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1
Lee, Chung-Sheng Brian. "Studies of SpoIIAA, the anti-anti-#sigma#'F factor of Bacillus subtilis." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365810.
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Andersson, C. Evalena. "Structure-Function Studies of Enzymes from Ribose Metabolism." Doctoral thesis, Uppsala University, Department of Cell and Molecular Biology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3999.
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In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors.
This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in Escherichia coli.
Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent Kd measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase.
Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or vice versa. Structural studies on the two genetically distinct isomerases in E. coli have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis. Sequence searches of completed genomes show ribose 5-phosphate isomerase B to be the sole isomerase in many bacteria, although ribose 5-phosphate isomerase A is a nearly universal enzyme. All genomes contain at least one of the two enzymes. These results confirm that both enzymes must be independently capable of supporting ribose metabolism, a fact that had not previously been established.
3
Lee, Gui-in. "Structure and dynamics of the receptor kinase interacting FHA domain of kinase associated protein kinase from arabidopsis." Free to MU campus, others may purchase, 2003. http://wwwlib.umi.com/cr/mo/fullcit?p3100058.
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Ding, Zhaofeng. "Kinase-interacting FHA domain of kinase associated protein phosphatase phosphopeptide interactions and NMR-detected dynamics /." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4729.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on September 24, 2007) Vita. Includes bibliographical references.
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Dehghani, Hesam. "Embryonic alkaline phosphatase and protein kinase C in preimplantation mouse development." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ58304.pdf.
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Gruninger, Robert J., and University of Lethbridge Faculty of Arts and Science. "Structure and mechanism of protein tyrosine phosphatase-like phytases." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry, c2009, 2009. http://hdl.handle.net/10133/2473.
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The structure and mechanism of the Protein Tyrosine Phosphatase-like Phytases (PTPLPs) from Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) were investigated using a combination of enzyme kinetics, site-directed mutagenesis, and X-ray crystallography. I show that PTPLPs use a classical protein tyrosine phosphatase catalytic mechanism and adopt a core PTP fold. Several unique structural features of PTPLPs confer specificity for inositol phosphates. The effect of ionic strength and oxidation on the kinetics and structure of PTPLPs was investigated. The structural consequences of reversible and irreversible oxidation on PTPLPs and PTPs are compared and discussed. We determine the structural basis of substrate specificity in PTPLPs and propose a novel reaction mechanism for the hydrolysis of inositol polyphosphates by PTPLPs. Finally, the structure and function of a unique tandemly repeated phytase has been determined. We show that the active sites of the tandem repeat possess significantly different specificities for inositol polyphosphate.xix, 148 leaves : ill. (some col.) ; 29 cm
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Sloss, Callum. "Control of subcellular distribution of the MAP kinase phosphatase, MKP-2." Thesis, University of Strathclyde, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288715.
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Lam, Hiu-chor, and 林曉初. "Functional characterization of tyrosine phosphatase non-receptor 21, anovel modulator of ErbB4/NRG3." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44229288.
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Davies, Elizabeth Louise. "The role of mitogen-activated protein kinase phosphatase-1 in cardiac hypertrophy." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391714.
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Kodituwakku, Jayanie Subhashi. "Mechanisms regulating mitogen-activated protein kinase phosphatase-2 expression in cardiac myocytes." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429111.
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Wu, Ming. "Averaging and Monotonicity Analysis of Ca2+/Calmodulin-Dependent Protein Kinase-Phosphatase System." Ohio University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1299866038.
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Wancket, Lyn Miller. "Mitogen-activated protein kinase phosphatase (MKP)-1 in immunology, physiology, and disease." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1385390100.
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Lam, Hiu-chor. "Functional characterization of tyrosine phosphatase non-receptor 21, a novel modulator of ErbB4/NRG3." Click to view the E-thesis via HKUTO View the Table of Contents & Abstract, 2010. http://sunzi.lib.hku.hk/hkuto/record/B44229288.
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Keshwani, Malik M. "Human p70 Ribosomal S6 Protein Kinase-1 (S6K1): Mechanism of Substrate Catalysis, Activation and Inhibition." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/213.
