Centrosome is the major microtubule organizing center in mammalian cells. The centrosome plays key roles in the formation of the mitotic spindle, cell polarity and cell locomotion. In a typical somatic cell, the centrosome is composed of a pair of centrioles that are surrounded by a mass of amorphous pericentriolar material(PCM). The centrosomal-associated protein, hNinein, has been identified as a microtubules minus end capping, centrioles position, centrosome maturation and anchoring protein. Recently data reveal ninein CCII domain is associated with centrosomal targeting signal, regulating signal and phosphorylation sites, suggesting that this protein could contribute to centrosomal targeting signal as well as regulate asymmetry centrosomes. In this report, we therefore examine whether four C-terminal of hNinein splicing isoforms, including isoform 1, isoform 2, isoform 5 and a newly finding isoform 6, represent differential characterization, expression and functional aspects. To investigate the expression level of hNinein isoform 6 in variant cancer cell lines and astrocytoma patients. Isoform 6 only exist in IMR32 cancer cell line ( neuroblastoma cell line) and 42¢Mastrocytoma patients. We speculate that isoform 6 might play a role and exist in nerve cell or neuron. Using immunofluorescence assay, we show that all three hNinein isoforms are concentrated at centrosomes in HeLa cells, but not isoform 6. Overexpression of isoform 6 in HeLa cell might influence the distribution of

The TSG101 protein has been implicated in multiple biological functions including the regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. Previous reports indicated the steady-state level of TSG101 must be maintained in a narrow range. Either deprivation or overexpression of TSG101 protein could result in neoplastic transformation. However, cellular signals that control TSG101 functions are not clear. TSG101 protein contains many kinase phosphorylation sites including two GSK-3£] phosphorylation sites, S-172 (S172-P-Y-P-S176 ) and S202 (S202-Q-Y-P-S206). Our previous in vitro kinase assay result indicated TSG101 could be phosphorylated by GSK-3£]. In the present study, we demonstrated that 47 kDa TSG101 is monoubiquitination of 42 kDa TSG101. The GSK-3£] inhibitors, LiCl and TDZD8 could decrease TSG101 level in both HeLa and HEp-2 cells. On the contrary, activation of GSK-3£] by serum starvation or by transfection of a plasmid encodes for constitutive active GSK-3£] led to the increase of TSG101 level in a dose-dependent manner. The effect of LiCl and TDZD8 could be blocked by MG132, implying the involvement of proteaosome mediated mechanism. Expression of constitutive active GSK-3bmutS9A led to a dose-dependent increases of wildtype and HA-TSG101mutS172176A, but decrease of HA-TSG101mutS202206A protein. In addition, either wildtype or mutant HA-TSG101 could complex with GFP-GSK-3b. The mutation of S202 GSK-3b phosphorylation site of TSG101 compromised its ability to for complex with GSK-3b. In summary, our data support that GSK-3b is an important cellular signaling in regulation of monoubiquitinated TSG101 steady-state level. Whether it also affects the subcellular localization of TSG101 awaits further investigation.

Glycogen Synthase Kinase-3 (GSK-3) is a highly conserved serine/threonine kinase comprised of two mammalian homologues, GSK-3α and β, encoded by independent genes. This thesis reports the characterization of GSK-3-null primary mouse embryonic fibroblasts (MEFs) generated by gene targeting to gain insight into the physiological functions of this protein kinase. Combined inactivation of both alleles of GSK-3α and GSK-β led to elevated sensitivity to TNFα-induced apoptosis, altered organization of focal adhesion complexes, defects in cell spreading on fibronectin, decreased cell growth associated with altered cell cycle progression through the G2/M phase and increased spontaneous apoptosis. Future work will focus on unraveling the molecular mechanisms responsible for these effects and identifying the common and distinct cellular roles for GSK-3α and β, and specific variants of these isoforms.

