Rho GTPases and their regulators such as guanosine exchange factors (GEFs) and GTPase activating proteins (GAPs) represent an important class of molecules controlling dendritic spine plasticity. Although they are typically described as cytoskeletal modulators, roles for the GTPases in endocytosis and cell polarity establishment have also been defined. The neuronal proteins a1- and a2-chimaerins belong to a group of Rac and Cdc42 GAPs that inactivate these GTPases; in addition to a GAP domain, the a-chimaerins share a phosphokinase C (PKC)-like C1 domain but have distinct N-terminal domains (NTDs). My project has explored the importance of specific domains of a1-chimaerin both in induction of a morphological cellular protrusion collapse phenotype ('circularisation') and in interactions with partner proteins that may help to explain the phenotype. The results described in my thesis show that a1-chimaerin possesses a previously undescribed C-terminal domain (CTD) that is indispensable for the ability of the protein to induce collapse of protrusions, and consequent circularisation, in various cell types; moreover, an intact CTD is also important for association of a1-chimaerin with its known effector EphA4, and potentially with other undefined membrane proteins, in a C1-domain- dependent manner. In addition, my results show that a1-chimaerin associates via its NTD with the Src kinase Fyn, and via its C1 domain with the NR2A subunit of the NMDA receptor. Further experiments explored a1-chimaerin effects on EphA4 and NMDA receptor cell surface expression, as well as binding to other putative partners - including the adaptor protein p35 and the polarity protein PAR6. Finally, I have shown that inhibition of a pathway involving the Rho-associated coiled-coil containing protein kinase (ROCK) reverts circularisation induced by a1- chimaerin, and that a blocking peptide based on the CTD may be employed to partially counteract the phenotype. These results uncover a novel domain in a1-chimaerin that may have a crucial importance for the induction of cellular process collapse by a1-chimaerin with a potential relevance to the EphA4-induced dendritic spine retraction, EphA4 receptor endocytosis, and cell surface expression of NR2A-containing NMDA receptors. This suggests a model of a multi-protein signalling complex involving a1-chimaerin that coordinates cellular process remodelling, and that is likely to be important both for adult neuronal circuit plasticity and for neurodegenerative diseases.

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.

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