Transforming growth factor-£] (TGF-£])-activated kinase 1(TAK1), a member of the MAPKKK family, was identified as a protein kinase that involved in c-Jun N-terminal kinase(JNK)/p38 MAPKs and NF-£eB signaling pathways. This kinase also participated in inflammatory and innate immune responses as well as the aggressiveness in several types of cancer. Association with its specific activator protein, TAK1 binding protein1 (TAB1), triggers TAK1 kinase activity. Recent evidences suggest that the interaction between chemokines and their cognate receptors involved in cancer metastasis. Our previous results demonstrated that CC chemokine receptor 7 (CCR7), the receptor for the two major chemokines CCL19 and CCL21, is up-regulated in breast cancer cells and may play an important role in the trafficking and homing of breast cancer cells to lymph nodes. In addition, CCR7 expression is positively correlated with lymphatic metastasis in the primary tumors of patients with breast cancer. Here, we show that TAK1 is constitutively activated in MDA-MB-231 breast cancer cells and suppression of TAK1 signaling by using TAK1 shRNA or TAB1 shRNA significantly reduces expression of CCR7 in these cells. In addition, the TAK1 inhibitor 5Z-7-oxozeaenol, a resorcylic lactone of fungal origin, also inhibited TAK1 kinase activity and CCR7 expression. Promoter deletion assay identified a responsive element localized between -500/-223 bp from transcription start site is critical for TAK1 to activate CCR7 transcription. Furthermore, Chromatin immunoprecipitation assay(ChIP assay)identified two transcription factors, c-Jun and NF£eB participate in TAK1 regulating CCR7 expression. In addition, our study in breast tumor tissues indicated that CCR7 was significantly associated with p-TAK1 expression as found in breast cancer cell lines.

The TAK1 and canonical IKK complexes are the two master protein kinases of the innate immune system that control the production of inflammatory mediators, but the mechanisms by which they are activated in this system are still unclear. In this thesis, I present the research I have carried out to solve these problems. The IKKb component of the canonical IKK complex is required to activate the transcription factors NF-kB and IRF5 and the protein kinase Tpl2, but how IKKβ itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more ‘upstream’ protein kinases in some reports, but by autophosphorylation in others. In the first part of this thesis, I describe my work that has resolved this controversy by demonstrating that the activation of IKKb induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 catalysed phosphorylation of Ser177 and, secondly, the IKKb-catalysed autophosphorylation of Ser181. The phosphorylation of Ser177 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser181, which enables IKKk to phosphorylate exogenous substrates. I also present genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of Met1-linked/linear ubiquitin (Met1-Ub) chains and their interaction with the NEMO (NF-kB essential modulator) component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKb at Ser177 and IKKa at Ser176. These findings may be of general significance for the activation of other protein kinases. The activation of the TAK1 complex by inflammatory stimuli is thought to be triggered by the binding of Lys63-linked ubiquitin chains to the TAB2 or TAB3 components of the TAB1-TAK1-TAB2 and TAB1-TAK1-TAB3 complexes. In the second part of the thesis I tested whether this broadly accepted model was correct by knocking out the genes encoding TAK1 and its regulatory subunits TAB1, TAB2 and TAB3 by CRISPR/Cas9 gene-editing technology, alone and in combination, in an IL-1 receptor expressing human cell line. These genetic studies led me to discover that the IL-1-dependent activation of TAK1 occurs by two different mechanisms. The first, involves the previously described interaction of Lys63-linked ubiquitin chains with TAB2 and TAB3, while the second can take place in the complete absence of TAB2 and TAB3. The second mechanism, which involves activation of the TAB1-TAK1 heterodimer is more transient than the first, but is sufficient for the IL-1-dependent transcription of immediate early genes (A20, IkBa). I show that the activation of the TAB1-TAK1 complex requires the expression of the E3 ubiquitin ligase TRAF6 and the TRAF6-generated formation of Lys63-linked ubiquitin chains, which leads to the phosphorylation of TAK1 at Thr187 and activation. However, neither TAB1 nor TAK1 bind directly to Lys63-linked ubiquitin chains. I identify one novel IL-1-dependent phosphorylation site on TAB1 and two on TAK1 and propose that Lys63-linked ubiquitin chains activate an as yet unidentified protein kinase, which phosphorylates one or more of the novel phosphorylation sites on the TAB1-TAK1 heterodimer inducing a conformational change that permits TAK1 to autophosphorylate Thr187.