Glycogen Synthase Kinase-3 (GSK-3) is a highly conserved serine/threonine kinase comprised of two mammalian homologues, GSK-3α and β, encoded by independent genes. This thesis reports the characterization of GSK-3-null primary mouse embryonic fibroblasts (MEFs) generated by gene targeting to gain insight into the physiological functions of this protein kinase. Combined inactivation of both alleles of GSK-3α and GSK-β led to elevated sensitivity to TNFα-induced apoptosis, altered organization of focal adhesion complexes, defects in cell spreading on fibronectin, decreased cell growth associated with altered cell cycle progression through the G2/M phase and increased spontaneous apoptosis. Future work will focus on unraveling the molecular mechanisms responsible for these effects and identifying the common and distinct cellular roles for GSK-3α and β, and specific variants of these isoforms.

No description available.

No description available.

Abstract¡G Tumor susceptibility gene 101, TSG101, is a protein exhibits multiple biological functions. For most protein, its specific function or structure stability can be regulated through protein phosphorylation or modification. The analysis of the amino acid sequence of TSG101 revealed that it has two GSK-3£] phosphorylation concensus sequences. Our previous data of in vitro kinase assay have demonstrated that TSG101 can be phosphorylated by GSK-3£], a wellknown protein kinase that regulates the stability and function of it¡¦s target protein. To investigate the effect of GSK-3£] phosphorylation on the stability and the function of TSG101 protein, we first exploited the effect of GSK-3£]inhibitor, LiCl, on endogenous TSG101 protein in COS1 cells. The results suggested that inhibition of GSK-3£] phosphorylation could impact on the stability of TSG101 protein. Upon the transfection of an active form GSK-3£] expression plasmid GSK-3£]/pEGFP, additional protein products of 40, 50-80 kD were detected, suggesting that GSK-3£] phosphorylation might induce modification or degradation of TSG101 protein. GSK-3£] phosphorylation site mutant TSG101 protein expression plasmids were constructed using site-directed mutagenesis, and were transfected into COS1 cells to evaluate the effect of GSK-3£] on TSG101 level. The results showed that GSK-3£] phosphorylation site mutant TSG101 protein is more stable then wild type TSG101 due to the lack of GSK-3£] phosphorylation site. The inhibition of GSK-3£] activity by LiCl treatment resulted in the increase of wildtype as well as the S172AS176 and S172AS176AS202AS206A mutant TSG101 proteins, whereas the S202AS206A mutant TSG101 protein level was not affected by LiCl treatment. The above data indicated that GSK-3£] might regulate the stability and biological activity of TSG101 protein through phosphorylation of serine residue at position 202, which is worthy of further investigation.

Glioblastoma multiforme (GBM) is the most malignant form of brain cancer. Therapies targeting glioblastoma have not consistently been able to give those diagnosed the best prognosis. Treatments that directly infiltrate into the tumor are highly sought after. Indirubins have been used to treat various types of cancers and are a promising avenue for future glioma research. In the current study, we further researched several key GSK-3 inhibitors, BIO (an indirubin) and CHIR99021, in addition to LiCl, to see their effects on the translocation of β-catenin to the nucleus, and the invasion and migration of cells in both a sphere assay and an aortic ring assay. Here we studied anti-invasive therapies that may have a future role in GBM treatment. It is thought that combining conventional treatments with anti-invasive therapies will create cytotoxicity in and reduce migration of the tumor. Three types of cells were used throughout the experiments: HBMEC, HUVEC, and U251 glioma cells. We reported that GSK-3 inhibitors might have a valuable role in the treatment of GBM. The selected inhibitors (BIO, CHIR99021, and LiCl) all were shown to lessen cell migration and invasion in vitro in a range of assays and in all cell lines tested. All inhibitors tested cause a dose-dependent, reversible inhibition of glioma cell invasion in spheroid assays. BIO was shown to cause a rapid upregulation of total and nuclear β-catenin. BIO, at higher concentrations, also created a toxic environment for cells, sometimes killing them. This shows that a more in-depth experiment involving different BIO concentrations is needed to test the optimal concentration for treatment. Each of the experimented GSK-3 inhibitors also showed a change in the junctions between cells. NaCl as a control showed normal, spikey, junctions, while CHIR99021 and BIO caused the junctions to become more smooth. This suggests that GSK-3 inhibition has a role in either maintaining the ECM and/or in communication between cells. Also in this assay, there was a heterogeneity between cells treated with the same inhibitor and in the same dish, indicating that not all cells respond to each drug the same way. The reasons for this are not known and further investigation is required. A new construct was also made to report β-catenin transcriptional coactivation using luciferase expression as the reporter in response to these selected GSK-3 inhibitors.With the combined results of these experiments, we concluded that GSK-3 inhibitors may be a promising approach to the treatment of GBM. Further investigation is required before any treatments can be administered to those diagnosed.

