5 mutations responsables de retard mental ont été identifiées dans le gène p21-activated kinase 3 (pak3). Nous avons récemment identifiés dans pak3 deux exons alternatifs très conservés appelés b et c. Ainsi, en plus du variants PAK3a (dépourvu des inserts b ou c), le gène pak3 code pour 3 nouveaux variants d’épissage PAK3b, PAK3c et PAK3cb qui sont constitutivement actifs et insensibles aux GTPases. De plus, contrairement à PAK1 et PAK3a, leur domaine d’auto-inhibition est incapable d’inhiber un domaine kinase. Ainsi, le but de ce projet était de comprendre le mécanisme de régulation de la kinase PAK3. Un modèle de régulation a récemment été proposé dans lequel PAK1 forme des homodimères pouvant être dissociés par les GTPases, permettant ainsi l’activation de la kinase. En se basant sur ces observations j’ai cherché à identifier les dimères PAK3 et j’ai montré que les kinases PAK3a, b, c et cb forment préférentiellement des hétérodimères avec PAK1. J’ai démontré l’existence de ces dimères dans le cerveau et j’ai mis en évidence que ces hétérodimères permettent à chaque monomère de réguler l’activité kinase de son partenaire in vitro. Ce travail permet de proposer un modèle de régulation symétrique pour PAK3a qui forme des hétérodimères avec PAK1 et un nouveau modèle de régulation asymétrique pour les variants d’épissage, également basé sur leur hétérodimérisation avec PAK1. Mes résultats montrant une corégulation des kinases PAK neuronales suggèrent d'une part que leur activation puisse être synchronisée et d'autre part que dans certaines situations physiopathologiques (Cancer et maladies neurologiques) leur dérèglement puissent interférer. / 5 mutations responsible for mental retardation have been identified in p21-activated kinase 3 (pak3) gene. We recently identified in pak3, two highly conserved alternative exons called b and c. In addition to the classical PAK3a variant (without any alternative exon), the pak3 gene encodes 3 new splice variants PAK3b, PAK3c and PAK3cb which are constitutively active and insensitive to GTPase activation. Moreover, unlike PAK1 or PAK3a, their autoinhibitory domain is unable to inhibit a kinase domain. The aim of this project was to understand how PAK3 regulation occurs. A model of regulation was recently proposed in which PAK1 forms homodimers that can be dissociated through GTPase binding, leading to kinase activation. Given these observations, I searched to identify PAK3 dimers and I showed that PAK3a, b, c and cb preferentially form heterodimers with PAK1. I demonstrated the existence of such dimers in the brain and that the different heterodimers allow each monomer to regulate the kinase activity of its partner. Through this study, I propose a symmetric regulation model for PAK3a which heterodimerizes with PAK1 and a new asymmetric regulation model for splice variants, also based on heterodimerization with PAK1. My results showing a co-regulation of neuronal PAK kinases suggest that their activation may be synchronized but also that, in some physiopathological situations (cancers and neurologic diseases), their misregulation may interfere.

