京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24272号 / 生博第486号 / 新制||生||64(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松本 智裕, 教授 高田 穣, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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The mitotic spindle is a bipolar microtubule-based structure that mediates proper cell division by segregating the genetic material and by positioning the cytokinesis cleavage plane. Spindle assembly is a complex process, involving the modulation of microtubule dynamics, microtubule focusing at spindle poles and the formation of stable microtubule attachments to chromosomes. The cellular events leading to spindle formation are highly regulated, and mitotic kinases have been implicated in many aspects of this process. However, little is known about their counteracting phosphatases. A screen for genes required for early embryonic cell divisions in C. elegans identified rsa-1 (for regulator of spindle assembly 1), a putative Protein Phosphatase 2A (PP2A) regulatory subunit whose silencing causes defects in spindle formation. Upon rsa-1(RNAi), spindle poles collapse onto each other and microtubule amounts are strongly reduced. My thesis work demonstrates that RSA-1 indeed functions as a PP2A regulatory subunit. RSA-1 associates with the PP2A enzyme and recruits it to centrosomes. The centrosome binding of PP2A furthermore requires the new protein RSA-2 as well as the core centrosomal protein SPD-5 and is based on a hierarchical protein-protein interaction pathway. When PP2A is lacking at centrosomes after rsa-1(RNAi), the centrosomal amounts of two critical mitotic effectors, the microtubule destabilizer KLP-7 and the kinetochore microtubule stabilizer TPXL-1, are altered. KLP-7 is increased, which may account for the reduction of microtubule outgrowth from centrosomes in rsa-1(RNAi) embryos. TPXL-1 is lost from centrosomes, which may explain why spindle poles collapse in the absence of RSA-1. TPXL-1 physically associates with RSA-1 and RSA-2, suggesting that it is a direct target of PP2A. In summary, this work defines the role of a novel PP2A complex in mitotic spindle assembly and suggests a model for how different microtubule re-organization steps might be coordinated during spindle formation.

As proteínas fosfatases são enzimas responsáveis pela desfosforilação de resíduos de fosfoaminoácidos, principalmente fosfotirosina e fosfoserina/treonina, o que divide esta classe de proteínas nas famílias PTP (proteína tirosina fosfatase) e PP (proteína serina/treonina fosfatase). Diversos membros da família das PPs, em particular da subfamília PPP (Phosphoprotein Phosphatase), existem como holoenzimas compostas de uma subunidade catalítica associada a uma ou mais subunidades reguladoras, que lhes conferem diversidade funcional. Neste trabalho tivemos como objetivo identificar, utilizando ensaios no sistema de duplo híbrido em leveduras, proteínas que interagem com as subunidades catalíticas das serina/treonina fosfatases do tipo 1 (DdPP1c) e do tipo 4 (DdPP4c) da ameba social D. discoideum. Varreduras de bibliotecas de cDNA das fases de crescimento e desenvolvimento da linhagem AX4 de D. discoideum com as iscas DdPP1c e uma variante mutante da DdPP1c (DdPP1cF269C) possibilitaram a identificação de pelo menos 30 genes com evidência de interação com a PP1c. A varredura das bibliotecas com a isca DdPP4 propiciou a identificação de 10 potenciais genes candidatos com evidência de interação com a PP4c. Várias dos candidatos identificados nas varreduras correspondem a genes que codificam para proteínas hipotéticas que não apresentam similaridade significativa com proteínas de função conhecida. Entre essas, identificamos e caracterizamos DdI-3, um ortólogo do inibidor-3 da PP1c de mamíferos. A interação de DdI-3 com DdPP1c foi confirmada através de ensaios independentes no sistema do duplo híbrido em leveduras. Demonstramos que DdI-3 recombinante expresso em bactérias possui atividade inibidora da DdPP1c in vitro, sendo que esta enzima tem 50% de sua atividade de fosforilase fosfatase inibida por cerca de 0,55 nM de rDdI-3. Estes dados indicam que DdI-3 é 50 vezes mais potente do que DdI-2, um ortólogo do inibidor-2 previamente caracterizado em D. discoideum. Neste trabalho, também iniciamos a