Glioblastoma is the most malignant form of brain cancer. Due to its aggressive nature, extensive research has been performed, but little progress has been made in identifying effective treatment options. Glycogen synthase kinase-3 (GSK-3) is a ubiquitous, multifaceted protein kinase. Previous studies have shown that small molecule inhibitors of GSK-3 block the migration of glioblastoma cells and may prevent spread of tumor in the brain. However, these studies were performed using non-selective GSK-3 inhibitors (LiCl and an indirubin derivative, BIO); thus, it was unclear whether GSK-3 was the most important target. In this study, we used recently generated highly selective GSK-3 inhibitors (CHIR99021, AZD1080, and AZD2858, as well as BIO) to investigate these questions. These were applied to four glioblastoma cell lines: G30, G9, U251, and U1242, in three migration assays: transwell, spheroid, and wound healing (scratch) assay to further assess the suitability of GSK-3 as a target in glioblastoma. We also utilized the ATP Luciferase reporter assay for cell viability to assess the influence of our panel of drugs on cell migration versus viability. In addition, the TOPFlash Luciferase reporter assay was performed as an indicator of the level of GSK-3 inhibition. The TOPFlash assay showed that all GSK-3 inhibitors were able to increase luciferase levels. This indicates that GSK-3 was inhibited in our cells after drug treatment. The transwell assays showed us that the GSK-3 inhibitors were able to block migration significantly in all cell lines tested in a dose-dependent manner. The effectiveness of GSK-3 inhibition in the three-dimensional collagen spheroid assays was cell line-dependent, with the non-selective GSK-3 inhibitor BIO showing the most potent effects. Cell migration was not blocked by any of the three selective GSK-3 inhibitors in the wound healing scratch assay. Thus we have found that the three distinct highly selective inhibitors of GSK-3 block glioblastoma cell migration, but only work consistently in the transwell assay. Therefore, we conclude that GSK-3 might be important in the contraction and morphological changes necessary for glioblastoma cells to migrate through the 8 micron pores in the transwell. Further investigation into this observation is necessary. Though results were variable between assays, we conclude that the inhibition of GSK-3 is a promising potential therapeutic strategy for glioblastoma treatment.