Les gliomes sont les tumeurs cérébrales les plus fréquentes chez l’adulte. Il s’agit d’un groupe de tumeurs hétérogène pouvant être classées dans différents sous-groupes selon des critères moléculaires et histopathologiques, donnant une indication sur leur agressivité. Les p21-activated kinase (PAK) sont des sérine-thréonine kinases effectrices des GTPases Rac et Cdc42 et font ainsi partie de nombreuses voies de signalisation. Elles régulent de nombreuses voies d’intérêt dans la biologie des cancers, comme les voies Mek/Erk, PI3K/Akt et Wnt/b-caténine. PAK1 est fréquemment surexprimé et/ou suractivé dans plusieurs cancers, notamment du sein, des ovaires de la prostate et du cerveau, alors que PAK3 induit la sortie du cycle cellulaire et la différenciation des cellules au cours de la neurogenèse dans plusieurs modèles animaux. Durant ma thèse, j’ai pu observer que l’expression de PAK3 était de bon pronostic pour les patients atteints de gliomes, et était plus élevée dans un sous-type de tumeurs caractérisé par la codélétion 1p/19q. In vitro, l’expression de PAK3 était plus élevée dans une lignée de cellules de gliome de signature proneurale, non tumorigène après xenogreffe chez la souris. Au cours de la différenciation des cellules de gliome, l’augmentation de l’expression de PAK3 est associée avec des marqueurs de lignage neural et neuronal. L’inhibition de l’expression de PAK3 entraîne l’augmentation de la capacité d’auto-renouvellement et de la tumorigénicité d’une lignée de cellules de gliome, et favorise la différenciation gliale des cellules. / Gliomas are the most common and lethal adult primary brain tumors. Their complex heterogeneity is evidenced by numerous genomic studies showing distinct molecular entities in glioma. P21-activated-kinases (PAK) are serine threonine kinases involved in multiple signal transduction pathways as downstream effectors of Rac and Cdc42. They regulate several key cancer-relevant pathways like cell division and movement. PAK1 and PAK3 are highly expressed in the brain; PAK1 is frequently overexpressed and/or over-activated in several human cancers whereas PAK3 is involved in neural differentiation and the developmental proneural pathway. The role of these two kinases in brain tumor pathophysiology is unknown. We have observed that PAK3 expression was associated with a longer survival for patients with glioma and was higher in 1p/19q gliomas. In vitro, PAK3 was highly expressed in a glioma cell line with a proneural signature that did not induce tumor after xenograft. Its increasing expression upon a set of differentiation paradigms was correlated with those of neural and/or neuronal markers in glioma cell lines. Inhibition of PAK3 expression increased cell renewal and tumorigenicity. It impaired cell differentiation, promoting the glial lineage.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer predisposition in which loss of heterozygosity at the NF2 gene locus leads to the development of tumors of neural crest derived origin, most commonly bilateral vestibular schwannomas. There are currently no FDA approved chemotherapeutic agents for treatment in patients with NF2. Development of therapeutic agents has been hampered by our incomplete knowledge of how Merlin, the protein product of the NF2 gene, functions as a tumor suppressor. In order develop a deeper understanding for how loss of Merlin leads to oncogenic transformation in Schwann cells we have developed a genetically engineered mouse model (GEMM) of Neurofibromatosis Type 2 in which functional expression of Merlin is lost in Schwann cell precursors. In parallel studies utilizing these mice, we have sought to understand the pathophysiology driving tumor formation in Merlin deficient Schwann cells. In Chapter 1, we explore the role of Merlin as a negative regulator of the Group A p21 activated kinases, PAK1 and PAK2. We demonstrate that PAK1, a previously well established oncogene in solid tumors and Merlin binding partner, is hyperactivated in Merlin deficient schwannomas. Through therapeutic interventions and genetic manipulations we demonstrate that inhibition of PAK1 was capable of reducing tumor formation and alleviating sensorineural hearing loss in our NF2 GEMM. In Chapter 2, we investigate the role of NF-kB inducing kinase (NIK) and NF-kB signaling in the formation and growth of Merlin deficient Schwann cell tumors. Prior work in our lab as well as by others demonstrated elevated NF-kB signaling in Merlin deficient Schwann cell tumors. We observed accumulation of a catalytically active fragment of NF-kB inducing kinase and present data that accumulation of a 55Kd constitutively active fragment of NIK is sufficient trigger wild type Schwann cells to form tumors. In vivo however, Schwann cell intrinsic expression of NIK is not required for tumor formation or growth. / 2 years (2021-05-24)

