As serina/treonina fosfatases (PPs) são enzimas responsáveis pela desfosforilação de resíduos de fosfoserina e/ou fosfotreonina e estão subdivididas em duas famílias gênicas designadas PPP e PPM. A família PPP está dividida em cinco subfamílias, que compreendem as PPs do tipo 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) e 7 (PP7), de acordo com a similaridade entre as sequências de aminoácidos das subunidades ou domínios destas enzimas. Novas PPs estão sendo descobertas em diferentes organismos e classificadas nestas famílias ou subfamílias com base na análise comparada de suas sequências. Uma delas é a proteína fosfatase do tipo 4 (PP4), descoberta originalmente em coelhos e cujas funções biológicas vêm sendo progressivamente elucidadas. Neste estudo tivemos como objetivos a identificação e caracterização de uma nova serina/treonina fosfatase de Dictyostelium discoideum. Para isto, rastreamos uma biblioteca de cDNA e um cDNA completo que codifica a subunidade catalítica da fosfatase do tipo 4 (PP4c) foi isolado e seqüenciado. Verificamos que o gene da PP4c é essencial e está presente em cópia única localizada no cromossomo 2, originando um mRNA expresso ao longo de todo o ciclo de vida de D. discoideum. Imunodetecção da PP4c realizada com anticorpo específico indicou que os níveis desta proteína também são constitutivos. Observamos que a superexpressão da PP4c sob o controle de um promotor constitutivo não causa alterações fenotípicas detectáveis. A análise da localização celular da PP4c expressa em células de D. discoideum como proteína de fusão com GFP (Green Fluorescent Protein) revelou que a enzima está localizada no citossol, contrastando com outros organismos, onde ela se encontra enriquecida no centrossomo. Por fim, descrevemos neste trabalho, além da organização genômica da PP4, a estrutura dos genes de todas as serina/treonina fosfatases da família PPP encontradas no cromossomo 2 e apresentamos dados das relações entre estas enzimas de D. discoideum. / The serine/threonine protein phosphatases (PPs) are enzymes responsible for dephosphorylation of phosphoserine and/or phosphothreonine residues and are divided in two gene families designated as PPP and PPM. The PPP family is divided in five subfamilies, which comprise type 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) and 7 (PP7) phosphatases. This subdivision is based on aminoacid sequence similarity or enzyme domains. Novel PPs have been discovered in different organisms and classified in these families or subfamilies based on comparative sequence analysis. One of these is type 4 protein phosphatase (PP4), originally discovered in rabbit, which functions have been progressively uncovered. In this study our goal is to identify and characterize a novel serine/threonine phosphatase in Dictyostelium discoideum. We first screened a cDNA library and isolated a complete cDNA encoding the catalytic subunit of protein phosphatase 4 (PP4c), confirmed by manual DNA sequencing. The PP4c is an essential, single-copy gene located in chromosome 2, which encondes a mRNA constitutively expressed throughout D. discoideum life cycle. Immunodetection of PP4c performed with specific antibodies indicated corresponding protein levels. Overexpression of PP4c under a strong promoter caused no detectable phenotype. Subcellular localization of PP4c expressed as a GFP-fusion protein revealed its cytosolic location, in contrast to other organisms, where it has been reported to be enriched in centrosomes. We also describe here the genetic organization of PP4c, the genetic structure of all serine/threonine protein phosphatases belonging to the PPP family found in chromosome 2, and a phylogenetic analysis indicating relationships among these enzymes in D. discoideum and other selected organisms.

