MOB (Mps One Binder) is a conserved gene family found in all major kingdoms. The MOB genes are essential components acting in mitotic exit and cytokinesis in both budding and fission yeasts. They are further identified as tumor suppressors in Drosophila (D.) melanogaster. Recently, they are found to be involved in the emerging Drosophila Hippo-LATS tumor suppressor pathway. Seven human homologs of yeast MOB (hMOB1A, 1B, 2A, 2B, 3, 4, 5) have been discovered. The hMOB1B is the gene that has been extensively studied and is reported to be required for the activation of LATS (Large Tumor Suppressor)/NDR (Nuclear Dbf2-related) protein kinase family, however, the functional significance of the gene remains unknown. This study is the first to elucidate the biological and biochemical functions of all seven human MOBs. By examining hMOB mRNA expression in various human tissues, we found that the hMOBs have exhibited different expression patterns. We also investigated the subcellular localization of hMOBs during interphase through immunofluorescent analysis. While hMOB2A is localized in the cytoplasm, hMOB4 is exclusively found in the nucleus. All of the other hMOBs are localized in both cytoplasm and nucleus. Furthermore, we identified hMOB1A and hMOB1B as the main binding partners of LATS and NDR in vitro. Additionally, we successfully identified a region on hMOB1B for the interaction with LATS or NDR and determined the crucial residue that is responsible for the binding of LATS2 with hMOB1B. Most significantly, we found that over-expression of hMOB1B in human cancer cells inhibits cell proliferation and induces cell death. Moreover, hMOB1B when targeted to the plasma membrane dramatically enhances the phenotype. Conversely, small interfering (si) RNA-mediated suppression of either endogenous hMOB1A or hMOB1B causes increased cell proliferation, whereas suppression of both hMOB1A and hMOB1B demonstrates a more significant enhancement in tumor cell growth. Moreover, co-expression of both LATS and hMOB1B targeted to the plasma membrane completely abolishes cell proliferation. Our findings provide convincing evidence that hMOB1A and hMOB1B function as negative regulators of cell proliferation and as pro-apoptotic proteins. Understanding hMOBs functions in the cell and their possible role in tumorigenesis can provide important information for the diagnosis and treatment of human cancers. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-23 14:18:43.896

Living apart together (LATs) refers to individuals (couples) who are in intimate relationships and do not share the same household. Over the last two decades, LATs have become a new emerging family form in Western societies. Previous research on LATs is generally limited to small-scale surveys and qualitative studies. There are virtually no national studies on this topic using representative data. In this study, I document the incidence and trends of LATs, and provide a national profile of the individual-level characteristics that are associated with those who live in LAT relationships, as well as examine how people from a LAT union differ from those who are married, cohabiting or single. Data from the Canadian General Social Survey (Cycle 25), conducted by Statistics Canada in 2011 is used. The regression results show that LATs are not an alternative to co-residential relationships nor to singlehood. The rate of LATs is associated with age, presence of children, income, main activity, education, place of residence, religiosity and religious affiliation. The implications of these finding are discussed in the context of future families. / Graduate

The Hippo pathway is frequently deregulated in human cancers, but mutations and deletions of core signalome members are rare, suggesting that our understanding of its upstream regulators remains incomplete. A focused RNAi-based kinome screen identified novel candidate regulators of Hippo signaling, including STK25 and MST4. Here, we characterize the kinase STK25 as a novel upstream activator of LATS signaling. Depletion of STK25 was found to significantly reduce YAP phosphorylation in response to Hippo-activating stimuli, with consequent increases in YAP/TAZ activity and increased proliferation and resistance to cell cycle arrest. Mechanistically, STK25 activates LATS independently of the canonical MST/MAP4K axis, wherein STK25 directly promotes the phosphorylation of the LATS kinase activation loop in the absence of a preceding hydrophobic motif phosphorylation event. This differentiates STK25 from all other identified Hippo kinases to date, which may explain why singular loss of this kinase cannot be compensated for by the presence of other Hippo kinases. We also find that loss of STK25 increases YAP/TAZ signaling in vivo and that this promotes organ overgrowth in murine models. Interestingly, STK25 is frequently focally deleted in a spectrum of human cancers, suggesting that its loss might represent a way by which cancer cells functionally inactivate Hippo signaling. We also report that STK25 may be a novel regulator of mTOR signaling, as loss of STK25 hyper-activates mTOR signaling in response to amino acids and growth factors, but not to energy stresses. Interestingly, we find that MST4, a kinase closely related to STK25, appears to have highly context-specific Hippo regulatory functions; loss of MST4 was found to modulate Hippo signaling only in non-polarized cell lines, suggesting that polarity-responsive subcellular localization of MST4 may dictate its ability to interact with Hippo signaling. Lastly, we describe a novel role for Hippo signaling as a surveillance system for abnormal prolongation of mitosis, in which the LATS kinases regulate cell fate following abnormal mitosis via its control over p53-p21 signaling and YAP/TAZ signaling. This work thus identifies new roles and mechanisms by which kinases interact in the context of this major tumor suppressor pathway to control cellular processes critical to physiologic homeostasis. / 2021-10-07T00:00:00Z