construção do catálogo (The Dictyostelium Phosphatome) que irá conter todas as subunidades catalíticas e reguladoras das proteínas fosfatases codificadas no genoma de D. discoideum. Até o momento, 101 genes foram catalogados e classificados nas diferentes famílias das proteínas fosfatases, sendo 16 na família das PTPs, 26 na família das DSPs (proteína fosfatase de dupla especificidade), 15 na família das PPMs (Phosphoprotein Phosphatase Magnesium-dependent) e 31 na família das PPPs, incluindo genes codificadores de subunidades catalíticas e reguladoras. / Protein phosphatases are responsible for dephosphorylating phosphoaminoacids residues, notably phosphotyrosine and phosphoserine/threonine, thus dividing these enzymes into PTP (protein tyrosine phosphatase) and PP (protein serine/threonine phosphatase) families. Several members of the PP family, in particular those belonging to PPP (Phosphoprotein Phosphatase) are composed of one catalytic subunit and one or more regulatory subunits that provide functional diversity to the holoenzyme. In this work our goal was to identify protein interactors to type 1 (DdPP1c) and type 4 (DdPP4c) phosphatase that might behave as potential regulatory subunits of these enzymes in the social amoeba Dictyostelium discoideum. For this intent, DdPP1c, a mutant isoform of DdPP1 (DdPP1cF269C) and DdPP4c were used as baits in yeast two-hybrid based screening of D. discoideum (AX-4 strain) cDNA libraries from growth as well as developmental stages. At least 30 genes were identified as potential DdPP1c interactors while 10 genes were selected as candidates to interact to DdPP4c. Most of them are currently annotated in D. discoideum genome as hypothetical proteins of unknown function. Among the potential PP1c interactors we selected DdI-3, an ortholog of mammalian inhibitor-3. Interaction of DdI-3 and DdPP1c was confirmed by independent yeast two-hybrid assays. We demonstrated that bacterial expressed recombinant DdI-3 is effective as an inhibitor of DdPP1c in vitro, since 50% of DdPP1c phosphorylase phosphatase activity is inhibited by circa 0,55 nM of purified rDdI-3. Our results also showed that DdI-3 is 50 times more effective than DdI-2, a previously characterized PP1c inhibitor in D. discoideum. In this work we began to organize The Dictyostelium Phosphatome, a catalog of all protein phosphatases, including their catalytic and regulatory subunits, encoded in D. discoideum genome. Until now, we have classified 101 genes into the protein phosphatase families, of which 16 were classified as classic PTP, 26 as DSP (dual-specificity phosphatases), 15 as PPM (Phosphoprotein Phosphatase Magnesium-dependent) and 31 as PPP, including genes for catalytic as well as regulatory subunits.

A serina/treonina fosfatase do tipo 1 (PP1) é uma enzima ubíqua nas células e nos tecidos das várias espécies em que foi pesquisada e regula vários processos como metabolismo intermediário, processamento de mRNA, transcrição e apoptose. Geralmente a holoenzima PP1 é encontrada como um dímero constituído por uma subunidade catalítica conservada (PP1c) e uma ou mais subunidades reguladoras variáveis. Em mamíferos, já foram identificados mais de 50 polipeptídeos que se associam direta ou indiretamente a PP1c, gerando holoenzimas com localizações celulares e especificidades distintas. Entre estas proteínas estão inibidores citosólicos de PP1c, tais como o inibidor-1 (I-1), o inibidor-2 (I-2) e o inibidor nuclear da PP1 (NIPP-1). Ortólogos do I-2 foram descritos em microorganismos como Saccharomyces cerevisiae e Neurospora crassa. Neste trabalho nós demonstramos que o genoma da ameba social Dictyostelium discoideum possui uma única cópia do gene que codifica um ortólogo do I-2 (DdI-2). Análise através de Northern blot mostrou que o mRNA de DdI-2 é expresso durante o crescimento e ao longo de todo o ciclo de desenvolvimento, com níveis variáveis. Também demonstramos que o gene DdI-2 codifica uma verdadeira proteína inibidora da PP1c uma vez que seu produto recombinante em bactéria é capaz de inibir, com eficácia equivalente, as atividades de fosforilase fosfatase das PP1c recombinantes selvagem (DdPP1c) e mutante (DdPP1cF269C) de D. discoideum e NcPP1c de N. crassa in vitro. A proteína DdPP1cF269C apresenta características distintas da DdPP1c