During stress-induced apoptosis, the initiator caspase-9 is activated by the Apaf-1 apoptosome and must remain bound in order to retain significant catalytic activity. Nevertheless, in apoptotic cells, the vast majority of processed caspase-9 is paradoxically observed outside of the complex. We demonstrate herein that apoptosome-mediated cleavage of procaspase-9 occurs exclusively through a CARD-displacement mechanism, so that unlike the effector procaspase-3, procaspase-9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase-9 possessed higher affinity for the apoptosome and could displace processed caspase-9 from the complex, thereby facilitating a continuous cycle of procaspase-9 recruitment/activation, processing, and release from the complex. Due to its rapid autocatalytic cleavage, however, procaspase-9 per se contributed little to the activation of procaspase-3. Thus, the Apaf-1 apoptosome functions as a proteolytic-based “molecular timer”, wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, procaspase-9 autoprocessing activates the timer, and the rate at which processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer “ticks” over. To understand the physiological relevance of molecular timer in vivo, we are currently generating caspase-9 knock-in mouse models. These mouse models will enhance our understanding of the importance of caspase-9 processing within the apoptosome. / text

<p>Apoptosis is a cellular suicide program that can be initiated by various genotoxic and cytotoxic stimuli. In many cases, such cell damaging agents promote cell death through the intrinsic apoptotic pathway by triggering mitochondrial cytochrome <italic>c</italic> release and subsequent caspase activation. Cytosolic cytochrome <italic>c</italic> is directly responsible for initiating formation of the caspase-activating apoptosome, which plays a crucial role in the apoptotic process. Given the importance of cellular fate, apoptosis is tightly controlled by a balance between survival and death signals. It has been shown that activated cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade and the PI3K/Akt signaling, enhance cell viability by conferring resistance to apoptotic cell death. However, the underlying mechanism(s) that lead to inhibition of functional apoptosome formation (and caspase activation) has yet to be elucidated. In the studies that are described in this dissertation, I have investigated the regulation of apoptosis downstream of mitochondrial cytochrome <italic>c</italic> release with the goal of understanding how survival signaling can alter the apoptotic program, contributing to human malignancies. </p><p>First, we describe a mechanism for the inhibition of cytochrome <italic>c</italic>-induced caspase activation by MAPK signaling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). We have found that recruitment of 14-3-3&epsilon; to phosphorylated Ser268 impedes the ability of cytochrome <italic>c</italic> to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14-3-3&epsilon; binding to Apaf-1 and rendered the cells insensitive to cytochrome <italic>c</italic>, suggesting a role for Rsk signaling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. These results identify a novel locus of apoptosomal regulation wherein MAPK signaling promotes Rsk-catalyzed Apaf-1 phosphorylation and consequent binding of 14-3-3&epsilon;, resulting in decreased cellular responsiveness to cytochrome <italic>c</italic>. </p><p>In the second part, we examine how apoptosis is inhibited by oncogenic tyrosine kinase signaling by using leukemogenic tyrosine kinase-induced leukemia model systems. We have demonstrated that protein phosphatase 5 (PP5) is responsible for Hsp90&beta; hypophosphorylation, which can contribute to impaired cell death in leukemia expressing oncogenic tyrosine kinases. Loss of PP5 results in an increase of Hsp90&beta; phosphorylation, raising leukemic cells' responsiveness to imatinib, a BCR-ABL kinase inhibitor. Further we have discovered that acetylation regulates PP5 activity on Hsp90&beta;. Mutational study showed that K144 acetylation on PP5, which was diminished in leukemic conditions, inhibited PP5 binding to Hsp90&beta;, causing Hsp90&beta; hyperphosphorylation and subsequently potentiating cells to apoptosis. These studies reveal a molecular mechanism by which agents enhancing PP5 acetylation may be a potential treatment for leukemias. Collectively, this work provides new insight into mechanisms of regulation of apoptosome formation/function, helping us understand how the evasion of apoptotic cell death contributes to cancer cell survival. Further, this finding implicates cytochrome <italic>c</italic>-induced apoptotic signaling in the context of cancer cell responsiveness to chemotherapeutic treatments.</p> / Dissertation