5 mutations responsables de retard mental ont été identifiées dans le gène p21-activated kinase 3 (pak3). Nous avons récemment identifiés dans pak3 deux exons alternatifs très conservés appelés b et c. Ainsi, en plus du variants PAK3a (dépourvu des inserts b ou c), le gène pak3 code pour 3 nouveaux variants d'épissage PAK3b, PAK3c et PAK3cb qui sont constitutivement actifs et insensibles aux GTPases. De plus, contrairement à PAK1 et PAK3a, leur domaine d'auto-inhibition est incapable d'inhiber un domaine kinase. Ainsi, le but de ce projet était de comprendre le mécanisme de régulation de la kinase PAK3. Un modèle de régulation a récemment été proposé dans lequel PAK1 forme des homodimères pouvant être dissociés par les GTPases, permettant ainsi l'activation de la kinase. En se basant sur ces observations j'ai cherché à identifier les dimères PAK3 et j'ai montré que les kinases PAK3a, b, c et cb forment préférentiellement des hétérodimères avec PAK1. J'ai démontré l'existence de ces dimères dans le cerveau et j'ai mis en évidence que ces hétérodimères permettent à chaque monomère de réguler l'activité kinase de son partenaire in vitro. Ce travail permet de proposer un modèle de régulation symétrique pour PAK3a qui forme des hétérodimères avec PAK1 et un nouveau modèle de régulation asymétrique pour les variants d'épissage, également basé sur leur hétérodimérisation avec PAK1. Mes résultats montrant une corégulation des kinases PAK neuronales suggèrent d'une part que leur activation puisse être synchronisée et d'autre part que dans certaines situations physiopathologiques (Cancer et maladies neurologiques) leur dérèglement puissent interférer.

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No description available.

<p>Glomerulosclerosis (GS) is the irreversible scarring of glomerular tissue which underlies the development of chronic kidney disease (CKD). Increased intraglomerular capillary pressure (P_gc) is a major contributor to the development of GS and can occur in both hypertensive and diabetic patients. With elevated P_gc, <em>in vitro</em> and <em>in vivo</em> evidence suggest that mesangial cells (MC) experience cyclic stretch and secrete pro-fibrotic factors such as connective tissue growth factor (CTGF) which contributes to GS. The signaling pathways that are activated in response to elevated P_gc and lead to extracellular matrix (ECM) production in MCs are the main focus of this thesis.</p> <p>Previous data demonstrated activation of the Rho GTPase, Rac1, with cyclic stretch in MCs. Furthermore, the most characterized effector of Rac1, p21-activated kinase (PAK), has been observed to have a role in endothelial cells (ECs) exposed to mechanical stress. We thus proposed that the Rac1-PAK signaling pathway is involved in mechanical stress signaling in MCs.</p> <p>Our data demonstrate that Rac1-PAK signaling was activated in response to cyclic stretch and required for stretch-induced CTGF production in MCs. RhoA activation was also regulated by Rac1-PAK signaling, and RhoA/ROCK were observed to mediate CTGF upregulation with stretch. Further investigation on the role of Rac1-PAK signaling and how it regulates CTGF in MCs exposed to stretch, will provide insight into potential therapeutic targets to delay the progression of hypertension-mediated CKD.</p> / Master of Science in Medical Sciences (MSMS)

The c-Myc transcription factor is a key player in cell homeostasis, being commonly deregulated in human carcinogenesis. In this PhD thesis we have addressed the question how regulatory mechanisms restrain the oncogenic activity of c-Myc and its impact on cell differentiation. In the first half, we report that PML promotes destabilization of c-Myc protein and re-activation of c-Myc-repressed target genes. The consequent re-expression of the cell cycle inhibitor CDKN1A/p21 mediates differentiation of leukemic cells. In the second half of the thesis we identified a novel mechanism of gene regulation by c-Myc, which is mediated through its interaction with DNA-bound RARα. In undifferentiated cells, c-Myc/Max dimers cooperate with RARα in the repression of genes required for differentiation. Upon phosphorylation of c-Myc by the previously identified Pak2, the complex switches from a repressive to an activating function by releasing Max and recruiting transcriptional coactivators. These