The mechanisms through which a cell controls its proliferation, differentiation, metabolism, motility, and ultimate survival in response to extracellular cues are largely controlled by the Ras-extracellular signal-regulated kinase (Ras-ERK) and phosphatidylinositol 3-kinase mammalian target of rapamycin (PI3K-mTOR) signaling pathways. Originally delineated as two separate and linear signaling pathways, multitudes of evidence through experimentation have shown that these pathways can co-regulate downstream targets and cellular outcomes. Here, we provide evidence for an additional point of pathway convergence the serine/arginine protein kinase 2 (SRPK2). Originally identified as a target of the mTORC1/S6K signaling pathway, we have shown SRPK2 to be a target of the Ras-ERK-Rsk pathway, as well as the PI3K-AKT. We discovered the S6K, AKT and RSK all phosphorylate SRPK2 at serine 494 in a cell-type, stimulus dependent manner, emphasizing the redundant nature of the AGC kinases. SRPK2 regulates the phosphorylation of the constitutive and alternative splicing factors the SR proteins. This led us to question mTORC1 involvement in splice site selection, and we discovered several alternative splicing events downstream of mTORC1 signaling. We found that the protein levels of the splicing factors ASF/SF2 and hnRNPa2b1 are regulated by mTORC1 signaling, and we hypothesize this is through regulated unproductive splicing and translation (RUST). Interestingly, we found that BIN1, a target of both ASF/SF2 and hnRNPa2b1, is alternatively spliced, following modulations in mTORC1 signaling. These biochemical studies and knowledge gleaned from them will lead to a better understanding of how the cell can regulate protein expression by controlling alternative splicing.

No description available.

