Neutrophil degranulation involves a number of well-orchestrated structural and biochemical events. We have investigated the mechanism of intracellular signalling involved in neutrophil degranulation that was mediated by the high affinity leukotriene (LT)B[subscript 4] receptor, BLT1. The model systems used were consisted of Peripheral blood neutrophils as well as promyeloid PLB-985 cells, stably transfected with human BLT1 cDNA (PLB-BLT) or a substitution mutant (2L(304-305)/A) of the distal dileucine motif in helix VIII of BLT1, and differentiated into a neutrophil-like phenotype. The degranulation of these cells was measured in the presence and absence of factors that would affect the signaling pathway. The results show that Degranulation responses to LTB[subscript 4] were similar for differentiated PLB-BLT1 and neutrophils. However, the degranulation response of cells bearing the dileucine mutation in helix VIII of BLT1 was significantly reduced in response to LTB[subscript 4]. Pretreatment of differentiated PLB-BLT1 cells and neutrophils with Y-27632, a pharmacological inhibitor of p160-ROCK, the down-stream effector of the small GTPase RhoA, abrogated their degranulation in response to LTB[subscript 4]. The degranulation defect observed with the dileucine mutation was corrected by transient transfection of the cells bearing the mutation with a constitutively active form of RhoA. Taken together, our results suggest an essential role for the distal dileucine motif in helix VIII of BLT1 involving RhoA which allows normal neutrophil degranulation in response to LTB[subscript 4].

Thesis advisor: David Burgess / This doctoral thesis addresses two central topics divided into separate chapters. In Chapter 1: The cortical response to RhoA is regulated during mitosis, experimental findings using sea urchin embryos are presented that demonstrate that the small GTPase RhoA participates in positive signaling for cell division and that this activity is negatively regulated prior to anaphase. In a second series of experiments, myosin phosphatase is shown to be a central negative regulator of myosin activity during the cell cycle through metaphase of mitosis and experimental findings support the conclusion that myosin phosphatase opposes RhoA signaling until anaphase onset. These experiments also reveal that myosin activation alone is insufficient to stimulate cortical contractions during S phase and during metaphase arrest following activation of the spindle checkpoint. In Chapter 2: Annotation of cytoskeletal and motility proteins in the sea urchin genome assembly, as part of a collaborative project, homologs of cytoskeletal genes and gene families were derived and annotated from the sea urchin genome assembly. In addition, phylogenetic analysis of multiple gene families is presented based on these findings. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

No description available.

Deregulated cell proliferation, resulting from disruption of cell cycle control, is characteristic of many cancers. In normal cells, cell cycle progression is mediated by a family of cyclin dependent kinases (Cdks) that are positively regulated by associated cyclins. The activities of these cyclin-Cdk complexes are regulated by two protein families: the inhibitors of Cdk4 (INK4) and the kinase inhibitor proteins (KIP). p27 is a KIP family member that can inhibit cyclin E-Cdk2 activity. It also plays a role in the assembly and nuclear import of cyclin D-Cdk4 in early G1. p27 has been shown to be deregulated in human cancers by accelerated proteolysis, sequestration in cyclin D-Cdk complexes, and mislocalization to the cytoplasm. The causes of these alterations are not fully understood, but result, at least in part, from changes in signal transduction pathways that alter p27 phosphorylation and function. Activation of both the Ras/Raf/ mitogen activated protein kinase (MAPK) and the phospho-inositol 3' kinase (PI3K) pathways have been shown to alter p27 function and to activate p27 degradation in different cell types. In this thesis, I have investigated the roles played by two kinases downstream of PI3K, protein kinase B (PKB) and p90 ribosomal S6 kinase (RSK1), in regulation of p27 function. I observed that PKB-mediated phosphorylation of p27 promotes p27-cyclin D1-Cdk4 assembly. p27 phosphorylation by RSK1 alters the interaction of p27 with cytoskeleton proteins to promote cell motility. I observed that PKB activation and the appearance of p27pT157 and p27pT198 in early G1 precede p27-cyclin D1-Cdk4 assembly. PI3K/PKB inhibition dissociates cellular p27-cyclin D1-Cdk4 and p27T157A, p27T198A and p27T157A/T198A bind cellular cyclin D1 and Cdk4 poorly. Cellular p27pT157 and p27pT198 co-precipitate with Cdk4 but not Cdk2. p27 phosphorylation by PKB increases the ability of p27 to assemble cyclin D1-Cdk4 in vitro, but yields inactive Cdk4. While Src does not affect p27's ability to assemble cyclin D1-Cdk4, Src treatment yields catalytically active p27-cyclin D1-Cdk4. Thus, while PKB dependent p27 phosphorylation promotes p27-cyclin D1-Cdk4 assembly, tyrosine phosphorylation of p27 is required for activation of p27-cyclin D1-Cdk4 complexes. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process. While RSK1 has been shown to phosphorylate p27, the key phosphorylation sites and the consequence of this phosphorylation event were not fully elucidated. I have shown that RSK1 activation in early G1 precedes p27 phosphorylation at T157 and T198 in synchronized cell populations. Overexpression of RSK1 causes resistance to G1 arrest by TGF-â. Moreover, cells overexpressing RSK1 show an increase in p27 phosphorylation at T198, increased p27 stability, and an increase in p27 binding to Cdk4. In addition, RSK1-transfectants have increased cytoplasmic p27, associated with increased cell motility and inhibition of RhoA. p27 phosphorylation by recombinant RSK1 increases p27 binding to RhoA, while p27T157A/T198A shows reduced association with RhoA in cells. Thus, phosphorylation of p27 at T198 by RSK1 promotes its binding to RhoA and loss of actin stress fiber stability. Oncogenic RSK1 activation may promote increased cancer cell migration and cancer metastasis. Taken together our results indicate that oncogenic activation of the PI3K pathway can contribute to loss of cyclin E-Cdk2 inhibitory action of p27 by at least two mechanisms. Activation of PKB and RSK1 signaling would promote cytoplasmic mislocalization of p27, p27-RhoA binding and inhibition of the RhoA pathway to augment cell motility. In addition, these phosphorylation events on p27 would increase the assembly of p27-cyclin D1-Cdk4 as a first step in a chain of events that would promote that nuclear import and activation of D-type cyclin Cdk complexes, shifting the equilibrium away from the Cdk2 inhibitory action of p27.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Thesis (Ph.D.). / Written for the Dept. of Neurology and Neurosurgery. Title from title page of PDF (viewed 2008/05/12). Includes bibliographical references.

