Protein phosphorylation constitutes an important mechanism for cellular regulation in both Eucarya and Bacteria. All living organisms evolved from a common progenitor; this implies that protein phosphorylation as a means of regulation also exists in Archaea. Previously, in the sulfur-dependent archaeon Sulfolobus solfataricus a gene was cloned encoding a protein-serine/threonine phosphatase that was similar to eucaryal protein-serine/threonine phosphatases type 1, 2A, and 2B. To identify protein phosphatases in other archaeons, oligonucleotides encoding conserved regions of eucaryal protein-serine/threonine phosphatases were used in the polymerase chain reaction to amplify genomic DNA from the methanogenic archaeon Methanosarcina thermophila. From the PCR reaction a fragment of DNA was isolated that encoded a portion of a protein phosphatase. Using this DNA fragment as a probe, the entire phosphatase gene was isolated. The amino acid sequence of the phosphatase encoded by this gene displayed greater than 30% identity with eucaryal protein-serine/threonine phosphatase type 1. The gene encoding the Methanosarcina phosphatase was expressed in Escherichia coli. The expressed protein exhibited protein serine phosphatase activity that was sensitive to inhibitors of eucaryal phosphatases such as okadaic acid, microcystin, calyculin, and tautomycin. In order to identify potential endogenous substrates of archaeal protein-serine/threonine phosphatases and kinases, a study was initiated to characterize the most prominent phosphoproteins in S. solfataricus. Cell extracts were incubated with [γ-³²P] ATP, MgCl₂, and MnCl₂, and the proteins in the extracts were separated by SDS-PAGE. Autoradiography of the gels revealed four prominent phosphoproteins with apparent molecular masses of 35, 46, and 50 kDa. N-terminal sequence analysis and enzymatic assays of the 35 kDa phosphoprotein identified this phosphoprotein as the a-subunit of succinyl-CoA synthetase. N-terminal sequence analysis and enzymatic assays revealed that the 50 kDa phosphoprotein was a hexosephosphate mutase. Neither the 50 kDa nor the 35 kDa phosphoprotein appeared to be the target of protein kinases or phosphatases. Therefore, while protein-serine phosphatases exist in Archaea, the targets of these phosphatases have yet to be determined. / Ph. D.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Background: Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. Methods and results: We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1(-/-)mice and Pim1(-/-)2(-/-)3(-/-) triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 (-/-) and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit S-35 methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Conclusions: Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

In order to generate healthy daughter cells, nuclear division and cytokinesis need to be coordinated. Premature division of the cytoplasm in the absence of chromosome segregation or nuclear proliferation without cytokinesis might lead to aneuploidy and cancer. The cyclin dependent kinases, CDKs, are a main regulator of the cell cycle. Timely increase and decrease in their activity is required for cell cycle progression. To enter mitosis, mitotic CDK activity needs to rise. CDK activity stays elevated until chromosome segregation is completed and exit from mitosis requires decrease in CDK activity. Observations in several experimental systems suggest that coordination of cytokinesis with the nuclear cycle is regulated through CDK activity. Prolonged high CDK activity, as it occurs when chromosome segregation is delayed, was found to oppose cytokinesis. Prevention of cytokinesis through high CDK activity may therefore provide a mechanism to prevent precocious cell division in the absence of chromosome segregation. To prevent polyploidy when cell division is delayed, progression through the next nuclear cycle should be inhibited until cytokinesis is completed, presumably by the inhibition of CDK activity. In the fission yeast Schizosaccharomyces pombe, a signaling cascade called Septation Initiation Network (SIN) is required for the coordination of cytokinesis with the nuclear cycle. The SIN is essential for cytokinesis, triggering the execution of cell division through constriction of the actomyosin ring. The activation of the SIN signaling cascade, and thus cytokinesis, is opposed by high CDK activity, preventing precocious cytokinesis. S. pombe delay entry into the next nuclear division in response to delayed cytokinesis due to defects in the contractile ring until cytokinesis is completed thereby preventing the accumulation