DNA mismatch repair (MMR) is a critical genome-maintenance system. It ensures genome stability by correcting mismatches generated during DNA replication, suppressing homologous recombination, and inducing apoptosis in response to severe DNA damage. As a result, defects in MMR lead to genome-wide mutations and susceptibility to both hereditary and sporadic cancer syndromes. The hallmark of cancer cells defective in MMR is their ability to display frequent instability in simple repetitive DNA sequences, a phenomenon called microsatellite instability (MSI). However, only ~70% of the MSI-positive tumors have identifiable MMR gene mutations, indicating that additional factor(s) are responsible for the MSI phenotype in the remaining 30% MSI-tumors. We demonstrate here that phosphorylation of proliferating cell nuclear antigen (PCNA), an MMR component required for the initiation and resynthesis steps of the repair reactions, blocks in vitro MMR. We found that nuclear extracts derived from colorectal cell lines containing high levels of phosphorylated PCNA are not only defective in MMR, but also inhibitory to MMR activity in HeLa extracts. To determine if PCNA phosphorylation inhibits MMR, several PCNA isoforms that mimic phosphorylated or non-phosphorylated PCNA were examined for their effects on MMR activity. We show that all phosphorylated PCNA mimics block MMR at the initiation step but MMR was not affected by the non-phosphorylated mimetic PCNA. In vitro gap-filling experiments reveal that the phosphorylated PCNA induces a mutational frequency several fold higher than non-phosphorylated PCNA. Since PCNA has been shown to interact with MMR initiation factors MutSα and MutLα, we examined the interactions of phosphorylated PCNA with these two initiation factors. Interestingly, PCNA phosphorylation reduces the PCNA-MutSα interaction, but not the PCNA-MutLα interaction. Since PCNA is proposed to transfer MutSα to the mismatch site, the simplest explanation of the result is that PCNA phosphorylation inhibits MMR by blocking MutSα-mismatch binding activity. Taken together, our results reveal that PCNA phosphorylation induces genetic instability by inhibiting MMR at the initiation step and by promoting DNA polymerase-catalyzed mis-incorporations. This study provides a novel mechanism by which posttranslational modifications inhibit MMR, leading to genome instability and tumorigenesis. A second part of the study is to determine MMR function of several MutLα mutants associated with relapse leukemia patients. One of the mutants contains a phenylalanine99 to leucine substitution in the MLH1 subunit of MutLα. We show that this mutation inhibits MMR by blocking both the ATPase activity and the endonuclease activity associated with MutLα, supporting the importance of the MutLα ATPase and the endonuclease activities in MMR.

Tip growth, the mechanism by which hyphae, pollen tubes, root hairs, and algal rhizoids extend, is a complex and dynamic process that is characterised by localised extension at the extreme apex of the cell and morphological polarity. Its complexity suggests that high degree of regulation is needed to ensure that the characteristics of a particular cell type are maintained during growth. Regulation is likely to come about through bidirectional interplay between the cell wall and cytoplasm, although the mechanisms by which such cross-talk might occur are unknown. Results of this thesis present immunocytochemical data that indicate the presence of, and a close association between β4 integrin subunit-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall-membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the possible action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. In addition, results show A. bisexualis contains proteins epitopically similar to the rod domain of animal talin. However, these proteins do not co-localise with F-actin, and mainly locate at the sub-apical region in hyphae. For comparative purposes, Saccharomyces cerevisiae was also used to investigate the presence of β4 integrin subunit-like proteins and tyrosine phosphorylation. Immunoblotting showed that S. cereviaise contains a protein, which is found in the microsomal pellet fraction, that cross reacts with anti-β4 integrin subunit antibody. Furthermore, there are a number of proteins containing phosphotyrosine residues. Immunocytochemistry shows that this anti-β4 integrin staining is at the cortical site but anti-phosphotyrosine residues are distributed throughout cells. On the basis of an ELISA and a coupled enzyme assay, it is suggested that a soluble fraction of S. cerevisiae contains tyrosine kinase activity. This activity is strongly inhibited by tyrphostins.

No description available.

No description available.