La voie de signalisation TGF-β joue un rôle biphasique durant la cancérogenèse. Mon laboratoire a identifié une nouvelle protéine inhibitrice de la voie TGF-β, WWP1. WWP1 est une E3 ubiquitine ligase qui induit la polyubiquitination et la dégradation du récepteur de type I au TGF-β. De plus, le gène WWP1 est amplifié dans une large proportion de cancers mammaires et prostatiques, suggérant que WWP1 pourrait jouer un rôle clé dans les processus de cancérogenèse liés au TGF-β. Mon projet de thèse était donc de caractériser la régulation de l’activité catalytique de WWP1 ainsi que son mécanisme d’action dans la cellule. Mes résultats montrent qu’à l’état basal, WWP1 est monoubiquitinée, son activité de polyubiquitination étant réduite par l’effet inhibiteur qu’exercent les domaines C2 et/ou WW sur son domaine HECT. En présence de substrats, la protéine WWP1 « s’ouvre » et peut alors induire la polyubiquitination et la dégradation de ses substrats. De plus, nous avons observé qu’un mutant de WWP1, détecté dans un cancer de la prostate, est incapable de s’autoréguler selon ce modèle. Il présente une plus forte activité ligase envers lui-même et ses substrats, ce qui entraîne une atténuation de la réponse cytostatique du TGF-β pouvant conférer une activité oncogénique à WWP1. De plus, nous avons identifié STARD13 comme un nouveau partenaire de WWP1. STARD13 est une protéine à activité RhoGAP, considérée comme un suppresseur de tumeur. Nous avons montré que STARD13 permet l’association de WWP1 avec la GTPase RhoA, entraînant ainsi la polyubiquitination et la dégradation de RhoA. De façon intéressante, le complexe WWP1/STARD13 est impliqué dans le remodelage de l’architecture du cytosquelette en dégradant préférentiellement la forme activée de RhoA. Ces résultats ont permis d’identifier un nouveau rôle de WWP1 qui pourrait jouer un rôle essentiel durant la migration des cellules cancéreuses lors du processus métastatique. La caractérisation de nouveaux mécanismes de régulation et d’action de WWP1 devrait permettre à terme d’identifier si WWP1 est un marqueur diagnostique dans le cancer et/ou une nouvelle cible thérapeutique pour le développement de médicaments anticancéreux. / The TGF-β pathway plays a biphasic role during cancerogenesis. My laboratory identified a new protein, WWP1, as a negative regulator of TGF-β signaling. WWP1 is an E3 ubiquitin ligase that triggers polyubiquitination and degradation of TGF-β type I receptor. A genomic amplification of WWP1 is found in a large portion of mammary and prostatic tumors, suggesting a key role for WWP1 during carcinogenesis related to TGF-β. My thesis project was to determine the regulation of the catalytic activity of WWP1 and a new molecular mechanism of action of WWP1 whose deregulation can be implicated in cancerogenesis. My results indicate that at steady states, WWP1 is monoubiquitinated, its polyubiquitination activity being silenced due to the inhibitory effects of C2 or/and WW domains on its Hect domain. In presence of substrates, WWP1 is « opened » and induces polyubiquitination and degradation of its substrates. Moreover, a WWP1 mutation found in prostate cancer disrupts this regulatory mechanism. It possesses an increased ligase activity towards itself and its substrates, which leads to the attenuation of TGF-β cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1. We also identified STARD13 as a novel WWP1 interacting partner. STARD13 has a RhoGAP activity, and is considered as a tumor suppressor. We have shown that STARD13 mediates the association of WWP1 with the GTPase RhoA, ultimately leading to RhoA polyubiquitination and degradation. Interestingly, the WWP1/STARD13 complex is involved in the actin cytoskeleton rearrangement by preferentially targeting the active form of RhoA for degradation. These results reveal a previously unrecognized role for WWP1, which could play a key role in the migration of cancer cells during metastasis. Characterization of new regulation and action mechanisms for WWP1 should allow identifying whether WWP1 is a diagnosis biomarker in cancer and/or a new therapeutic target for the development of anticancer drugs.

The struggle for cultural intelligibility can be clearly articulated through intersections between race, class, and socioeconomic status. Judith Butler demystifies the societal symbols responsible for denoting gender through a discussion of a stable “reality” in relation to performativity. When superimposed over Butler’s gender work, class stratifications and their relevance to cultural intelligibility reflect similar concerns presented in Butler’s work. In this work, I argue that through subversive use of black female archetypes presented by Patricia Hill Collins, strategic language, and flamboyant displays of tangible wealth, characters on Bravo’s The Real Housewives of Atlanta consciously perform class to resist the policing of social boundaries and to highlight their position within liminal social spaces. However, as a result of their performativity, these women violate the liminal space by patrolling class boundaries from within their social circle.