 is mediated by a functional nuclear localisation signal (NLS) and overexpression of a cytoplasmic NLS mutant of PME-1 resulted in demethylation of PP2AC at the ‘wrong’ place, i.e. in the cytoplasm. Therefore, targeting of PME-1 to the nucleus can provide a mechanism for spatial regulation of PP2A methylation as it restricts demethylation to nuclear PP2AC.
I also established an additional function for PME-1 in the regulation of PP2A, since PME-1 demethylates not only PP2AC, but also stabilizes an inactivated PP2A pool. This inactive form of PP2A is not due to demethylation of the phosphatase, since LCMT1 could not reactivate this inactive PP2A pool. In contrast, reactivation of the Ser/Thr phosphatase activity of the inactive PP2A-PME-1 complex is achieved by addition of the phosphatase two A phosphatase activator (PTPA), leading to a dissociation of PME-1 from PP2A and a stable activation of PP2A. Since PME-1 could not inactivate active PP2A and an inactive PME-1 S156A mutant also bound inactive PP2A, it seems that the catalytic activity of PME-1 itself does not cause PP2A inactivation. Instead, PME-1 stabilizes an inactivated PP2A pool which is - given the nuclear localisation of PME-1 - presumably present in the nucleus. However, the function and precise nature of this inactive nuclear PP2A pool still remains to be determined.
Finally, I have investigated the substrate specificity of LCMT1 and PME-1, so far known to be specific for PP2A. I demonstrated by an in vitro assay that both PP4C and PP6C can be reversibly methylated on their C-terminal leucine residue by the same LCMT1 and PME-1. These findings show that the PP2A-like phosphatase family can share, at least in vitro, the same (de)methylating enzymes and establish reversible methylation as a common regulatory mechanism for all PP2A-like phosphatases.status: Publishe
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
Fysiologische functie van de PR130 B" subeenheid van proteïne fosfatase 2A
No full textOmkeerbare fosforylering is een uiterst belangrijk mechanisme dat tussenkomt in de regulatie van vele cellulaire processen. Deze modificatie wordt bewerkstelligd door proteïne kinasen en fosfatasen. Proteïne fosfatase 2A (PP2A) is verantwoordelijk voor een groot deel van de serine/threonine defosforylerende activiteit in een cel en komt zo tussen in tal van signalisatie transductiewegen, waaronder celcyclus regulatie, cytoskelet organisatie, gen transcriptie en proteïne synthese. PP2A komt hoofdzakelijk voor als een heterotrimeer, opgebouwd uit een katalytische C subeenheid, een structurele A subeenheid en een regulerende B subeenheid. Tot op heden zijn er twee A subeenheden, twee C subeenheden en meer dan 20 B subeenheden geïdentificeerd. Bijgevolg bestaan er een groot aantal verschillende PP2A complexen, elk met een mogelijk specifieke functie. In deze studie hebben we onze aandacht gevestigd op de PP2A complexen die PR130 als regulerende B subeenheid bevatten (PP2AT130). PR130 is het grootste proteïne van de PR72/B familie van regulerende subeenheden. De PR72/B leden bevatten allen een goed geconserveerd centraal gedeelte met daarin twee Ca2+-bindende EF hand motieven.In het eerste gedeelte van dit werk hebben we de sequentie en genomische organisatie van de verschillende PR72/B isovormen afgeleid bij de muis en dit als basis gebruikt om een aangepaste naamgeving voor de PR72/B familie te introduceren. We vonden 4 transcripten (PR130/Ba1,a3; PR72/Ba2,a4) afgeleid van Ppp2r3a (waarvan PR130/Ba1 en PR72/Ba2 de belangrijkste zijn), 4 transcripten (PR59/Bd1,2,3,4) afgeleid van Ppp2r3d (waarvan PR59/Bd3 niet kan binden met PP2A en PR59/Bd4 in heel geringe mate aanwezig is) en 1 transcript (G5PR/Bg) afgeleid van Ppp2r3c. Alhoewel we PR70 isovormen terugvinden bij de mens, hond en kikker, is er bij de muis geen PR70 ortholoog aanwezig. Ook vonden we geen humaan ortholoog van de verschillende muize PR59/Bd isovormen. Hieruit kunnen we afleiden dat de PR72/B familie, in vergelijking met PR55/B en PR61/B, een grotere diversiteit vertoont op evolutionair vlak. Via Northern blotting hebben we de weefselspecificiteit en de embryonale expressie van de belangrijkste muize PR72/B isovormen onderzocht. Allen komen hoog tot expressie in het hartweefsel. Mogelijk hebben zij daar een belangrijke functie. Immunohistochemische analyse van hart en skeletspier toonde een gestreept expressiepatroon, de zogenaamde A band, aan voor PR130 en PR72. Daarnaast zagen we een epitheliale expressie voor PR130 in de blaas en nier. Voorts hebben we ook de subcellulaire lokalisatie en de impact op de celcyclus voor de verschillende B/PR72 isovormen bepaald. Deze vertoonden zowel verschillen als gelijkenissen. Bovenstaande studie verstrekt waardevolle informatie voor de generatie en karakterisatie van toekomstige PR72/B knock-out muizen.In het tweede gedeelte van dit werk hebben we geprobeerd meer inzicht te krijgen in de fysiologische functies die door PP2AT130 beïnvloed worden. In een eerste luik hebben we de capaciteit van IQ-1 om PP2AT130 en PP2AT72 in vitro en in vivo te inhiberen, nagekeken. Na IQ-1 toevoeging zagen we een reductie van de protamine