Les neutrophiles sont d'une grande importance dans la première ligne de défense de l'organisme contre les pathogènes. Ils participent activement par leurs actions antimicrobiennes, comme la phagocytose et la relâche de granules, mais influencent également la réponse immunitaire par les différentes cytokines et chimiokines qu'ils produisent. L'étude des différentes fonctions du neutrophile a permis d'établir les étapes clés de la signalisation intracellulaire qui mène à ces différentes fonctions. De plus, les études dé signalisation, dans différents organismes, ont placé TAK1, une MAP3K, à l'avant-plan dans l'activation des sentiers MAP kinase et des facteurs de transcription NF-kB. Nos efforts pour élucider les sentiers métaboliques du neutrophile nous ont fait nous pencher sur le rôle que TAK1 pouvait y jouer. Nous avons donc découvert que TAK1 était la kinase d'importance dans le contrôle des fonctions du neutrophile avec le LPS et le TNF[alpha], deux stimuli activateurs de NF-kB. Dans cette étude, nous nous sommes penchés sur le rôle de TAK1 chez le neutrophile avec des stimuli dont les réponses cellulaires ne passent pas par l'activation de NF-kB. C'est dans cette perspective que nous avons utilisé un chimioattractant, le fMLP, et un facteur de croissance, le GM-CSF. Ce sont deux stimuli physiologiques fréquemment retrouvés aux sites inflammatoires. Le fMLP et le GM-CSF activent rapidement TAK1 et celle-ci se retrouve en amont de la voie MEK/ERK, mais pas des voies p38 MAPK et PI3K/AKT. L'inhibition de TAK1 diminue l'expression et la sécrétion d'IL-8 et d'IL-1RA. L'inhibition de MEK/ERK et de PI3K/AKT a le même effet. De plus, l'inhibition de TAK1 empêche l'effet antiapoptotique du GM-CSF ainsi que diminue la production de leucotriènes par le fMLP. En conclusion, les travaux présentés montrent que TAK1 est une MAP3K essentielle dans les réponses fonctionnelles du neutrophile au fMLP et au GM-CSF. Cette découverte ouvre la porte à de nouvelles cibles thérapeutiques, particulièrement dans le cas de maladies chroniques impliquant le GM-CSF.

Indiana University-Purdue University Indianapolis (IUPUI) / Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) plays important roles in EBV-mediated B cell transformation, development of EBV-associated malignancies, and exacerbation of certain autoimmune conditions. LMP1 functionally mimics tumor necrosis factor receptor (TNFR) superfamily member CD40, but LMP1 signals are amplified and sustained compared to those induced by CD40. CD40 and LMP1 rely on TNFR-associated factors (TRAFs) to mediate signaling, but use TRAFs differently. TRAF6 is important for CD40 signaling, and was implicated in LMP1 signaling in non-B cells. Here, we addressed the hypothesis that TRAF6 is a critical regulator of a subset of LMP1 signals in B cells. We found that TRAF6 was required for LMP1-mediated kinase activation and costimulatory molecule upregulation, and associated with the LMP1 TRAF1/2/3/5 binding site (TBS). Additionally, TRAF6 and the TBS contributed to LMP1-induced autoreactivity and antibody (Ab) production in vivo. Finally, in contrast to CD40, LMP1 required the TRAF6 receptor-binding domain to mediate TRAF6-dependent pathways. Thus, TRAF6 is critical for LMP1 signaling and requires LMP1 interaction to propagate signals. Importantly, TRAF6 associates with LMP1 in a manner distinct from CD40, raising the possibility of disrupting LMP1 functions while leaving normal CD40 signaling intact. We next investigated roles of the kinase TAK1 in TRAF6-dependent LMP1 functions. TAK1 was required for CD40- and LMP1-mediated JNK activation in B cells, leading to IL-6 and Ab production. Understanding mechanisms of CD40 and LMP1 signaling provides important insights into normal regulatory control of CD40 functions and how LMP1-mediated pathogenesis escapes or subverts these regulatory mechanisms. LMP1 itself may be a difficult therapeutic target, because it lacks an extracellular domain and is continually processed from the cell surface. Thus, it is important to elucidate similarities and differences between CD40 and LMP1 signals to identify therapeutic targets to block LMP1-mediated pathogenesis. Comparing and contrasting CD40 and LMP1 also increases our understanding of the critical mechanisms used to regulate normal CD40 signaling.