Orientador: Nilson Ivo Tonin Zanchin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T12:40:52Z (GMT). No. of bitstreams: 1 KosckyPaier_CarlosRoberto_M.pdf: 3760581 bytes, checksum: 99331529324819b59a4360d60efd9b9a (MD5) Previous issue date: 2009 / Resumo: A quinase de 70 kDa da proteína ribossomal S6, isoforma 1 (S6K1), é uma fosfoproteína implicada na regulação de genes relacionados ao controle da tradução em mamíferos e possui uma forma nuclear (a1) e uma citoplasmática (a2). A fosforilação do seu principal alvo, a proteína RPS6, tem sido comumente associada ao recrutamento seletivo dos 5'-TOP (5' tract of oligopyrimidine) mRNAs pela maquinaria de tradução, embora haja estudos contrariando esta hipótese. Devido às funções de seus demais alvos, S6K1 tem sido implicada na sobrevivência celular e em diversos outros processos, como crescimento, câncer e resistência à insulina. S6K1 é ativada por um mecanismo que envolve fosforilação seqüencial através da ativação das vias mTORC1 (complexo 1 do alvo da rapamicina em mamíferos) e PI3K (fosfoinositol-3 quinase). Como uma quinase da família AGC, S6K1 deve ser fosforilada por mTORC1 no resíduo Thr389 do domínio hidrofóbico e, em seguida, por PDPK1 (proteína quinase 1 dependente de fosfoinositol) no resíduo Thr229 da alça T do domínio catalítico. Estes eventos ocorrem somente após a fosforilação em diversos sítios do domínio auto-inibitório carboxiterminal, por mTORC1. O objetivo deste trabalho foi desenvolver um ensaio modelo para análise da função da S6K1 in vitro e utilizá-lo como ferramenta na elucidação do papel de proteínas adaptadoras da via de mTOR em interações com a S6K1. Para isso foi necessário produzir as proteínas recombinantes para ensaios de interação e para realização de um ensaio de atividade para a S6K1. Foram testados vários sistemas de expressão para Escherichia coli para produção das construções GST-S6K1a1-His6, GST-S6K1a2-His6 e GST-S6K1a2T389E?CT (forma a2 de S6K1 com a substituição T389E e o carboxiterminal truncado), GST-PDPK1 e GST-CDPDPK1 (domínio catalítico de PDPK1 fusionado a GST). A expressão das formas truncadas de S6K1 e PDPK1 foi mais eficiente em E. coli. Embora o rendimento tenha ficado muito aquém do esperado, foi suficiente para os ensaios de interação in vitro. Também foi feita a expressão em E. coli da região C-terminal da proteína RPS6, que é o substrato da S6K1, em fusão com a proteína D do fago ?. Posteriormente, foram montados sistemas de expressão das construções His6-S6K1a2T389E?CT e His6-CDPDPK1 em células de inseto, a partir de vetor de baculovírus. Constatou-se que essas construções são expressas na forma de fosfoproteínas em células de inseto. Ensaios de GST pull-down com GST-S6K1a2-His6 e GST-S6K1a2T389E?CT contra as duas isoformas da subunidade catalítica da PP2AC, His6-PP2ACa(maior) e His6-PP2ACa(menor), revelaram que His6-PP2ACa(maior) não interage com GST-S6K1a2-His6, embora interaja fortemente com GST-S6K1a2T389E?CT. Já a construção His6-PP2ACa(menor) interage fracamente com as construções GST-S6K1a2-His6 e GST-S6K1a2T389E?CT. Tomados em conjunto, os resultados sugerem que a presença do C-terminal não fosforilado de S6K1a2 impede a interação com PP2ACa(maior). PP2ACa(menor) comporta-se de forma completamente diferente da isoforma maior, pois a interação entre PP2ACa(menor) e S6K1a2 parece ser independente do carboxiterminal da quinase, visto que as quantidades de S6K1a2T389E?CT e de S6K1a2 inteira que interagem com PP2ACa(menor) são semelhantes. Esses resultados necessitam ainda serem confirmados in vivo. Outros experimentos de GST pull-down confirmaram que as construções de S6K1 não interagem com a4, embora interajam com TIPRL1. Se confirmado in vivo, esse resultado compõe um novo quadro na regulação coordenada entre mTOR1 e PP2A, do qual TIPRL1 parece participar. As construções genéticas e os sistemas de expressão gerados neste trabalho possibilitaram a obtenção dos reagentes necessários para analisar o mecanismo de regulação da quinase S6K1, mediado por proteínas regulatórias. Permitem também desenvolver uma série de experimentos, como busca de inibidores específicos para a S6K1, que dependem da reconstituição de ensaios de atividade in vitro com a S6K1 ativada. Contudo, o ensaio de atividade realizado não apresentou resultados satisfatórios e precisa ser desenvolvido. / Abstract: The 70kDa ribosomal S6 protein kinase 1 (S6K1) is a phosphoprotein involved in the regulation of genes related to translational control in mammals. S6K1 shows distinct nuclear (a1) and cytoplasmic (a2) forms. Phosphorylation of the S6K1 best characterized target, the protein of the small ribosomal subunit (RPS6), has been generally associated to the selective recruitment of the 5'-TOP mRNAs (5' tract of oligopyrimidine) by the translational machinery, although there is still some controversy on this issue. Due to the function of its targets, S6K1 has been implicated in several cellular processes including cell growth, cancer and insulin resistance. S6K1 is activated by a mechanism of sequential phosphorylation following activation of the mTORC1 (mammalian target of rapamycin complex 1) and PI3K (phosphoinositide-3-kinase) pathways. As a kinase of the AGC family, S6K1 activation requires mTORC1 phosphorylation of residue Thr389 of the hydrophobic domain followed by PDPK1 (phosphoinositide dependent protein kinase 1) phosphorylation of residue Thr229 at the T loop of the catalytic domain. These take place only after phosphorylation by mTORC1 of several residues of the autoinhibitory C-terminal domain. The objective of this work was to develop an assay to analyze the function of S6K1 in vitro and use it as a tool in the discovering of the functions of regulators proteins of the mTOR cascade in interactions with S6K1. For these purposes, expression systems were constructed to produce the various recombinant proteins to be used in the interaction and activity assays. Several genetic constructions were tested in Escherichia coli for the production of GST-S6K1a1-His6, GST-S6K1a2-His6 and GST-S6K1a2T389E?CT (a2 form of S6K1 with the T389E substitution and truncated carboxiterminus), GST-PDPK1 and GST-CDPDPK1 (GST fusion protein of the catalytic domain of PDPK1). The truncated forms were expressed more efficiently in E. coli. Although the yield in E. coli was lower than expected, it was sufficient to perform interaction assays. The C-terminal domain of RPS6, a substrate for S6K1, was successfully expressed in E. coli as a fusion protein with the phage ? protein D. Subsequently, expression systems for production of His6-S6K1a2T389E?CT and His6-CDPDPK1 in insect cells were constructed using baculovirus vectors. It was found that these constructs are expressed in the form of phosphoproteins in insect cells. GST pull-down assays using GST-S6K1a2-His6 e GST-S6K1a2T389E?CT to test interaction with the PP2AC isoforms His6-PP2ACa(major) and His6-PP2ACa(minor) revealed that His6-PP2ACa(major) does not interact with GST-S6K1a2-His6, although it interacts strongly with GST-S6K1a2T389E?CT. On the other hand, His6-PP2ACa(minor) interacts weakly with both GST- S6K1a2-His6 and GST-S6K1a2T389E?CT. This finding suggests that the unphosphorylated C-terminal of S6K1a2 inhibits interaction with PP2ACa(major). His6-PP2ACa(minor) behaves differently form His6-PP2ACa(major). Its interaction with S6K1a2 seems to be independent of the C-terminal since the amounts of S6K1a2T389E?CT and S6K1a2 that interact with His6-PP2ACa(minor) are similar. Future work in vivo is required to confirm these results. GST pull-down assays confirmed that a4 does not interact with the constructions of S6K1, while TIPRL1 interacts with them. If confirmed in vivo, these results provides a new perspective for the coordinated regulation between mTOR1 and PP2A, which apparently involves also TIPRL1. The genetic constructions and expression systems established in this work allow the production of the reagents required to study the mechanism of S6K1 regulation mediated by adaptor proteins. They will also allow the development of experiments such as screening for specific S6K1 inhibitors, which depend on reconstitution of S6K1 activity assays using activated S6K1. Nevertheless, the activity assay performed did not yield satisfactory outcomes and must be improved. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