findings add a new and at least partially Max-independent mechanism for transcriptional regulation by c-Myc and also discover an unexpected function of c-Myc in inhibiting and promoting cellular differentiation. Taken together, our results describe two new mechanisms that counteract the oncogenic activity of c-Myc. Both PML and Pak2 can be considered as tumor suppressors since they modulate c-Myc function in a way that ultimately promotes differentiation of leukemic cells. This knowledge provides the basis for novel approaches to be exploited for the development of c-Myc-targeted therapies. / El factor de transcripció c-Myc juga un paper clau en l’homeòstasi cel·lular, essent freqüentment desregulat en la carcinogènesi humana. En aquesta tesi s’ha estudiat com diferents mecanismes reguladors poden frenar l’activitat oncogènica de c-Myc i el subsegüent impacte en la diferenciació cel·lular. A la primera meitat de la tesi, es demostra que PML promou la desestabilització de la proteïna c-Myc i, en conseqüència, la reactivació dels genes diana reprimits per c-Myc. Entre aquests gens diana es troba l’inhibidor del cicle cel·lular CDKN1A/p21, la reexpressió del qual provoca la diferenciació de cèl·lules leucèmiques induïda per PML. En la segona meitat, s’identifica un nou mecanisme de regulació transcripcional per part de c-Myc a través de la interacció amb RARα, el qual està unit a l’ADN. En cèl·lules indiferenciades, els dimers c-Myc/Max cooperen amb RARα en la repressió de gens essencials per a la diferenciació. Un cop c-Myc és fosforil·lat per la kinasa Pak2, el complex de c-Myc amb RARα esdevé activador mitjançant la pèrdua de Max i el reclutament de coactivadors transcripcionals. Aquest descobriment suposa un nou mecanisme mitjançant el qual c-Myc pot exercicir la regulació gènica almenys en part independentment de Max, i també revela una funció desconeguda de c-Myc en la inhibició i promoció de la diferenciació cel·lular. En conjunt, aquests resultats descriuen dos nous mecanismes que contrarestren l’activitat oncogènica de c-Myc. PML i Pak2 poden ser considerats supressors de tumors ja que modulen la funció de c-Myc per a promoure la diferenciació de les cèl·lules leucèmiques. Aquests descobriments poden utilitzar-se com a base pel desenvolupament de noves teràpies anti-tumorals que tinguin com a diana la proteïna c-Myc.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder that affects roughly 1 in 3500 individuals. In addition to physical features (e.g., neurofibromas), developmental disorders are also common that can affect cognition, learning, attention and social function. The NF1 gene encodes neurofibromin, a GTPase activating protein (GAP)-like protein that negatively regulates Ras GTPase activation. Mutation at the NF1 locus increases the output of MAPK and PI3K signal transduction from the cellular membrane to the nucleus. Similar to humans, Nf1+/- mice show spatial learning abnormalities that are potentially correlated with increases in GABA-mediated inhibition and deficits in long-term potentiation in the hippocampus. Here, we demonstrate for the first time that Nf1+/- mice exhibit a selective loss of long-term social learning / memory and increased GABAergic inhibition in the basolateral amygdala, a critical brain region for regulating social behaviors. Next, utilizing a genetic intercross, we show that the co-deletion of p21-activated kinase type 1 (Pak1-/-), which positively regulates MAPK activation, restores Nf1+/--dependent MAPK hyperactivation in neurons cultured from the frontal cortex. We found that the co-deletion of Pak1 in Nf1+/- mice (Nf1+/- / Pak1-/-) also restores the deficits in long-term social learning / memory seen in Nf1+/- mice and normalizes the increases in GABA-mediated inhibition in the BLA, as compared to Nf1+/- mice. Together, these findings establish a role for Nf1 and Pak1 genes in the regulation of social learning in Nf1-deficient mice. Furthermore, proteomic studies identify dysregulation of F-actin and microtubule dynamics in the prefrontal cortex, and implicate proteins associated with vesicular release as well as neurite formation and outgrowth (e.g., LSAMP, STXBP1, DREB). In the BLA, disintegrin and metalloproteinase domain-containing protein 22 (ADAM22) was identified, and ADAM22 may play a role in the regulation of AMPA receptors. Finally, due to the increased co-occurrence of NF1 and autism, these findings may also have important implications for the pathology and treatment of NF1-related social deficits and some forms of autism.

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