As serina/treonina fosfatases (PPs) são enzimas responsáveis pela desfosforilação de resíduos de fosfoserina e/ou fosfotreonina e estão subdivididas em duas famílias gênicas designadas PPP e PPM. A família PPP está dividida em cinco subfamílias, que compreendem as PPs do tipo 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) e 7 (PP7), de acordo com a similaridade entre as sequências de aminoácidos das subunidades ou domínios destas enzimas. Novas PPs estão sendo descobertas em diferentes organismos e classificadas nestas famílias ou subfamílias com base na análise comparada de suas sequências. Uma delas é a proteína fosfatase do tipo 4 (PP4), descoberta originalmente em coelhos e cujas funções biológicas vêm sendo progressivamente elucidadas. Neste estudo tivemos como objetivos a identificação e caracterização de uma nova serina/treonina fosfatase de Dictyostelium discoideum. Para isto, rastreamos uma biblioteca de cDNA e um cDNA completo que codifica a subunidade catalítica da fosfatase do tipo 4 (PP4c) foi isolado e seqüenciado. Verificamos que o gene da PP4c é essencial e está presente em cópia única localizada no cromossomo 2, originando um mRNA expresso ao longo de todo o ciclo de vida de D. discoideum. Imunodetecção da PP4c realizada com anticorpo específico indicou que os níveis desta proteína também são constitutivos. Observamos que a superexpressão da PP4c sob o controle de um promotor constitutivo não causa alterações fenotípicas detectáveis. A análise da localização celular da PP4c expressa em células de D. discoideum como proteína de fusão com GFP (Green Fluorescent Protein) revelou que a enzima está localizada no citossol, contrastando com outros organismos, onde ela se encontra enriquecida no centrossomo. Por fim, descrevemos neste trabalho, além da organização genômica da PP4, a estrutura dos genes de todas as serina/treonina fosfatases da família PPP encontradas no cromossomo 2 e apresentamos dados das relações entre estas enzimas de D. discoideum. / The serine/threonine protein phosphatases (PPs) are enzymes responsible for dephosphorylation of phosphoserine and/or phosphothreonine residues and are divided in two gene families designated as PPP and PPM. The PPP family is divided in five subfamilies, which comprise type 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) and 7 (PP7) phosphatases. This subdivision is based on aminoacid sequence similarity or enzyme domains. Novel PPs have been discovered in different organisms and classified in these families or subfamilies based on comparative sequence analysis. One of these is type 4 protein phosphatase (PP4), originally discovered in rabbit, which functions have been progressively uncovered. In this study our goal is to identify and characterize a novel serine/threonine phosphatase in Dictyostelium discoideum. We first screened a cDNA library and isolated a complete cDNA encoding the catalytic subunit of protein phosphatase 4 (PP4c), confirmed by manual DNA sequencing. The PP4c is an essential, single-copy gene located in chromosome 2, which encondes a mRNA constitutively expressed throughout D. discoideum life cycle. Immunodetection of PP4c performed with specific antibodies indicated corresponding protein levels. Overexpression of PP4c under a strong promoter caused no detectable phenotype. Subcellular localization of PP4c expressed as a GFP-fusion protein revealed its cytosolic location, in contrast to other organisms, where it has been reported to be enriched in centrosomes. We also describe here the genetic organization of PP4c, the genetic structure of all serine/threonine protein phosphatases belonging to the PPP family found in chromosome 2, and a phylogenetic analysis indicating relationships among these enzymes in D. discoideum and other selected organisms.