Prostate cancer is the most common invasive cancer diagnosed in men and a major and growing health problem in Western countries. Deregulation of different pathways has been implicated in progression of prostate cancer, namely nuclear factor kappa enhancer binding protein (NF-κB), transforming growth factor β (TGFβ), phosphoinositide 3ʹ-kinase/AKT (PI3K/AKT) and Src kinase pathways. However, the detailed mechanisms by which TGFβ activates these pathways to contribute in tumorigenesis and invasive behavior of prostate cancer cells have not been elucidated. We have demonstrated (paper I) that the E3 ligase activity of TRAF6 is crucial for recruitment of the regulatory subunit of PI3K, p85α, to TβRI and for TGFβ-induced Lys63-linked polyubiquitination of p85α. TRAF6 is required for the TGFβ-induced recruitment of AKT to the complex of PI3K and TβRI, where the polyubiquitination and activation of AKT occurs. When activated, AKT promotes TGFβ-induced cell migration which is dependent on p85 and PI3K activity, as well as on TRAF6, but not on TβRI kinase activity. Thus, TGFβ-induced activation of PI3K/AKT induces cell motility contributing to the progression of cancer. We have demonstrated (paper II) a pivotal role of TAK1 polyubiquitination in three different pathways, including TNFR, IL-1R, and TLR4 signaling. Lys63-linked polyubiquitination of TAK1 at Lys34 is essential for downstream signaling to NF-κB-mediated target gene expression in both cancer and immune cells. These findings are of importance for the understanding of the mechanism of activation of NF-κB in inflammation and may aid in the development of new therapeutic strategies to treat chronic inflammation and cancer. We have also shown (paper III) that TGFβ activates the tyrosine kinase Src via formation of a complex between TβRI and Src. The E3 ligase TRAF6 promotes the formation of the complex in a manner not dependent on its ubiquitin ligase activity, suggesting that TRAF6 acts as an adaptor. Moreover, the activation of Src is not dependent on the kinase activity of TβRI. On a functional level, Src activity was found to be necessary for TGFβ-induced chemotaxis. In conclusion, we have elucidated molecular mechanisms whereby TGFβ activates non-Smad pathways, i.e. PI3K and Src. Our findings shed light on the pro-tumorigenesis mechanisms of TGFβ. In addition, we have demonstrated how the activation of TAK1, an important component of the TGFβ non-Smad pathway, by TGFβ and other stimuli leads to the activation of NF-κB and thereby induction of inflammation which likely contributes to prostate cancer progression.