of multinucleate, non viable cells. This safeguard against multinucleate cells is termed the cytokinesis checkpoint. The cytokinesis checkpoint keeps CDK activity low, preventing nuclear cycle progression. The SIN is required for the cytokinesis checkpoint and therefore is a key coordinator between nuclear cycle and cytokinesis. How the SIN functions in the cytokinesis checkpoint was not known. Cdc14-family phosphatases are highly conserved from yeast to humans, but were only characterized in Saccharomyces cerevisiae at the time this thesis was initiated. Cdc14 had been identified as the effector of a signaling cascade homologous to the SIN, called the mitotic exit network (MEN), which is required for exit from mitosis. This thesis describes the identification of the S. pombe Cdc14-like phosphatase Clp1p as a component of the cytokinesis checkpoint. Clp1p opposes CDK activity, and Clp1p and the SIN activate each other in a positive feedback loop. This maintains an active cytokinesis checkpoint and delays mitotic entry. We further found that Clp1p regulates chromosome segregation. Concluding, this thesis describes discoveries adding to the characterization of the cytokinesis checkpoint and the function of Clp1p. While others found that Cdc14-family phosphatases, including Clp1p, have similar catalytic functions, we show that their biological function may be quite different between organisms, possibly due to different biological challenges.

A bactéria Listeria monocytogenes é reconhecida como um importante patógeno humano estando amplamente distribuída no ambiente e é responsável pela contaminação de alimentos crus e processados. O mecanismo de patogenicidade é determinado pela presença de genes no cromossomo da bactéria e entre eles estão os genes iap e hly que são essenciais para o mecanismo de invasão e atividade hemolítica do microorganismo, respectivamente. O obejtivo do presente estudo foi confirmar as cepas de L. monocytogenes usando a ténica de PCR para o gene hly e análise da variação nucleotídica do domínio central do gene iap, que é caracterizado pela presença de seqüências repetidas dos aminoácidos treonina e asparagina. Vinte e seis cepas de L. mnocytogenes, previamente isoladas de produtos lácteos e identificdas por métodos clássicos, se mostraram positivas para a PCR espécie-específico e então submetidas à determinação da seqüência nucleotídica. Os resultados mostraram variações na seqüência nucleotídica contendo substituições, inserções, deleções e um número de seqüências similares entre as cepas isoladas e a cepa controle EGD-e. Vinte e três cepas exibiram a mesma deleção que compreende 24 pares de bases dentro da seqüência de repetição e alterações similares na proteína traduzida. Apenas três cepas mostraram tamanhos diferentes de deleções e diferentes alterações na proteína. De acordo com estes resultados, a maioria das cepas apresentou uma característica molecular comum, diferentes da cepa padrão e este perfil predominante pode ser considerado como a característica das L. monocytogenes isoladas de produtos lácteos no Sul do Brasil. / The bacterium Listeria monocytogenes is recognized as an important human pathogen being omnipresent in the environment and is responsible for contamination in raw and processed foods. The mechanism of pathogenicity is established by presence of some genes on chromosome of bacteria between them, iap and hly genes that are essential to the invasion mechanism and hemolytic activity of microorganism, respectively. The aim of present study was confirm the strain L. monocytogenes using PCR to hly gene and analysis the nucleotide variability of central domain of iap gene characterized by the presence of similar sequences of threonine and asparagine amino acids. Twenty-six strains previously isolated from dairy products and classified by classic methods to L. monocytogenes were positive to specie-specific PCR and than submitted to nucleotide sequence determination. The results showed a sequence variation containing nucleotide substitutions, insertions, deletions and a number of repeated sequences among the isolates and control strain EGD-e. Twenty-three strains exhibited the same gap that includes a deletion of 24 base pairs inside of the repeated sequence and similar alterations in the translated protein. Just three strains showed different sizes of gaps and different protein alterations. According to these results, the majority strains displayed a common molecular characteristic different from the strain pattern and this predominant profile can be considerate as characteristic to L. monocytogenes isolated from dairy products in South Brazil.