Los cítricos, al igual que muchas otras especies vegetales, poseen tres sistemas de transporte para la absorción del ión nitrato, separados genéticamente, y que actúan y se regulan en función de diferentes factores para hacer frente a las demandas nutricionales de la planta. Existen dos sistemas de transporte de alta afinidad, uno constitutivo (cHATS) y otro inducible (iHATS) que operan a bajas concentraciones externas de nitrato y que siguen una cinética de Michaelis-Menten. El sistema de transporte de baja afinidad (LATS) opera a altas concentraciones externas de nitrato y sigue una cinética lineal. Los parámetros cinéticos de estos sistemas son más bajos que los de las especies herbáceas estudiadas, lo que podría indicar una menor capacidad de estos sistemas. Ambos necesitan energía metabólica en forma de ATP para poder realizar la absorción del ión nitrato. En lo referente al efecto de la salinidad, en todos los casos el ión cloruro reduce la absorción del ión nitrato, pero de forma diferente si se trata del HATS o el LATS. En el HATS se da una inhibición competitiva entre ambos iones, mientras que para el LATS, es necesario que se acumule cloruro en los tejidos, para que se de la reducción de la absorción del ión nitrato, por un mecanismo no conocido, independiente de la reducción de la transpiración y en el que podría estara implicada la enzima nitrato reductasa.

The Hippo-LATS signaling pathway originally identified in Drosophila is conserved in mammalian systems and serves essential roles in mediating size control as well as tumorigenesis. In humans, the core kinase cassette consisting of adaptor proteins WW45 and MOB1, and Ser/Thr kinases MST1/2 and LATS1/2 signal by phosphorylating and inactivating transcriptional co-activators YAP and TAZ, causing cell growth arrest. As the central kinases within the Hippo-LATS pathway, examining the cellular and molecular phenotypes of LATS1 and LATS2 (LATS) will provide insight into the role of this pathway in tumorigenesis. By simultaneously knocking down both LATS1 and LATS2, genes that were differentially expressed were identified through a whole human genome microarray screen. The multitude of genes identified including CYR61, MYLK, CDKN1A, SLIT2, and TP53INP1 not only provide further evidence for the role of LATS in cell proliferation and apoptosis, but also implicate LATS in novel functions such as cell motility. Loss of LATS1 and/or LATS2 enhances cell migration whereas overexpression of LATS1 dramatically inhibits cell migration in multiple cell lines. The ability of LATS to regulate cell migration occurs through two potential mechanisms. Firstly, LATS functions through its kinase substrates YAP and/or TAZ, or alternatively, LATS1 directly binds actin and inhibits actin polyermization. Thus, through loss of functions studies, we identified a novel role for LATS in regulating cell migration as well as novel mechanisms of LATS function. As an important signaling molecule within the cell, LATS and the Hippo-LATS pathway are tightly regulated. Using clues from the Drosophila pathway, we examined how the previously uncharacterized gene, hEx, functions within this pathway. Importantly, this thesis characterizes hEx as a putative tumor suppressor showing that it can inhibit cell proliferation, sensitize cancer cells to Taxol treatment as well as inhibit tumor growth in nude mice. However, unlike Drosophila expanded, hEx functions independently of the Hippo-LATS pathway, suggesting that the mammalian signaling pathway is more complicated. The research findings from this thesis enhance our knowledge of the Hippo-LATS pathway in tumorigenesis by elucidating new functions and mechanisms of LATS functions as well as by exploring how upstream components function in relation to this pathway. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-09-23 10:06:54.687