Palmitoylation is a post-translational modification that has been implicated in the control of multiple proteins, including ion channels. S-Palmitoylation is a lipophilic modification that involves the attachment of palmitate through a thioester linkage to a cysteine residue in a target protein. By increasing the hydrophobicity of the target region, palmitoylation can promote membrane targeting. Here, palmitoylation is shown to play an important role in regulating large conductance calcium- and voltage- activated (BK) potassium channels. The STREX splice variant of the BK channel contains a 58 amino acid insert at the splice site C2 within the intracellular C-terminal RCK1-RCK2 linker that confers increased calcium sensitivity to the channel and determines PKA inhibition of channel activity. The cysteine rich STREX domain was predicted to be palmitoylated, and using an imaging assay STREX was shown to act as a membrane targeting domain through palmitoylation of a di-cysteine motif (C645:C645). A membrane potential assay and electrophysiological analysis demonstrates that palmitoylation at the C645:C646 site in STREX is important in mediating the increased calcium sensitive properties inherent to the STREX channel. Palmitoylation is also shown to modulate PKA channel inhibition. The stability of palmitoylation can often be reliant on the local environment within the protein. Generally in most proteins; lipidated regions, basic domains or transmembrane domains are found adjacent to a palmitoylation site. In STREX, a polybasic domain composed of 11 basic residues just upstream from the C645:C646 palmitoylation site, functions to control the palmitoylation status of the STREX insert. A site directed mutagenesis approach to disrupt the polybasic domain revealed an important role in controlling membrane targeting of the STREX C-terminus, mediating the increased calcium sensitivity inherent to STREX channels and controlling the palmitoylation status of the C645:C646 palmitoylation site using multiple techniques involving electrophysiology, fluorescent imaging and biochemical assays. Further to this, using imaging to examine the membrane association of fluorescently tagged C-terminal proteins, phosphorylation is shown to function as a physiological electrostatic switch to regulate the polybasic region in controlling palmitoylation of the STREX insert. Finally, an additional palmitoylation site that is constitutively expressed in all BK channels was identified to be located in the S0-S1 linker (C53:C54:C56). Mutation of the C53:C54:C56 palmitoylation site in the S0-S1 linker was shown to abolish all palmitoylation in BK channels that did not contain the STREX insert. Palmitoylation allows the S0-S1 linker to associate with the plasma membrane however the mutated de-palmitoylated channels did not affect channel conductance or the calcium/voltage sensitivity of the channel. Palmitoylation of the S0-S1 linker was shown to be a critical determinant of cell surface expression of BK channels, as steady state surface expression levels were reduced by ~55% in the C53:C54:C56 mutant. STREX channels that could not be palmitoylated in the S0-S1 linker also showed decreased surface expression even through STREX insert palmitoylation was unaffected. Palmitoylation is rapidly emerging as an important post-translational mechanism to control ion channel behaviour. This work reveals that palmitoylation of the BK channel can control channel function of the STREX splice variant channel and can regulate cell surface expression in all other channel variants. Palmitoylation appears to be functionally independent at these two distinct sites expressed within the same channel protein.

A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, was shown to be a protein of Mr of 60,000 as estimated by SDS-PAGE and could catalyze the phosphorylation of the synthetic peptide S6-21, the histone H4, and myelin basic protein. Mild digestion of the inactive S6/H4 kinase with trypsin was necessary, but not sufficient, to activate the kinase fully

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Dietary phosphate is essential for normal fracture healing and bone growth. Previous studies have established that mice given a phosphate deficient diet after a fracture demonstrate delayed cartilage maturation and callus mineralization, as well as changes in gene expression consistent with oxidative phosphorylation dysfunction. This study was undertaken in order to examine the role of inorganic and organic phosphate availability on chondrocyte differentiation and mineralization, and to define the relationship between these processes and changes in chondrocyte metabolic function. ATDC5 murine chondroprogenitor cell line, which has been shown to undergo in vitro differentiation and extracellular matrix mineralization, was cultured under both differentiating and non-differentiating media conditions under conditions in 1mM -0.25mM sodium phosphate monobasic (inorganic phosphate) in the presence or absence of 4mM β-glycerol phosphate (organic phosphate). In the first series of studies, overall cell growth (total DNA and protein contents), mineralization (calcium accumulation), and cell-normalized oxidative metabolism (basal respiration, maximal respiration, ATP turnover, spare capacity, proton leak, and non-mitochondrial respiration rates) were measured over a 28 day time course in cultures grown in differentiating (ascorbic acid, insulin-transferrin-selenium, and β-glycerol phosphate) conditions in 1mM phosphate. These studies found that when the cells were induced to differentiate, there was a measurable increase in protein content while DNA content decreased by 30%, indicating a fraction of the cells underwent cell death. Differentiation was further associated with an overall two-fold increase in oxidative respiration. Next we assessed how differentiation, the promotion of matrix mineralization, and inorganic phosphate availability affected oxidative respiration. When differentiation was not induced with ascorbic acid and β-glycerol phosphate, there was no over growth in the cultures nor any change in total extracellular matrix mineralization or oxidative respiration. In the absence only of β-glycerol phosphate, differentiation proceeded but matrix mineralization did not occur. However, overall protein content and oxidative respiration were statistically two- and 1.5-fold higher, respectively, independent of the inorganic phosphate contents of the growth media. These results suggest that both differentiation and overall protein accumulation are strongly associated with increased oxidative metabolism while mineralization of the matrix decreased oxidative function. Only at the lowest phosphate levels were changes in basal oxidative function observed. These results are consistent with previous in vivo findings suggesting that diminished expression of mitochondrial associated genes in callus tissues from hypophosphatemic mice were associated with an overall decrease in chondrocyte differentiation.