<p>Integrins are transmembrane glycoproteins primarily mediating interactions of cells with the extracellular matrix. Each receptor is a complex of one α- and one β-subunit with affinity for a diverse set of ligands. A prerequisite for ligand binding, and subsequent events, is the activation of integrins by cytoplasmic signals that confer a large conformational change to the extracellular domain.</p><p>In this thesis, the role of a cytoplasmic threonine-cluster, conserved in several β subunits, in β1-integrin activation was investigated. Phosphorylation of these residues is postulated to regulate β2 and β3 integrin affinity for ligands, but it has not been shown so far to occur for β1. Residue T788, but not T789, was established as a site of critical importance for inside-out activation of β1 integrins by mutagenesis to alanine. In contrast to β1-T788A, a phospho-mimicking mutation, β1-T788D, expressed the conformation sensitive 9EG7-epitope and mediated normal cell adhesion. In addition, the T788D mutation did not interfere with binding of the talin head domain, an interaction important for integrin activation. Thus, phosphorylation of T788 in integrin β1 was concluded to be compatible with inside-out receptor activation, in line with β2 and β3 integrin regulation. </p><p>Focal adhesion kinase (FAK) is activated after integrin ligation and is, together with Src, one of the central players in integrin-mediated events. Phosphatidylinositol 3-kinase (PI3K) is thought to be activated by binding to FAK. However, a novel, major β1-integrin signalling pathway to activate PI3K was identified, which is FAK- and Src-independent.</p><p>Growth factor induced stimulation of extracellular signal-regulated kinase (Erk) is largely dependent on signals from integrin mediated adhesion to pass checkpoints downstream of Ras. The mechanisms by which β1-integrins mediate Erk-activation were characterized by pin-pointing what phosphorylation sites on the mitogen-activated protein (MAP) kinases and their effector proteins were FAK-dependent. The results indicated that β1 integrins can promote Erk activation by FAK-dependent mechanisms at the levels of both cRaf and Mek, and in addition, a FAK-independent checkpoint at the level of Mek activation.</p>

Integrins are transmembrane glycoproteins primarily mediating interactions of cells with the extracellular matrix. Each receptor is a complex of one α- and one β-subunit with affinity for a diverse set of ligands. A prerequisite for ligand binding, and subsequent events, is the activation of integrins by cytoplasmic signals that confer a large conformational change to the extracellular domain. In this thesis, the role of a cytoplasmic threonine-cluster, conserved in several β subunits, in β1-integrin activation was investigated. Phosphorylation of these residues is postulated to regulate β2 and β3 integrin affinity for ligands, but it has not been shown so far to occur for β1. Residue T788, but not T789, was established as a site of critical importance for inside-out activation of β1 integrins by mutagenesis to alanine. In contrast to β1-T788A, a phospho-mimicking mutation, β1-T788D, expressed the conformation sensitive 9EG7-epitope and mediated normal cell adhesion. In addition, the T788D mutation did not interfere with binding of the talin head domain, an interaction important for integrin activation. Thus, phosphorylation of T788 in integrin β1 was concluded to be compatible with inside-out receptor activation, in line with β2 and β3 integrin regulation. Focal adhesion kinase (FAK) is activated after integrin ligation and is, together with Src, one of the central players in integrin-mediated events. Phosphatidylinositol 3-kinase (PI3K) is thought to be activated by binding to FAK. However, a novel, major β1-integrin signalling pathway to activate PI3K was identified, which is FAK- and Src-independent. Growth factor induced stimulation of extracellular signal-regulated kinase (Erk) is largely dependent on signals from integrin mediated adhesion to pass checkpoints downstream of Ras. The mechanisms by which β1-integrins mediate Erk-activation were characterized by pin-pointing what phosphorylation sites on the mitogen-activated protein (MAP) kinases and their effector proteins were FAK-dependent. The results indicated that β1 integrins can promote Erk activation by FAK-dependent mechanisms at the levels of both cRaf and Mek, and in addition, a FAK-independent checkpoint at the level of Mek activation.