gestimuleerde PP2AT130/72 activiteit, niettegenstaande we geen verschil in binding van PR130/72 met PP2A zagen. Verdere analyse is dus nodig vooraleer we deze stof kunnen gebruiken om in vivo specifiek PP2AT130 te inhiberen. In een tweede luik hebben we PR130-interagerende proteinen geïdentificeerd via de gist dubbel-hybride methode met PR130N als aas en via massaspectrometrische identificatie van proteïnen die coïmmunoprecipiteren met PR130. Beide strategieën hebben geleid tot de identificatie van LPP, een proteïne belangrijk voor de communicatie tussen celadhesieplaatsen en de kern. Hoewel LPP en PR130 zich beide bevinden in focale contacten en aan de frontale celmembraan van migrerende cellen, is enkel LPP aanwezig in focale adhesies. We konden aantonen dat PR130 een negatieve invloed heeft op cel-matrix adhesie en dat het LPP-PP2AT130 complex noodzakelijk is voor een efficiënte celmigratie. Daarnaast zagen we, afhankelijk van het celtype, een dynamische expressie van PR130 in de kern. De functie(s) van PR130 in de kern zijn voorlopig nog onbekend. SHIP2, een inositol en fosfoinositide 5-fosfatase werd ook geïdentificeerd als een PR130-interagerend proteïne. Dit proteïne bindt ondermeer aan de EGF receptor (EGFR) en heeft een negatief effect op EGFR degradatie. We hebben kunnen aantonen dat ook PR130 associeert met de EGFR en een negatieve invloed heeft op de EGF-geïnduceerde EGFR degradatie. Bovendien translokeert PR130 net als SHIP2 naar de celmembraan na EGF stimulatie. Momenteel zijn we de hypothese aan het onderzoeken dat PR130 een brug vormt tussen SHIP2 en de EGFR en zo SHIP2 helpt om de EGFR degradatie te beïnvloeden.Uit bovenstaande gegevens kunnen we besluiten dat PR130 een negatieve invloed heeft op cel adhesie en EGFR degradatie en een positieve rol speelt tijdens cel migratie. Cel adhesie en migratie spelen een belangrijke rol tijdens de ontwikkeling van kanker. Daarnaast is geweten dat EGFR hyperactivatie correleert met verschillende soorten kanker. Bijgevolg suggereren deze data dat PR130 mogelijks een tumor promoverende functie vervult.status: Publishe
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
Karakterisering van de tumorigenesis in de PP2A B'delta knockout muis: Een model voor hepatocarcinogenesis
No full textTo fully transform immortalized human cells into malignant cells, Protein Phosphatase type 2A (PP2A) needs to be inactivated, identifying PP2A as a major cellular tumor suppressor. It remained however an open question, whether in vivo loss of PP2A might also predispose mice tocancer development. In this work, we found that Ppp2r5d KO mice, deficient for the ubiquitously expressed PP2A B'delta subunit, spontaneously develop several primary tumors, with a high incidence of Hepatocellular Carcinoma (HCC) and lymphoma. Hence, we demonstrated for the first time that loss of one specific PP2A holoenzyme, is without doubt, sufficient to promote tumorigenesis in vivo. Because of the high incidence of HCC and the rarity of spontaneous HCC development in mice, we focused on the further characterization of this phenotype. B'delta-null HCCs feature all histopathological stages seen in human HCC, rangingfrom the most benign dysplastic nodules to the most malignant, poorly-differentiated HCCs, without any correlation with increased inflammation or steatosis. Furthermore, we observed intracellular fibrinogen deposits, another feature of human HCC, and an unbiased 2D DIGE analysis confirmed differential expression of several proteins/markers of human HCC. In order to define the exact role of B'delta in hepatocarcinogenesis, we performed targeted western blot studies in protein lysates of several independent samples of HCCs, healthy surrounding liver tissues and WT liversto analyze potential activation of signaling pathways known to be disturbed in HCC and to be regulated by PP2A. In general, these results reflected the enormous diversity, so notoriously associated with HCC. However, amidst all this heterogeneity, two consistent alterations were observed in all HCC samples tested: an increase in S62 phosphorylation of the c-myc oncogene and an increase in S9 phosphorylation of GSK-3, the c-myc T58 kinase. These findings are consistent with increased c-myc oncogenicactivity. In accordance, we found increased expression of several established c-myc target genes. Because GSK-3 S9 hyperphosphorylation is already present in non-malignant KO liver, the lack of direct GSK-3 dephosphorylation is an integral part of the tumor predisposing mechanism in B'delta-null mice. In addition, we hypothesize that PP2A-B'delta may be themajor c-myc S62 phosphatase in liver, since expression of B'alpha, the established PP2A c-myc-targeting subunit, is barely detectable in this tissue. Interestingly, we identified several direct and indirect mechanisms, resulting in PP2A-B'delta malfunction in human cancers. Together, our data identify B'delta as a novel candidate tumor suppressor in HCC, and B'delta KO mice as a valuable novel animal model to study hepatocarcinogenesis.status: Publishe
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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