incluindo maior estabilidade, maior atividade de fosforilase fosfatase e maior sensibilidade frente ao inibidor caliculina A. Estas diferenças devem-se a substituição da cisteína conservada da posição 269 por uma fenilalanina, que é verificada na enzima selvagem. DdPP1c e DdPP1cF269C foram também ensaiadas na presença de INc-1L e INc-1 que são ortólogos de I-2 em N. crassa. Ambas as proteínas recombinantes purificadas exibiram efeito inibidor sobre a atividade de fosforilase fosfatase das DdPP1c recombinantes selvagem e mutante, sendo que INc-1 foi um inibidor duas vezes mais eficiente que INc-1L. Este efeito pode ser devido a um segmento de 38 aminoácidos codificado por um íntron em fase que é retido na isoforma INc-1L. Nossos dados indicam ainda que a mutação F269C não afetou a sensibilidade da DdPP1c recombinante a nenhum dos ortólogos de I-2 testados in vitro. Ensaios de duplo-híbrido utilizando a PP1c selvagem e mutante de D. discoideum (DdPP1c e DdPP1cF269C) e de N. crassa (NcPP1c) como iscas e DdI-2 como presa mostraram que estas proteínas interagiram in vivo. Quando a presa era o INc-1L ou INc-1 a interação ocorreu apenas com a NcPP1c, sendo mais forte no caso de INc-1. As regiões de DdI-2 envolvidas na interação física com a DdPP1c foram mapeadas através da expressão de proteínas truncadas no ensaio de duplo híbrido. Os experimentos apontaram que o carbóxi-terminal de ~100 aminoácidos não é essencial para a interação, mas que o somatório das diversas regiões responde pela integridade da interação. / The serine/threonine phosphatase of type-1 (PP1) is a ubiquous enzyme in the cells and tissues from several species studied and regulates numerous processes such as intermediate metabolism, mRNA splicing, transcription, and apoptosis. PP1 holoenzymes consist of a well-conserved catalytic subunit (PP1c) and one or more variable regulatory subunits. In mammals, more than fifty polypeptides that bind PP1c have been identified, originating holoenzymes with distinct cell locations and specificities. These proteins include cytosolic PP1c inhibitors such as inhibitor-1 (I-1), inhibitor-2 (I-2) and nuclear inhibitor of PP1 (NIPP-1). I-2 orthologs have also been described in Saccharomyces cerevisiae and Neurospora crassa. In the present work, we demonstrate that the genome of the social amoeba Dictyostelium discoideum has a single gene encoding for an I-2 ortholog (DdI-2). Northern blot analyses have shown that DdI-2 mRNA is expressed throughout Dictyostelium developmental cycle at variable levels. We also demonstrated that DdI-2 is a true PP1c inhibitor as its recombinant product is capable of inhibiting the phosphorylase phosphatase activity of wild-type PP1c (DdPP1c) and mutant (DdPP1cF269C) of D. discoideum and NcPP1c of N. crassa in vitro. DdPP1cF269C protein presents distint traits including higher stability, phosphorylase phosphatase activity and sensibility to calyculin A than the wild-type. These differences are originated from the replacement of a well conserved cisteine residue by a phenylalanine found in the wild-type. The wild-type and mutant DdPP1c have also been assayed in the presence of INc-1L and INc-1 which are orthologues to I-2 in N. crassa. Both purified recombinant proteins have shown inhibitory effects over phosphorylase phosphatase activities, with INc-1 being twice more potent than INc-1L. This might be due to the presence of an intron retention event in the latter that results in a insertion of 38 aminoacids. Our data also indicate that F269C mutation did not affect DdPP1c sensitivity to inhibition by all the three recombinant I-2 orthologues in vitro. Yeast two-hybrid assays using wild type (DdPP1c) and mutant (DdPP1cF269C) D. discoideum and N. crassa (NcPP1c) PP1c as preys and the putative inhibitor DdI-2 as a bait showed inequivocally that these proteins interacted in vivo. When the prey was INc-1 or INc-1L the interaction occured only with NcPP1c and was stronger with INc-1. The domains of DdI-2 involved in the interaction with DdPP1c were mapped by two-hybrid interaction assays with DdI-2 deleted mutants. These experiments have pointed out that the DdI-2 carboxi-terminus of ~100 aminoacids is not essential for the interaction but that the sum of all regions is responsible for the integrity of the interaction.