Orientador: André Sampaio Pupo / Banca: Fernando Maurício Francis Abdalla / Banca: Carlos Allan Cândido Dias Júnior / Banca: Maria de Fátima Magalhães Lázari / Banca: Flávia Heberler-Barbosa Trovão / Resumo: As catecolaminas endógenas adrenalina e noradrenalina controlam importantes funções fisiológicas através da ativação de 1A-ARs, que são receptores com 7 domínios transmembrana os quais ativam proteínas G. Muitos agonistas de 1-ARs são utilizados na terapêutica, principalmente por conta de seu efeito vasoconstritor, porém, a taquifilaxia observada com a oximetazolina, um agonista de 1-ARs da classe das imidazolinas, é um efeito adverso importante e pode ser observado principalmente com a utilização de descongestionantes nasais por períodos de tempo prolongado. No presente estudo, investigamos a participação de PKC e GRK2 nos processos de fosforilação, dessensibilização e internalização de 1A-ARs induzidos pelos agonistas noradrenalina e oximetazolina. Segundo os resultados obtidos, a oximetazolina, mas não a fenilefrina, induziu taquifilaxia nas contrações de artéria caudal de rato. Além disso, 1A-ARs ativados pela oximetazolina são fosforilados principalmente pela GRK2, seguida de rápida dessensibilização e internalização. Já 1A-ARs ativados pela noradrenalina são fosforilados principalmente pela PKC, sem dessensibilização nem rápida internalização. Esses resultados em conjunto demonstram que a oximetazolina e a noradrenalina regulam os 1A-ARs através de diferentes mecanismos, envolvendo diferentes quinases / Abstract: The endogenous cathecolamines epinephrine and norepinephrine control important physiological functions through activation of 1A-ARs, which are 7-transmembrane receptors that activate G proteins. Several 1-ARs agonists are therapeutically useful in reason of its vasoconstrictor effects; however, tachyphylaxis in the vasoconstrictor effects of nasal decongestant containing oxymetazoline, an imidazoline 1-ARs agonist, is an important adverse effect observed after prolonged treatment. On the present study, we investigated the roles of PKC and GRK2 on the 1A-AR phosphorylation, desensitization and internalization process after receptor activation by norepinephrine and oxymetazoline. The results show that oxymetazoline, but not phenylephrine, induce tachyphylaxis in contractions of rat tail artery. Moreover, 1A-ARs activated by oxymetazoline are phosphorylated predominantly by GRK2, followed by rapid desensitization and internalization whereas 1A-ARs activated by norepinephrine are phosphorylated predominantly by PKC and not followed by desensitization or rapid internalization. These results show that norepinephrine and oxymetazoline regulate 1A-ARs through different mechanisms, involving different protein kinases / Doutor

The ability to sense and respond to environmental cues is crucial for the survival of all organisms. This response is often manifested by exerting control at different levels of gene expression, i.e. transcription, translation and post translation levels. Global control of protein synthesis is frequently exercised at the initial step of translation initiation and is generally achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them. The formation of ternary complex (TC) is considered first step of translation initiation and depends on the recycling of inactive eIF2-GDP to active eIF2-GTP form. This nucleotide exchange reaction is catalyzed by the eukaryotic initiation factor-2B (eIF2B). eIF2B is composed of a regulatory sub-complex of alphaβdelta subunits and a catalytic sub-complex of the γε subunits. The guanine nucleotide exchange activity of eIF2B is regulated by phosphorylation of eIF2alpha and additionally in mammalian cells, by direct phosphorylation of eIF2B at multiple sites in ε subunit, where most of the catalytic activity of eIF2B resides. Recent unpublished studies in the Pavitt laboratory identified novel phosphorylation sites by Mass Spectrometry in γ and ε subunits of eIF2B catalytic sub-complex. In order to study the functional significance of these phospho-sites for translation initiation, Site Directed Mutagenesis (SDM) was performed to generate Ser to Ala mutants. All mutations are viable and have no significant growth defect on rich or minimal media; however the significance of these sites in yeast growth became apparent by growing yeast in different stress conditions (e.g. Rapamycin, Torin1, amino acid starvation and 1-butanol). Effects on the phosphorylation pattern at these sites were monitored by using custom generated phospho-specific antibodies. All phosphorylation events appear independent of the eIF2alpha kinase (Gcn2p in yeast). The phosphorylation of ε-S528 depends on the presence of ε-S525. This study finds that addition of rapamycin, Torin1, amino acid starvation and butanol, which each inhibits global translation initiation, alters the phosphorylation pattern at ε-S435, ε-S525 and ε-S528 sites. Linking growth to phosphorylation, it appears that phosphorylation at ε-S435 and ε-S525 is directly proportional to growth. Phosphorylation of ε-S435 is necessary for effect of eIF2alpha-Ser51 phosphorylation on protein synthesis while phosphorylation of ε-S528 seems to be a target of various mechanisms. This study also suggests that eIF2Bε may be a key player of the cell cycle progression and phosphorylation changes can serve as marker for the regulation of eIF2B activity. The kinases responsible for phosphorylation at these sites are not yet known in yeast. Further investigation is required to find the functional significance of alterations in phosphorylation pattern to definitively establish eIF2Bε phosphorylation as a mechanism for regulating eIF2B activity in yeast. Models are presented to account for the results obtained that show how phosphorylation of eIF2Bε at these sites may contribute to the control of protein synthesis.