The Hippo signalling pathway is an evolutionarily conserved kinase cascade responsible for the cell proliferation, tissue growth and apoptosis during development and its dysregulation contributes to tumourigenesis. This signalling pathway was initially discovered in Drosophila and soon after that, it was shown to be highly conserved in mammals. The core Lats kinases of this tumour suppressive pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Latest reports revealed various E3 ubiquitin ligases to negatively regulate the Hippo pathway through ubiquitination, yet few deubiquitinating enzymes have been described. In the present study, we report USP9X deubiquitinating enzyme as an essential regulator of the central components of this pathway. USP9X interacted strongly with Lats2 kinase and to a lesser extent with WW45, Kibra and Angiomotin family proteins. The knockdown of USP9X resulted in notable downregulation and destabilisation of Lats kinase and to lesser extents WW45, Kibra and Amot. This resulted in enhanced nuclear localisation of YAP and TAZ accompanied with activation of their target genes, CTGF and CYR61. USP9X was shown to stabilise Hippo components through its deubiquitinating activity. USP9X enzyme defective mutant lost the activity to stabilise Lats2, WW45, Kibra and Angiomotins through deubiquitination, leading to their ubiquitination. In the absence of USP9X, cells exhibited epithelial to mesenchymal transition phenotype and additionally gained anchorage-independent growth in soft agar. Moreover, USP9X knockdown disrupted acinar organisation of breast cells in three-dimensional acini cultures. In addition, YAP/TAZ target gene activation in USP9X knockdown cells could be rescued by knockdown of YAP, TAZ and TEAD2. Lastly, USP9X protein expression showed a positive correlation with Lats kinases, but negative correlation with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. The results strongly indicate that USP9X cooperates with Lats2 and other important Hippo components to suppress tumour growth.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

Hippo signaling is a tumor suppressive signaling pathway that controls organ size by regulating cellular proliferation, apoptosis, and differentiation during development, regeneration, and homeostasis. The Hippo pathway inhibits transcriptional co-activators and Hippo pathway effectors YAP/TAZ, activation of which is often seen in cancer. Within the adult mammalian intestine, homeostasis of which requires intricate reciprocal interaction between the gut epithelium and adjacent mesenchyme, the Hippo-YAP pathway is crucial for intestinal epithelial homeostasis and regeneration. However, its role in adult mesenchymal homeostasis remains poorly understood. Here, I genetically dissect the role of mesenchymal Hippo-YAP signaling in adult intestinal homeostasis. I find that deletion of core kinases LATS1/2 or YAP activation in mesenchymal progenitor cells, but not terminally differentiated cells, disrupts signaling in the stem cell niche and mesenchymal homeostasis by inducing mesenchymal overgrowth and suppressing smooth muscle actin expression. Furthermore, inhibition of Hippo signaling in Gli1+ mesenchymal progenitors, the main source of Wnt ligands within the stem cell niche, stimulates Wnt ligand production and subsequent epithelial Wnt pathway activation, thereby driving epithelial regeneration following DSS-mediated injury as well as exacerbating APC-mediated tumorigenesis. Altogether, our data reveal a previously underappreciated requirement and the underlying mechanism for stromal Hippo-YAP signaling in adult intestinal homeostasis.

Mechanical tension is an important regulator of cell proliferation, differentiation, migration and cell death. It is involved in the control of tissue architecture and wound repair and its improper sensing can contribute to cancer. The Hippo tumor suppressor pathway was recently shown to be involved in regulating cell proliferation in response to mechanical tension. The core of the pathway consists of the kinases MST1/2 and LATS1/2, which regulate the target of the pathway, the transcription co-activator YAP/ TAZ (hereafter referred to as YAP). When the Hippo pathway is inactive, YAP remains in the nucleus and promotes cell proliferation and stem cell maintenance. When the Hippo signaling pathway is turned on, MST1/2 phosphorylate and activates LATS1/2. LATS1/2 phosphorylates and inactivates YAP in the cytoplasm which is sequestered and degraded, stopping cell proliferation and promoting differentiation of stem cells. Mechanical forces are transmitted across cells and tissues through the cell-cell junctions and the actin cytoskeleton. However, the factors that connect cell-cell junctions to the Hippo signaling pathway were not clearly known. We identified a LIM domain protein called TRIP6 that functions at the adherens junctions to regulate the Hippo signaling pathway in a tension-dependent manner. TRIP6 responds to mechanical tension at adherens junctions and regulates LATS1/2 activity. Under high mechanical tension, TRIP6 sequesters and inhibits LATS1/2 at adherens junctions to promote YAP activity. Conditions that reduce tension at adherens junctions by inhibition of actin stress fibers or disruption of cell-cell junctions reduce TRIP6-LATS1/2 binding, which activates LATS1/2 to inhibit YAP. Vinculin has been shown to act as part of a mechanosensory complex at adherens junctions. We show that vinculin promotes TRIP6 inhibition of LATS1/2 in response to mechanical tension. Furthermore, we show that TRIP6 competitively inhibits MOB1 (a known LATS1/2 activator) from binding and activating LATS1/2. Together these findings reveal TRIP6 responds to mechanical signals at adherens junctions to regulate the Hippo signaling pathway in mammalian cells.