Gene expression profiles obtained by means of semi-quantitative RT-PCR showed that both PPP1R1B and PPP1R3C were frequently silenced in multiple carcinomas. Bisulfite treated tumor DNA was subjected to Methylation-specific PCR (MSP) using primers flanking across the ∼130bp CpG island of the promoter of the particular gene of interest. It was revealed that PPP1R1B and PPP1R3C gene silencing in the carcinoma cell lines were due to promoter CpG island hypermethylation. Such claim was further confirmed by bisulfite genomic sequencing (BGS). Treatment with 5' azacytidine and TSA restored PPP1R1B and PPP1R3C expression in carcinoma cells through demethylating the hypermethylated promoter. In terms of cancer growth inhibition, ectopic expression of PPP1R1B and PPP1R3C could significantly inhibit the proliferation of carcinoma cell lines by 40--50% and 50--60%, respectively, according to the result of anchorage-dependent colony formation assay. / Overall, we believed that PPP1R1B and PPP1R3C are the putative tumor suppressor genes in which their expression silencing through promoter CpG island hypermethylation may be strongly linked to the development of cancer. / Protein Phosphatase 1 regulatory subunits are a family of small molecules which define the substrate specificity and subcellular localization of protein phosphatase-1 upon their interactions. Downregulation of Protein Phosphatase 1 regulatory subunits were often associated with tumor initiation and progression, for example, ASPP family (PPP1R13A and PPP1R13B). In the present study, PPP1R1B and PPP1R3C were identified in which their tumor suppressor functions had been investigated. / Reduction in the level of p-ser473 Akt and p-ser552 beta-catenin could be observed when PPP1R1B expression was restored in respective carcinoma cells. In addition, the transcription activity of AP-1 decreased in the presence of full-length PPP1R1B expression as determined by Dual-Luciferase reporter assay system. Ectopic expression of PPP1R3C increased the amount of inactive pSer9-GSK-3beta as shown in the western blot analysis and a concomitant increased in p53 level was observed in colorectal carcinoma HCT116 cells. Transcription activity of NF-kappaB in HCT116 cells was increased but decreased in KYSE150 cells (ESCC) in the presence of PPP1R3C expression. Subcellular localization study using the GFP-fusion protein revealed that PPP1R1B protein was distributed throughout the cytoplasm while PPP1R3C protein was mainly localized around the nuclear membrane. / Leung, Ching Hei. / Adviser: Tak Cheung Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 160-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.

Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.

A serina/treonina fosfatase do tipo 1 (PP1) é uma enzima ubíqua nas células e nos tecidos das várias espécies em que foi pesquisada e regula vários processos como metabolismo intermediário, processamento de mRNA, transcrição e apoptose. Geralmente a holoenzima PP1 é encontrada como um dímero constituído por uma subunidade catalítica conservada (PP1c) e uma ou mais subunidades reguladoras variáveis. Em mamíferos, já foram identificados mais de 50 polipeptídeos que se associam direta ou indiretamente a PP1c, gerando holoenzimas com localizações celulares e especificidades distintas. Entre estas proteínas estão inibidores citosólicos de PP1c, tais como o inibidor-1 (I-1), o inibidor-2 (I-2) e o inibidor nuclear da PP1 (NIPP-1). Ortólogos do I-2 foram descritos em microorganismos como Saccharomyces cerevisiae e Neurospora crassa. Neste trabalho nós demonstramos que o genoma da ameba social Dictyostelium discoideum possui uma única cópia do gene que codifica um ortólogo do I-2 (DdI-2). Análise através de Northern blot mostrou que o mRNA de DdI-2 é expresso durante o crescimento e ao longo de todo o ciclo de desenvolvimento, com níveis variáveis. Também demonstramos que o gene DdI-2 codifica uma verdadeira proteína inibidora da PP1c uma vez que seu produto recombinante em bactéria é capaz de inibir, com eficácia equivalente, as atividades de fosforilase fosfatase das PP1c recombinantes selvagem (DdPP1c) e mutante (DdPP1cF269C) de D. discoideum e NcPP1c de N. crassa in vitro. A proteína DdPP1cF269C apresenta características distintas da DdPP1c