La cohésion des chromatides sœurs est assurée par un complexe protéique en forme d’anneau assurant leur capture topologique. Ce complexe est constitué par des protéines conservées de la levure à l’Homme regroupées sous le terme « cohésine » : Smc1, Smc3 et la phosphoprotéine Scc1 fermant l’anneau (respectivement Psm1, Psm3 et Rad21 chez Schizosaccharomyces pombe). Les protéines régulatrices Rad61-Wapl, Pds5 et Scc3 (Wpl1,Pds5 et Psc3 respectivement chez S. pombe) interagissent avec l’anneau via Scc1. Il a été proposé que la capture de l’ADN par les cohésines nécessite l’ouverture transitoire de l’interface Smc1/Smc3. La réaction de dissociation fait quant à elle intervenir le sous-complexe Wapl/Pds5/Scc3 entraînant vraisemblablement l’ouverture de l’interface Scc1/Smc3. Le mécanisme par lequel la cohésion est créée et celui par lequel Wapl promeut la dissociation des cohésines des chromosomes, sont encore inconnus. Parmi les mutants de cohésion chez Saccharomyces cerevisiae, la mutation thermosensible eco1-1 affecte le gène ECO1 codant une acétyl-transférase, essentielle à la viabilité cellulaire, conservée de la levure à l’Homme (Eco1 « Establishment of Cohesion » chez S. cerevisiae, Eso1 chez S.pombe, ESCO1-2 chez l’Homme) et ayant Smc3 pour substrat. Il a été montré que l’acétyl-transférase s’oppose à l’action de dissociation de Wapl. C’est un crible génétique réalisé par plusieurs équipes, visant à trouver des mutants suppresseurs d’eco1-1, qui a permis d’identifier les gènes codant les protéines Wapl, Pds5, Scc3 et Smc3 comme composants du mécanisme d’ouverture de l’anneau de cohésine. Un crible similaire a été réalisé chez S.pombe dans notre laboratoire, dans le but de trouver des suppresseurs de la mutation thermosensible eso1-H17. Ce crible a identifié les gènes orthologues à ceux trouvés chez la levure : wpl1, pds5, psc3 et psm3 mais aussi le gène codant la sous-unité catalytique du complexe sérine/thréonine phosphatase de type IV (PP4), noté pp4c. Nous avons alors mis en œuvre des expériences pour caractériser PP4c ainsi que sa sous-unité régulatrice Psy2 qui s’est révélée être également impliquée dans la cohésion des chromatides soeurs. Nous avons également identifié la protéine Rad21 comme substrat du complexe PP4, puis identifié les phosphosites potentiellement cibles de PP4, pour ensuite cribler et analyser des phosphomutants de Rad21 récapitulant l’effet suppresseur de la délétion de PP4. / Sister-chromatid cohesion is ensured by a ring shape protein complex which is in charge of their topological embrace. This complex consists of proteins which are conserved from yeast to human and grouped under the term “cohesin”: Smc1, Smc3 and the phosphoprotein Scc1 which closes the ring (respectively Psm1, Psm3 and Rad21 in Schizosaccharomyces pombe). The regulatory proteins Rad61-Wapl, Pds5 and Scc3 (Wpl1,Pds5 and Psc3 respectively in S. pombe) interact with the ring via Scc1. It has been suggested that DNA capture by the cohesin complex involves the transient opening of the Smc1/Smc3 interface. The dissociation reaction involves the sub-complex Wapl/Pds5/Scc3 which likely causes the opening of the Scc1/Smc3 interface. The mechanisms by which cohesion is created and by which Wapl promotes the cohesin dissociation from chromosomes are still unknown. Among the cohesion mutants in Saccharomyces cerevisiae the thermosensitive eco1-1 mutation affects the ECO1 gene encoding an acetyl-transferase essential for cell viability and conserved from yeast to human (Eco1 « Establishment of Cohesion » in S.cerevisiae, Eso1 in S. pombe and ESCO1-2 in human) and whose substrate is Smc3. It has been shown that the acetyl-transferase counteracts the dissociation action of Wapl. A genetic screen carried out by several teams in order to find suppressors of the eco1-1 mutation has led to the identification of the genes encoding the Wapl, Pds5, Scc3 and Smc3 proteins as components of the opening mechanism of the cohesin ring. A similar screen was carried out in S. pombe in our lab to find suppressors of the thermosensitive mutation eso1-H17. This screen identified the orthologous genes to those found in the budding yeast: wpl1,pds5, psc3 and psm3 and also the gene encoding the catalytic subunit of the type 4 serine/threonine phosphatase complex (PP4) named pp4c. We have therefore carried out experiments to characterize PP4c and its regulatory subunit Psy2 which has also been found to be involved in sister-chromatid cohesion. We have likewise identified the Rad21 subunit as a PP4 substrate and identified phosphosites as potential targets of PP4. We have then screened and analyzed Rad21 phosphomutants which were able to mimic the suppressor effect of the deletion of pp4c.