P68 RNA helicase is a RNA helicase and an ATPase belonging to the DEAD-box family. It is important for the growth of normal cells, and is implicated in diverse functions ranging from pre-mRNA splicing, transcriptional activation to cell proliferation, and early organ development. The protein is documented to be phosphorylated at several amino-acid residues. It was previously demonstrated in several cancer cell-lines that p68 gets phosphorylated at threonine residues during treatments with TNF-α and TRAIL. In this study, the role of threonine phosphorylation of p68 under the treatment of anti-cancer drug, oxaliplatin in the colon cancer cells is characterized. Oxaliplatin treatment activates p38 MAP-kinase, which subsequently phosphorylates p68 at T564 and/or T446. P68 phosphorylation, at least partially, influences the role of the drug on apoptosis induction. This study shows an important mechanism of action of the anti-cancer drug which could be used for improving cancer treatment. This study also shows that p68 is an important transcriptional regulator regulating transcription of the cytoskeletal gene TPPP/p25. Previous analyses revealed that p68 RNA helicase could regulate expression of genes responsible for controlling stability and dynamics of different cytoskeletons. P68 is found to regulate TPPP/p25 gene transcription by associating with the TPPP/p25 gene promoter. Expression of TPPP/p25 plays an important role in cellular differentiation while the involvement of p68 in the regulation of TPPP/p25 expression is an important event for neurite outgrowth. Loss of TPPP expression contributes to the development and progression of gliomas. Thus, our studies further enhance our understanding of the multiple cellular functions of p68 and its regulation of the cellular processes.

Spinal cord injury (SCI) results in a well-orchestrated inflammatory response which causes secondary tissue damage. Activated macrophages contribute to this cytotoxic response, which includes damage to neurons, glia and myelin, and tissue loss that worsens functional outcomes after SCI. However, activated macrophages in the spinal cord under other conditions are not cytotoxic, such as after intraspinal injection of lysophosphatidylcholine (LPC), a potent demyelinating agent. Recovery from SCI may be optimized by reducing the detrimental effects of macrophages while promoting their beneficial ones. Therefore, I compared spinal cord tissue, as well as purified macrophages, from mice after SCI (cytotoxic response) and intraspinal LPC injection (non-cytotoxic response). As a first step to carry out this work, I characterized the injury parameters for SCI contusion injury (i.e. injury force and spinal cord displacement) in mice using the Infinite Horizons impactor (Chapter 2). This lesioning model was used in other work for the thesis. The role T cells may play in mediating macrophage activation after LPC microinjection and SCI was also assessed using Nude mice (Chapter 3). Next, Affymetrix GeneChip analysis was carried out on spinal cord tissue obtained at the peak of the macrophage response after SCI and intraspinal LPC injection to identify potential candidate genes that may control the divergent inflammatory responses (Chapter 4). Several potential genes were identified. I next characterized the expression and role of one of these genes, MAPK activated protein kinase 2 (MK2), and showed that it mediates secondary tissue damage after SCI via several mechanisms (Chapter 5). The differences in gene expression profiles of macrophages purified from the spinal cord after SCI and LPC-injection were also assessed (Chapter 6). This microarray analysis of macrophages led to the identification of 10 novel candidate genes, two of which were validated at the protein level. Finally, I also examined the expression and role of secretory leukocyte protease inhibitor (SLPI) in SCI (Chapter 7). Using a combination of knockout/overexpressing transgenic mice and recombinant SLPI, I found that SLPI mediates protective anti-inflammatory effects after SCI. In conclusion, work done for this thesis has led to the identification of several novel molecules that influence the inflammatory response after injury and thus have led to the identification of potentially novel targets for the development of pharmacological approaches to treat acute SCI.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.