La protéine proapoptotique Bax joue un rôle fondamental au cours de la voie intrinsèque de l’apoptose. Elle participe au déclenchement de la mort en permettant la libération de facteurs apoptogéniques mitochondriaux vers le cytosol. Un des points-clé de la fonction de Bax est son activation, caractérisée par la transition entre une forme cytosolique, globulaire et inactive de la protéine et une conformation mitochondriale, membranaire et active. Les différentes étapes de l’activation de Bax sont relativement bien connues, toutefois un grand nombre de questions reste en suspens quant-à leur régulation.Ce travail s’est focalisé sur la régulation de l’activation de Bax par les kinases Akt et GSK-3β ainsi que par la protéine antiapoptotique Bcl-xL . Ces régulations ont été caractérisées en exprimant la protéine Bax humaine chez la levure Saccharomyces cerevisiae, un paradigme d’étude simplifié qui permet d’accéder aux composantes individuelles des mécanismes d’activation de Bax.Les données obtenues suggèrent qu’il existe deux étapes régulées indépendamment au cours de l’activation de Bax. Nous avons montré que la protéine kinase GSK-3β favorise l’adressage de Bax vers la mitochondrie mais qu’elle n’entraîne pas un changement de conformation suffisant à son activation complète et à la perméabilisation de la membrane mitochondriale externe. Des changements de conformations complémentaires de Bax sont requis pour conduire à une forme capable d’entraîner la libération des facteurs apoptogéniques mitochondriaux. La protéine kinase Akt est impliquée dans le contrôle de Bax via la phosphorylation de la sérine 184 et participe à l’inhibition de l’apoptose. Nous avons mis en évidence qu’une mutation phosphomimétique de la sérine 184 ou l’expression d’Akt, en l’absence de partenaires antiapoptotiques, stimulent un changement de conformation de Bax vers une forme active. Akt semble donc plus jouer un rôle sur la conformation de Bax qu’entraîner une inhibition directe. La présence de protéines antiapoptotiques serait ainsi requise pour l’inhibition de Bax en présence d’Akt.D’autre part, nous nous sommmes intéressés aux mécanismes d’action de la protéine antiapoptotique Bcl-xL . Nous avons déterminé que Bcl-xL pouvait favoriser l’adressage de Bax vers la membrane mitochondriale tout en exerçant un rôle antiapoptotique. Ceci suggère que Bcl-xL intervienne dans le contrôle des étapes tardives de l’activation de Bax. Ce contrôle est dépendant d’une interaction stable entre les deux protéines. Inversement, un variant de Bcl-xL n’interagissant que de façon transitoire avec Bax (Bcl-xL ∆C) entraîne l’activation de Bax. Cette observation est en faveur d’un modèle d’activation indirecte de Bax consécutive à la rupture de l’interaction avec Bcl-xL et dans lequel les protéines à BH3-seulement telles que Bad joueraient un rôle crucial. / Proapoptotic protein Bax plays a major role during apoptosis intrinsic pathway. Bax promotes cell death by inducing the release of apoptogenic factors from mitochondria to cytosol. Bax activation is a key step of its function which involves a change from a globular, cytosolic and inactive conformation to an active mitochondrial, membrane inserted conformation. Bax activation substeps are rather well known, however their regulation remains to be characterized.This work focuses on the study of the regulation of Bax activation by kinases Akt and GSK-3β and by antiapoptotic protein Bcl-xL . Human Bax regulations have been studied by expressing the protein in yeast Saccharomyces cerevisiae which represents a simplified paradigm for the understanding of the individual components of Bax activation mecha- nisms.Our data suggest that there are two independently regulated steps during Bax activation. We showed that GSK-3β expression led to Bax addressing to mitochondria but was not sufficent to promote a complete activation and mitochondrial outer membrane premeabilization. Further conformational changes are required to promote Bax full activation and the release of mitochondrial apoptotic factors. Protein kinase Akt is involved in Bax activation control through the phosphorylation of serine 184 and contributes to apoptosis inhibition. We observed that either a phosphomimetic mutation of serine 184 or coexpression of Akt, in the absence of antiapoptotic partners, were responsible of Bax conformational change into an active form. By itself Akt did not inhibit Bax but appeared more likely to control its conformational change. Thus, implication of antiapoptotic proteins seems to be critical in a model of Bax inhibition by Akt.Furthermore, we tried to understand the molecular mechanisms of antiapoptotic protein Bcl-xL inhibition on Bax. We determined that Bcl-xL could increase Bax mitochondrial localization while leading to its inhibition suggesting that Bcl-xL controled Bax late activation steps. Bax inhibition was dependent on a stable interaction with Bcl-xL . Conversely, a variant of Bcl-xL having a transitory interaction with Bax (Bcl-xL ∆C) was able to promote Bax activation. This supports a model of Bax indirect activation following the rupture of interaction with Bcl-xL in which BH3-only proteins like Bad would play an important role.