La mort cellulaire programmée ou apoptose est un mécanisme conservé chez les eucaryotes multicellulaires qui contribue au développement embryonnaire et à l'homéostasie cellulaire des organismes. Dans les cellules vivantes, l'activité des protéases qui exécutent le programme de mort cellulaire, les caspases, est contrôlée par des signaux de survie provenant de l'environnement cellulaire. Les caspases initiatrices de l'apoptose régulée par l'environnement, la caspase 9 et la caspase 8 sont activées respectivement par l'apoptosome et par les récepteurs de mort. Les signaux environnementaux, parmi lesquels le contact avec la matrice extracellulaire ou la présence de facteurs de croissance, activent des voies de signalisation contrôlant la machinerie de mort cellulaire. La voie des MAPK1/3 est une voie de signalisation contrôlée par le proto-oncogènes Ras et comportant les kinases Raf, MEK1/2 et MAPK1/3 (ERK2/1 ou p42/p44). La voie des MAPK1/3, qui est impliquée dans la prolifération et la différentiation cellulaire, joue un rôle essentiel dans la survie cellulaire. L'objectif de cette thèse a été de caractériser les mécanismes moléculaires impliqués dans le contrôle de la mort cellulaire par la voie des MAPK1/3. Ce travail est basé sur l'utilisation d'une forme active et inductible de la kinase Raf-1 (DRaf-1:ER) dont l'activation forte et prolongée correspond à une induction pathologique de la voie des MAPK1/3. Nous avons montré que, selon le type cellulaire, l'activation de deltaRaf-1:ER favorise la survie ou la mort cellulaire. Dans les cellules fibroblastiques CCL39, l'activation de deltaRaf-1:ER protège de la mort cellulaire mitochondriale induite par la privation en sérum du milieu de culture. Dans ces conditions, nous avons montré que la stimulation de Raf-1 :ER bloque l'activation de la caspase-9 mais n'empêche pas la délocalisation du cytochrome c, la multimérisation d'APAF1 ni le recrutement de la procaspase 9 dans l'apoptosome. Ce mécanisme post mitochondrial de protection contre la mort cellulaire dépend de la néo-synthèse des protéines et nécessite une activité continue de la kinase MEK. A l'inverse, dans les cellules HEK 293 issues de rein embryonnaire et présentant des caractéristiques neuronales, nous avons montré que l'activation soutenue de la voie des MAPK1/3 par DRaf1-ER induit une mort cellulaire massive. Celle-ci est caractérisée par l'activation des caspases et la fragmentation de l'ADN. La mort cellulaire est détectée plus de 24 heures après l'activation de Raf1-ER, elle est maximale à 48h. L'induction de la mort cellulaire ne requière la synthèse protéique que durant la phase précoce d'activation mais nécessite l'activité continue du module MEK/MAPK. La mort cellulaire résulte de l'activation de la caspase 8 et n'implique pas la voie mitochondriale, elle est caractérisée par une vacuolisation importante du cytoplasme des cellules qui l'apparente à une forme particulière d'apoptose. L'inactivation des fonctions du récepteur fas et de son adaptateur FADD indique que le processus d'activation de la caspase 8 est indépendant de la voie des récepteurs de mort. L'ensemble de ces travaux apporte des connaissances nouvelles sur le contrôle de la mort cellulaire par la voie Raf/MAPK1/3. Nous avons montré que la voie de signalisation peut, selon le contexte cellulaire, favoriser la survie cellulaire ou induire la mort. Dans les deux cas, le contrôle de la mort cellulaire dépend à la fois de la synthèse protéique et de mécanismes post-traductionnels. Les mécanismes moléculaires affectés par l'activation prolongée des MAPK1/3 seraient impliqués aussi bien dans la résistance des cellules tumorales aux traitements proapoptotiques que dans le développement des maladies neurodégénératives.

