A via de sinalização da GTPase RhoA atua em diversos processos celulares. Para avaliar o comportamento de RhoA, após estresse causado por radiação ultravioleta, foram gerados clones mutantes que expressam RhoA em seu estado constitutivamente ativo e dominante negativo. Após exposição das linhagens à radiação ultravioleta, observou-se uma maior sensibilidade e um maior tempo de recuperação das linhagens quando a atividade de RhoA é reduzida. Estes prejuízos no reparo prejudicaram a proliferação e sobrevivência celular quando da deficiência na atividade de RhoA. Em linhagens deficientes na via de NER, percebemos que estas linhagens possuem uma capacidade ainda mais reduzida de reparo quando a atividade de RhoA é inibida. / The RhoA GTPase signaling pathway acts on many cellular processes. To evaluate this possible RhoA function after stress caused by ultraviolet radiation, mutant clones expressing RhoA in its constitutively active or dominant negative forms were generated. After exposure of the cells to ultraviolet radiation, cell lines showed a higher sensitivity and a delayed recovery capacity when the RhoA activity is reduced. The impaired repair reduced the cells proliferation and survival under RhoA deficiency. In cell lines deficient in NER pathway, we notice that these cell lines, have a further reduced ability to repair damaged DNA under RhoA inhibition.

<p>The Rho GTPases, which are related to the Ras family of proto-oncogenes, have been found to have important roles in regulating the morphogenic and migratory properties of eukaryotic cells. In addition, these proteins have been shown to regulate aspects of cell signaling, cell growth, cell division and cell survival. The Rho GTPases cycle between inactive GDP-bound and active GTP-bound states. In resting cells, Rho GTPases are sequestered in the cytoplasm by forming an inactive complex with guanine dissociation inhibitors (GDIs), and are, thus, unable to exchange guanine nucleotides. Rho GTPases exchange guanine nucleotides at slow rates <i>in vivo</i>, and these reactions can be catalyzed by two different classes of proteins. Upon cell activation, guanine exchange factors stimulate the exchange of GTP for GDP and thereby activate the Rho GTPases, whereas the GTPase activating proteins turn off the Rho GTPase by stimulating their inherent GTP-hydrolysis activity. The active Rho GTPase associates with so-called effector proteins, which in turn mediate a plethora of responses.</p><p>In recent years a great number of Rho GTPase effectors have been identified. The Cdc42-interacting protein 4 (CIP4) is one such protein, and this thesis has focused on elucidating the role of this protein in Rho GTPase regulated activities resulting in changes in the organization of the actin filament system. Changes in actin dynamics are required for many cellular activities, such as cell migration, cytokinesis and membrane-trafficking. CIP4 is a member of the Pombe Cdc15 homology (PCH) family of proteins. Many PCH proteins been proposed to cooperate with so-called formin homology proteins to induce changes in actin dynamics resulting in cytokinesis. We show that CIP4 interacts with the diaphanous-related formin DAAM1 (Disheveled associated activator of morphogenesis 1). DAAM1 appeared to influence both changes in actin dynamics and microtubule dynamics, possibly by integrating signals from CIP4, Src and the Rho GTPases Rac, Cdc42</p><p>The RhoGAP domain-containing protein RICH-1 (Rho GAP interacting with CIP4 homologoues-1) was isolated in a yeast two hybrid screen for proteins binding to CIP4. RICH-1 was shown to down-regulate the Rho GTPases Cdc42 and Rac1. In addition to the RhoGAP domain, RICH-1 possesses a proline-rich motif which confers binding to a variety of Src homology 3 (SH3) domain-containing proteins including CIP4, FBP17, Src, Abl and CIN85. Furthermore, RICH-1 exhibits a BIN/amphiphysin/Rvsp (BAR) domain which associates with membrane lipids, and in addition this domain was shown to deform liposomes in an in vitro assay, which is thought to mimic the deformation of cellular lipid bilayers, for example the invagination of the plasma membrane during endocytosis. Our results suggest a role for RICH-1 in intracellular membrane-trafficking events. RICH-1 was in addition shown to interact with the SH3 domains of two BAR domain-containing proteins, endophilin A1 and amphiphysin, which induce deformation of the plasma membrane during the specialized clathrin-mediated endocytosis. In conclusion, our data supports the notion that RhoGAPs are multi-functional proteins, fulfilling not only the role as downregulators of Rho GTPase activity, but also as signal transducers of numerous vital cellular processes.</p>

<p>All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. </p><p>We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K.</p><p>The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway.</p><p>ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation.</p><p>Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.</p>