incluindo maior estabilidade, maior atividade de fosforilase fosfatase e maior sensibilidade frente ao inibidor caliculina A. Estas diferenças devem-se a substituição da cisteína conservada da posição 269 por uma fenilalanina, que é verificada na enzima selvagem. DdPP1c e DdPP1cF269C foram também ensaiadas na presença de INc-1L e INc-1 que são ortólogos de I-2 em N. crassa. Ambas as proteínas recombinantes purificadas exibiram efeito inibidor sobre a atividade de fosforilase fosfatase das DdPP1c recombinantes selvagem e mutante, sendo que INc-1 foi um inibidor duas vezes mais eficiente que INc-1L. Este efeito pode ser devido a um segmento de 38 aminoácidos codificado por um íntron em fase que é retido na isoforma INc-1L. Nossos dados indicam ainda que a mutação F269C não afetou a sensibilidade da DdPP1c recombinante a nenhum dos ortólogos de I-2 testados in vitro. Ensaios de duplo-híbrido utilizando a PP1c selvagem e mutante de D. discoideum (DdPP1c e DdPP1cF269C) e de N. crassa (NcPP1c) como iscas e DdI-2 como presa mostraram que estas proteínas interagiram in vivo. Quando a presa era o INc-1L ou INc-1 a interação ocorreu apenas com a NcPP1c, sendo mais forte no caso de INc-1. As regiões de DdI-2 envolvidas na interação física com a DdPP1c foram mapeadas através da expressão de proteínas truncadas no ensaio de duplo híbrido. Os experimentos apontaram que o carbóxi-terminal de ~100 aminoácidos não é essencial para a interação, mas que o somatório das diversas regiões responde pela integridade da interação. / The serine/threonine phosphatase of type-1 (PP1) is a ubiquous enzyme in the cells and tissues from several species studied and regulates numerous processes such as intermediate metabolism, mRNA splicing, transcription, and apoptosis. PP1 holoenzymes consist of a well-conserved catalytic subunit (PP1c) and one or more variable regulatory subunits. In mammals, more than fifty polypeptides that bind PP1c have been identified, originating holoenzymes with distinct cell locations and specificities. These proteins include cytosolic PP1c inhibitors such as inhibitor-1 (I-1), inhibitor-2 (I-2) and nuclear inhibitor of PP1 (NIPP-1). I-2 orthologs have also been described in Saccharomyces cerevisiae and Neurospora crassa. In the present work, we demonstrate that the genome of the social amoeba Dictyostelium discoideum has a single gene encoding for an I-2 ortholog (DdI-2). Northern blot analyses have shown that DdI-2 mRNA is expressed throughout Dictyostelium developmental cycle at variable levels. We also demonstrated that DdI-2 is a true PP1c inhibitor as its recombinant product is capable of inhibiting the phosphorylase phosphatase activity of wild-type PP1c (DdPP1c) and mutant (DdPP1cF269C) of D. discoideum and NcPP1c of N. crassa in vitro. DdPP1cF269C protein presents distint traits including higher stability, phosphorylase phosphatase activity and sensibility to calyculin A than the wild-type. These differences are originated from the replacement of a well conserved cisteine residue by a phenylalanine found in the wild-type. The wild-type and mutant DdPP1c have also been assayed in the presence of INc-1L and INc-1 which are orthologues to I-2 in N. crassa. Both purified recombinant proteins have shown inhibitory effects over phosphorylase phosphatase activities, with INc-1 being twice more potent than INc-1L. This might be due to the presence of an intron retention event in the latter that results in a insertion of 38 aminoacids. Our data also indicate that F269C mutation did not affect DdPP1c sensitivity to inhibition by all the three recombinant I-2 orthologues in vitro. Yeast two-hybrid assays using wild type (DdPP1c) and mutant (DdPP1cF269C) D. discoideum and N. crassa (NcPP1c) PP1c as preys and the putative inhibitor DdI-2 as a bait showed inequivocally that these proteins interacted in vivo. When the prey was INc-1 or INc-1L the interaction occured only with NcPP1c and was stronger with INc-1. The domains of DdI-2 involved in the interaction with DdPP1c were mapped by two-hybrid interaction assays with DdI-2 deleted mutants. These experiments have pointed out that the DdI-2 carboxi-terminus of ~100 aminoacids is not essential for the interaction but that the sum of all regions is responsible for the integrity of the interaction.