<p>Many pro-apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade, promote cell viability both by impeding mitochondrial cytochrome c release and by inhibiting subsequent activation of caspases. Cytosolic cytochrome c is directly responsible for initiating formation of the caspase-activating apoptosome, which, in many cell types, plays a crucial role in the apoptotic process. Given the important role of cytochrome c in dismantling the dying cell, we wanted to investigate the process of cytochrome c-induced apoptosis with the goal of understanding how this mechanism is altered in certain malignant conditions. </p> <p> First, we examined cytochrome c-induced caspase activation in normal and tumorigenic mammary epithelial cells. Although most tumor types have developed mechanisms for evading apoptosis, we surprisingly discovered that breast cancer cells were hypersensitive to cytochrome c when compared with their normal counterpart. Specifically, breast cancer cells show increased binding of caspase-9 to the Apaf-1 caspase recruitment domain. This altered apoptosome formation is mediated by overexpression of the protein PHAPI in the malignant mammary epithelial cells. Immunoblot analysis demonstrated that protein levels of PHAPI are also elevated in human breast tumors. These results suggest a novel paradigm where breast cancer cells are refractory to cytochrome c release in response to certain stimuli, but they are quite sensitive to apoptosis downstream of the mitochondria. </p> <p> Secondly, we describe a mechanism for the inhibition of cytochrome c-induced caspase activation by MAPK signaling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). This Apaf-1 phosphorylation results in impaired apoptosome formation, thereby inhibiting caspase activation. The Rsk effect on Apaf-1 is antagonized by protein phosphatase 1 (PP1), which promotes Apaf-1 dephosphorylation. High endogenous levels of Rsk in PC3 prostate cancer cells leads to Apaf-1 phosphorylation and renders them relatively insensitive to cytochrome c, suggesting a role for Rsk signaling in the apoptotic resistance of certain cancers. These results identify a novel locus of apoptosomal regulation wherein MAPK signaling promotes direct Rsk-catalyzed phosphorylation of Apaf-1, resulting in decreased cellular responsiveness to cytochrome c. Collectively, this work provides insight into novel mechanisms of regulation for cytochrome c-induced apoptosis.</p> / Dissertation

Apoptosis is a programmed cell death mechanism that is evolutionary conserved from worms to humans. Apoptosis is mediated by initiator and effector caspases. The initiator caspases carry long pro-domains for their interaction with scaffolding proteins to form a cell-death platform, which is essential for their activation. Activated initiator caspases then cleave effector caspases that execute cell death through cleaving downstream targets. In addition to their apoptotic function, caspases also participate in events where caspase activity is not required for cell killing, but for regulating other functions, so-called non-apoptotic functions of caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian caspase-2 and caspase-9 has a CARD domain that is essential for its interaction with the scaffolding protein Dark to form the apoptosome. Apoptosome formation is crucial for activation of Dronc. Activity of both initiator and effector caspases are further kept in control by the ubiquitin system to avoid inappropriate caspase activity. However, mechanistic details of how the ubiquitin system regulates activation of Dronc are not clear. Therefore, I investigated the ubiquitylation status of Dronc and its function in Drosophila. I found that Dronc is mono-ubiquitylated at Lys78 (K78) in its CARD domain, which blocks its interaction with Dark and formation of the apoptosome. Furthermore, I demonstrated that K78 mono-ubiquitylation plays an inhibitory role in Dronc’s non-apoptotic functions, which may not require its catalytic activity but may be important for the survival of the fly. This thesis study unveils the link between the ubiquitin system and caspases through a regulatory mechanism where a single mono-ubiquitylation event could inhibit both apoptotic and non-apoptotic functions of a caspase.