The control of growth is a poorly understood aspect of animal development. This thesis focuses on body size regulation in Caenorhabditis elegans, and in particular, how worms grow to a certain size. In C. elegans, a key regulator of size is the TGFβ homologue DBL-1. Mutations that deplete the worm of DBL-1 result in a small body size, whereas overexpression of the gene renders long animals. The small mutants have the same number of cells as wild type suggesting that some or all cells are smaller. DBL-1 activates a TGFβ receptor leading to the nuclear localization of three Smad proteins which then initiate a transcriptional program for size control whose targets are mainly unknown. In order to learn more about how body size in C. elegans is regulated, we set up EMS mutagenesis screens to identify new loci that caused a long phenotype. A subset of the genes we have identified might function in the TGFβ signaling pathway regulating growth while others likely function in parallel pathways. One gene that we found in this screen, lon-3, encodes a cuticle collagen that genetically lies downstream of the DBL-1 TGFβ signaling pathway. Interestingly, loss of function mutations in lon-3 result in a Lon phenotype, whereas increasing the amount of LON-3 protein cause the worms to be dumpy, i.e. shorter, but slightly fatter than wild type. LON-3 is expressed in the hypodermis, the tissue from which the cuticle is synthesized and in which TGFβ signaling, regulating body size, has its focus. This study and previous work have shown that DBL-1 may affect body volume via effects on hypodermal nuclear ploidy, however this is unaffected in lon-3 mutants. Consistent with this finding, the volume of lon-3 mutant worms is not different from wild type. Taken together, our results suggest that another mechanism, by which TGFβ signaling can regulate body length, is by altering the shape of the cuticle via its effect on lon-3 and possibly other cuticle collagens. Studies in worms, flies and mice show that body size and nutrient allocation are closely connected. p70 S6-kinase (S6K) is a known regulator of cell and body size that also plays a role in metabolism. In mice and flies S6K mutants are much smaller than wild type. Our work on the worm homolog, rsks-1, shows that in worms as well, this gene is important for growth regulation and cell size. However, this effect seems to be at least in part independent of DBL-1 TGFβ signaling. Furthermore, rsks-1mutants have a 50 % increase in the amount of stored fat. Fatty acid metabolism has been shown to play an important role in environmental adaptation, especially in regards to temperature changes. Consistent with this idea, rsks-1 mutants appear to have difficulties in adjusting to such changes, reflected in a much-decreased fecundity at 15 and 25 °C compared to their cultivation temperature (20 °C). Within the nervous system the gene is specifically expressed in a subset of the chemosensory neurons that, when nutrients are abundant, secrete signals that promote growth. Intriguingly, this expression seems to be negatively regulated by insulin- like signaling, in contrast to the positive regulation of S6K by insulin in Drosophila and mice. Taken together we show that rsks-1 is an important regulator of growth and fat metabolism in Caenorhabditis elegans.

No description available.

Investigou-se os efeitos da L-glutamina (1g/kg de massa corpórea) em camundongos jejuados por 24 horas. A L-glutamina atenuou a perda de massa muscular e a diminuição da área das fibras musculares esqueléticas causadas pelo jejum via Akt-mTOR. No sóleo, a L-glutamina estimulou a Akt (início da via), e no EDL ativou a S6 (final da via). Investigou-se também os efeitos da L-glutamina em miotubos (C2C12) cultivados por 48 horas. A diminuição de L-glutamina no meio (de 2 para zero mM) causou balanço proteico negativo e aumento do conteúdo dos aminoácidos livres, exceto dos produtos da glutaminólise, indicando estimulação de proteólise. O aumento de L-glutamina no meio (de 2 para 8 e 16 mM) não alterou o conteúdo intracelular de proteínas e dos aminoácidos livres. Na presença de 2 mM de glutamina no meio, a insulina teve efeito positivo no balanço proteico via Akt/mTOR/S6K, estimulando a S6K. Na ausência de glutamina, houve maior fosforilação de eIF2α estimulada por dexametasona e portanto menor síntese proteica. / We investigated the effects of L-glutamine (1g/kg of body mass) on 24 h fasted mice. L-Glutamine attenuated the loss of muscle mass and the reduction of the skeletal muscle fibers area caused by fasting. This attenuation occurred via Akt-mTOR, however, glutamine stimulated Akt (upstream) in the soleus, whereas it activated S6 (dowstream) in EDL. The effects of L-glutamine on myotubes (C2C12) cultured for 48 hours were also examined. The reduction of L-glutamine in the medium (from 2 to zero mM) decreased protein content and increased contents of all amino acids, except products of glutaminolysis, indicating stimulation of proteolysis. The increased L-glutamine levels in the medium (from 2 to 8 and 16 mM) did not change the intracellular contents of protein and free amino acids. In the presence of 2 mM glutamine, insulin had a positive effect on total protein content through Akt/mTOR/S6K pathway, stimulating S6K. In turn, in the absence of L-glutamine, there was increased eIF2α phosphorylation stimulated by dexamethasone and thus less protein synthesis.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.