No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

En premier lieu, en utilisant des inhibiteurs pharmacologiques du NF-[kappa]B, nous avons constaté que l'inhibition du facteur de transcription NF-[kappa]B chez ces cellules diminue de beaucoup l'expression génique et la sécrétion de diverses cytokines et chimiokines (TNF-[alpha], IL-8 ou CXCL8, Mip-1[alpha]/[bêta] induites par des stimuli tels que TNF-[alpha] ou LPS. Nous montrons ensuite que le complexe IKK (IKK[alpha], IKK[bêta], et IKK[gamma]) est aussi partiellement localisé dans le noyau, alors que les kinases reliées à IKK (IKK[epsilon] et TBK-1) sont cytoplasmiques; la kinase NIK, quant à elle, est strictement nucléaire. Suite à une activation des neutrophiles, IKK[bêta] et IKK[gamma] deviennent transitoirement phosphorylées dans le cytoplasme et le noyau, alors qu'IKK[alpha] disparaît temporairement de ces deux compartiments cellulaires d'une manière qui semble dépendante de IKK[bêta]. Ces réponses s'accompagnent, dans les deux compartiments, de la dégradation d'I[kappa]B[alpha] et de la phosphorylation du RelA sur la sérine 536. Bien que les deux protéines puissent être des substrats de IKK, l'inhibition de ce dernier empêche la dégradation d'I[kappa]B[alpha], tandis que le niveau de phosphorylation du RelA est essentiellement inchangé. Nous apportons enfin une preuve que des isoformes de IKK nucléaires s'associent à la chromatine suivant l'activation des neutrophiles, ce qui suggère un rôle potentiel dans la régulation de gènes. Deuxièmement, nous rapportons que les neutrophiles expriment la MAP3K, TAK1, ainsi que ses partenaires associés, TAB1/2, dans le cytoplasme et le noyau. La kinase TAK1 est associée de façon constitutive aux protéines TAB1 et TAB2, ainsi qu'au complexe IKK[alpha]/[bêta] dans les neutrophiles au repos. Le niveau d'interaction de ces complexes demeure inchangé suite au traitement des neutrophiles avec le TNF-[alpha] ou le LPS. La kinase TAK1 devient rapidement et transitoirement activée suite à une stimulation des cellules avec le TNF-[alpha] ou le LPS. L'inhibition de l'activité kinase de TAK1 avec un inhibiteur hautement sélectif (5z-7-oxozeaenol) a empêché la phosphorylation d'IKK[alpha]/[bêta], de RelA, et la dégradation de I[kappa]B[alpha] dans les fractions cytoplasmiques et nucléaires, ainsi que la liaison à l'ADN du NF-[kappa]B dans des neutrophiles activés.En conséquence, l'expression et la sécrétion de cytokines inflammatoires induites par le TNF-[alpha] ou le LPS ont été profondément altérées suivant une inhibition de TAK1.En revanche, la phosphorylation de IKK[gamma] induite par le LPS n'a pas été affectée par l'inhibition de TAK1. Finalement, nos résultats indiquent que l'activation du NF-[kappa]B et les réponses cellulaires dépendantes du NF-[kappa]B sont indépendantes des ROS endogènes dans les neutrophiles humains primaires ou dans la lignée promyélocytaire PLB-985, qui peut être différenciée en granulocytes et se comporte comme les neutrophiles. Parallèlement, nous avons optimisé les conditions de transfection des PLB-985 différenciées, ce qui nous a permis de montrer pour la première fois l'activation de promoteurs [kappa]B-dépendants chez des granulocytes humains. Ces travaux rendent par ailleurs possibles les études portant sur l'activation des promoteurs chez les granulocytes. Dans leur ensemble, ces observations démontrent l'importance du NF-[kappa]B dans la génération inductible de cytokines et chimiokines par les neutrophiles. Il s'agit de la première étude qui montre la présence et l'activation (phosphorylation) du complexe IKK et la phosphorylation des protéines NF-[kappa]B/Rel dans les neutrophiles humains. Plus important encore, nos résultats dévoilent un mode d'activation de la cascade de signalisation IKK/I[kappa]B/NF-[kappa]B dans le noyau de cellules primaires. Nos données établissent également le rôle central de TAK1 dans le contrôle de la cascade de signalisation IKK/I[kappa]B/NF-[kappa]B cytoplasmique et nucléaire dans les neutrophiles primaires humains, ce qui pourrait représenter une cible prometteuse pour une intervention thérapeutique considérant le rôle critique des neutrophiles dans plusieurs conditions inflammatoires.

<p>Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells [3, 4]. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.</p>