The Rho GTPases, which are related to the Ras family of proto-oncogenes, have been found to have important roles in regulating the morphogenic and migratory properties of eukaryotic cells. In addition, these proteins have been shown to regulate aspects of cell signaling, cell growth, cell division and cell survival. The Rho GTPases cycle between inactive GDP-bound and active GTP-bound states. In resting cells, Rho GTPases are sequestered in the cytoplasm by forming an inactive complex with guanine dissociation inhibitors (GDIs), and are, thus, unable to exchange guanine nucleotides. Rho GTPases exchange guanine nucleotides at slow rates in vivo, and these reactions can be catalyzed by two different classes of proteins. Upon cell activation, guanine exchange factors stimulate the exchange of GTP for GDP and thereby activate the Rho GTPases, whereas the GTPase activating proteins turn off the Rho GTPase by stimulating their inherent GTP-hydrolysis activity. The active Rho GTPase associates with so-called effector proteins, which in turn mediate a plethora of responses. In recent years a great number of Rho GTPase effectors have been identified. The Cdc42-interacting protein 4 (CIP4) is one such protein, and this thesis has focused on elucidating the role of this protein in Rho GTPase regulated activities resulting in changes in the organization of the actin filament system. Changes in actin dynamics are required for many cellular activities, such as cell migration, cytokinesis and membrane-trafficking. CIP4 is a member of the Pombe Cdc15 homology (PCH) family of proteins. Many PCH proteins been proposed to cooperate with so-called formin homology proteins to induce changes in actin dynamics resulting in cytokinesis. We show that CIP4 interacts with the diaphanous-related formin DAAM1 (Disheveled associated activator of morphogenesis 1). DAAM1 appeared to influence both changes in actin dynamics and microtubule dynamics, possibly by integrating signals from CIP4, Src and the Rho GTPases Rac, Cdc42 The RhoGAP domain-containing protein RICH-1 (Rho GAP interacting with CIP4 homologoues-1) was isolated in a yeast two hybrid screen for proteins binding to CIP4. RICH-1 was shown to down-regulate the Rho GTPases Cdc42 and Rac1. In addition to the RhoGAP domain, RICH-1 possesses a proline-rich motif which confers binding to a variety of Src homology 3 (SH3) domain-containing proteins including CIP4, FBP17, Src, Abl and CIN85. Furthermore, RICH-1 exhibits a BIN/amphiphysin/Rvsp (BAR) domain which associates with membrane lipids, and in addition this domain was shown to deform liposomes in an in vitro assay, which is thought to mimic the deformation of cellular lipid bilayers, for example the invagination of the plasma membrane during endocytosis. Our results suggest a role for RICH-1 in intracellular membrane-trafficking events. RICH-1 was in addition shown to interact with the SH3 domains of two BAR domain-containing proteins, endophilin A1 and amphiphysin, which induce deformation of the plasma membrane during the specialized clathrin-mediated endocytosis. In conclusion, our data supports the notion that RhoGAPs are multi-functional proteins, fulfilling not only the role as downregulators of Rho GTPase activity, but also as signal transducers of numerous vital cellular processes.

All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K. The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway. ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation. Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.

A via de sinalização da GTPase RhoA atua em diversos processos celulares. Para avaliar o comportamento de RhoA, após estresse causado por radiação ultravioleta, foram gerados clones mutantes que expressam RhoA em seu estado constitutivamente ativo e dominante negativo. Após exposição das linhagens à radiação ultravioleta, observou-se uma maior sensibilidade e um maior tempo de recuperação das linhagens quando a atividade de RhoA é reduzida. Estes prejuízos no reparo prejudicaram a proliferação e sobrevivência celular quando da deficiência na atividade de RhoA. Em linhagens deficientes na via de NER, percebemos que estas linhagens possuem uma capacidade ainda mais reduzida de reparo quando a atividade de RhoA é inibida. / The RhoA GTPase signaling pathway acts on many cellular processes. To evaluate this possible RhoA function after stress caused by ultraviolet radiation, mutant clones expressing RhoA in its constitutively active or dominant negative forms were generated. After exposure of the cells to ultraviolet radiation, cell lines showed a higher sensitivity and a delayed recovery capacity when the RhoA activity is reduced. The impaired repair reduced the cells proliferation and survival under RhoA deficiency. In cell lines deficient in NER pathway, we notice that these cell lines, have a further reduced ability to repair damaged DNA under RhoA inhibition.

Rho-GTPases are essential signalling proteins which regulate a multitude of central cellular processes that are vital for organisms to thrive and adapt to changing environments. Many regulatory networks involving Rho proteins have first been elucidated in the model yeast Saccharomyces cerevisiae, in which Rho5 emerges as a central hub connecting different signalling pathways, such as the responses to cell wall stress, high medium osmolarity, and oxidative stress. In this work, the rapid translocation of Rho5 to mitochondria as reaction to oxidants and glucose starvation was thoroughly investigated. The studies on structure-function relationships was focussed on the C-terminal region of the Rho5 which in other Rho-type GTPases determines their spatio-temporal distribution and contributes to their physiological function. The C-terminal end of these GTPases is considered to be a hypervariable region (HPR) that consists of a polybasic region (PBR) and its preceding amino acid residues, followed by the CAAX motif which becomes prenylated at its cysteine residue. These motifs are conserved in the yeast Rho5 where the PBR contains a serine residue as a putative phosphorylation target. Moreover, Rho5 of S. cerevisiae is characterized by an extension preceding the PBR that comprises 98 amino acid residues. While substitutions of the serine residue within the PBR for either phosphomimetic or non-phosphorylatable residues indicate that it is of minor physiological importance, deletion analyses of the yeast-specific extension showed that it is required for proper localization of Rho5 to the plasma membrane. As expected, substitution of the cysteine residue within the CAAX motif also prevented proper plasma membrane localization, accompanied by a loss of function both with respect to oxidative stress response and glucose starvation. Results from studies employing a trapping-device of GFP-Rho5 to the mitochondrial surface indicate that the GTPase needs to be activated at the plasma membrane by its dimeric GDP/GTP exchange factor (GEF) which is composed of Dck1 and Lmo1, in response to stress conditions. The trimeric DLR complex is then capable of rapidly translocate to mitochondria and fulfil its functions at the organelle. This view was supported by the finding that a constitutively active Rho5 variant restored function when trapped to mitochondria. Interestingly, Rho5 requires the dimeric GEF for the translocation process under oxidative stress while Dck1 and Lmo1 can reach the mitochondria independent from each other. Finally, the human Rho5 homolog Rac1 cannot complement the defects of a rho5 deletion and does not show a proper intracellular distribution, unless its C-terminal end is equipped with the yeast-specific extension. Taken together, the results of this thesis contributed to a better understanding of the structure-function relationships of Rho5 and its human homolog Rac1.

No description available.

No description available.

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga / The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination