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.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-11T08:45:17Z No. of bitstreams: 1 texto completo.pdf: 2070709 bytes, checksum: 8aca4e1bccb3e3a726679695fe3ab253 (MD5) / Made available in DSpace on 2015-11-11T08:45:17Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2070709 bytes, checksum: 8aca4e1bccb3e3a726679695fe3ab253 (MD5) Previous issue date: 2014-08-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Serine-Arginine Protein Kinase 2 - SRPK2 é uma cinase reguladora de fatores de splicing não-snRNPs (proteínas SR), e de fatores snRNPs (U4⁄U6-U5 tri-snRNP). SRPK2 regula as proteínas SR fosforilando-as em seus resíduos de serina e recrutando- as para a formação do spliceossomo, enquanto atua na formação do complexo U4⁄U6- U5 tri-snRNP, envolvido na seleção do sítio de splicing γ’. As interações com fatores snRNP e não-snRNP são conhecidas e bem estabelecidas funcionalmente, no entanto, pouco se sabe sobre proteínas que regulam o funcionamento da SRPK2, e sobre demais alvos de fosforilação. As proteínas SRPKs possuem seu domínio cinase divido em dois por uma região espaçadora de grande flexibilidade estrutural, passível de interação. Portanto, possível responsável pela seleção de substratos e/ou alvo de regulação. Nosso objetivo com este trabalho foi encontrar novas interações proteicas de SRPK2 através de sua região espaçadora. A técnica escolhida foi o duplo-híbrido em leveduras, no qual estas foram cotransformadas com o plasmídeo pBTM116K/S-SRPK2 trp1 e com uma biblioteca de cDNAs de leucócitos humanos em pACT2 leu2. O experimento foi processado em meio restritivo SD–TRP –LEU –HIS, acrescido de 30mM de 3AT e mantido em estufa 30°C por 120 horas. Ao final deste período, foi verificado o crescimento de 119 colônias de leveduras, submetidas a diferentes testes de ativação dos genes repórteres his3 e lacZ. Os resultados destes testes nos levaram a seleção de 42 clones, que tiveram seus DNAs plasmidiais extraídos, sequenciados e analisados pela ferramenta Blast do NCBI. As proteínas codificadas por estes genes estão funcionalmente relacionadas a processos celulares como biogênese ribossomal, migração, diferenciação, angiogênese, proliferação, sobrevivência e ciclo celular. Estes resultados sugerem novos mecanismos moleculares para a atuação da SRPK2 no processo tumorigênico. / Serine-Arginine Protein Kinase 2 - SRPK2 is a regulator kinase of non-snRNP splicing factors (SR proteins) and of snRNPs factors (U4⁄U6-U5 tri-snRNP). SRPK2 regulates SR proteins phosphorylating them on their serine and recruiting them for the formation of the spliceosome, while acts on the tri-snRNP U4/U6-U5 complex assembly, involved in the splicing γ’ site selection. The interactions with snRNP and non-snRNP factors are known and well-established functionally, however, little is known about the proteins that regulate the functioning of SRPK2, and about other targets of phosphorylation. The SRPKs proteins have their kinase domain divided into two by a spacer region of great structural flexibility, believable of interaction. Therefore, possible responsible for selection of substrates and/or aim of regulation. Our aim with this work was to find new protein interactions of SRPK2 by its space region. The technique chose was two-hybrid yeast, in which yeasts were cotransfected with pBTM116K/S-SRPK2 trp1 plasmid and a human leukocyte cDNA library in pACT2 leu2. The experiment was processed in restrictive medium SD –TRP –LEU –HIS, plus 30 mM of 3AT and keep in a incubator 30°C for 120 hours. At the end of this period, it was found the growth of 119 yeast colonies, subjected a different tests of activation of reporter genes his3 and lacZ. The results of these tests led to the selection of 42 clones, which had its plasmid DNA extracted, sequenced and analyzed by the NCBI Blast tool. The proteins encoded by these genes are functionally related in cellular processes as ribosome biogenesis, migration, differentiation, angiogenesis, proliferation, survival and cell cycle. These results suggest new molecular mechanisms for the performance of SRPK2 in tumorigenic process. / Sem lattes