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S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). The fully activated catalytic kinase domain construct, His6-S6K1 alphaII(∆AID)-T389E (activity = 250 nmol/min/mg) was generated by baculovirus-mediated expression and purification from Sf9 insect cells that were coinfected with recombinant baculovirus expressing the catalytic kinase domain of PDK1 [His6-PDK1(∆PH)]. The kinetic mechanism of fully active His6-S6K1 alphaII(∆AID)-T389E for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA) was determined. Two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, while pre-steady state kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the enzyme's affinity for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme. We propose that ordered binding of ATP causes partial unfolding of enzyme residues, which unmask the peptide substrate binding epitope. Next, the kinetic mechanism of PDK1 for catalyzing T229 phosphorylation of S6K1 (native and T389E mutant forms) was determined. Surprisingly, we found that His6-PDK1(∆PH) effectively and specifically phosphorylates T229 of His6-S6K1 alphaII(∆AID), regardless of whether a negative charge is localized at residue 389. Steady-state kinetic studies revealed S6K1 alphaII to be a competitive inhibitor of ATP, thereby enforcing an Ordered Bi Bi mechanism whereby ATP must bind first. Kinetic studies further revealed exceptionally slow bimolecular association of S6K1 alphaII substrate to form the productive ternary complex that catalyzes S6K1 alphaII T229 phosphorylation, indicating a high degree of nonproductive binding events. In this regard, the T389E mutant exhibited a two-fold increased efficiency of productive binding over native S6K1 alphaII. Finally, to investigate the regulatory role of C-terminal AID of S6K1, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D2ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation.
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Freeman, Alyson. "CHK2 is Negatively Regulated by Protein Phosphatase 2A." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/3443.
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Checkpoint kinase 2 (CHK2) is an effector kinase of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'α subunit of protein phosphatase 2A (PP2A), a major protein serine/threonine phosphatase of the cell, as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'α binds to the SQ/TQ cluster domain of CHK2, which is a target of ATM phosphorylation. CHK2 is able to bind to many B' subunits as well as the PP2A C subunit, indicating that it can bind to the active PP2A enzyme. The induction of DNA double-strand breaks by ionizing radiation (IR) as well as treatment with doxorubicin causes dissociation of the B'α and CHK2 proteins, however, it does not have an effect on the binding of B'α to CHK1. IR-induced dissociation is an early event and occurs in a dose-dependent manner. CHK2 and B'α can re-associate hours after DNA damage and this is not dependent upon the repair of the DNA. Dissociation is dependent on ATM activity and correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after IR. CHK2 is able to phosphorylate B'α in vitro; however, in vivo, irradiation has no effect on PP2A activity or localization. Alternatively, PP2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity and protein stability. These data reveal a novel mechanism for PP2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP2A/CHK2 complex in later time points after damage, which may help to attenuate the signal. This mechanism of CHK2 negative regulation by PP2A joins a growing list of negative regulations of DNA damage response proteins by protein serine/threonine phosphatases.
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Rahman, Nor Zaihana Abdul. "Investigating the role of mitogen activated protein kinase phosphatase-2 on CNS function." Thesis, University of Strathclyde, 2015. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=24835.
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Mitogen-activated protein kinase phosphatases (MKPs) belong to the dual-specificity phosphatase (DUSP) family and are negative regulators of mitogen-activated protein kinases (MAPKs). Recently, MKP-2 has been shown to play a novel role in development, the immune system and cancer. However, our knowledge in relation to its function in the central nervous system (CNS) is limited. Hence, we have utilised novel MKP-2 knockout mice to investigate the role of this phosphatase in CNS function. The effect of MKP-2 deletion on CNS was first investigated using primary hippocampal culture by examining astrocyte proliferation and neurite length using immunocytochemistry techniques performed on cultures 1-7 days in vitro (DIV). To investigate the functional consequence of MKP-2 deletion on CNS at 3, 7 and 11 DIV, intracellular calcium concentrations were determined by fluorescence imaging using Fluo-4 dye. Na+, K+ current, synaptic transmission was also investigated using patch clamp electrophysiology. To examine the effect MKP-2 deletion in whole tissue, standard western blotting techniques were utilised to examine the consequence of MKP-2 deletion on ERK activity in the hippocampus of 3 week old mice. The functional effect of MKP-2 deletion on basal synaptic transmission and paired pulse facilitation (PPF) on acute hippocampal slices, field excitatory postsynaptic potentials (fEPSPs) were recorded from acute hippocampal slices of 3 week old mice. Astrocytes number and neurite length were reduced in 1-3 DIV in MKP-2-/- compared to MKP-2+/+. However, there is no difference in astrocyte proliferation and neurite length in 4-7 DIV. Further investigation into the reduction of neurite length, it was due to impairment in astrocyte function and not account of reduction in astrocyte proliferation. Investigating further into the functional consequences, astrocytic intracellular calcium was reduced at 7 DIV however neuronal intracellular calcium was increased at 11 DIV in MKP-2-/- primary hippocampal culture. Furthermore, Na+ and K+ current were also reduced at 7 DIV. However, spontaneous excitatory postsynaptic current (sEPSC) and synapse number has been shown to increase at 7 and 11 DIV. The mechanism underlying this seems to be not related to ERK phosphorylation as no difference in ERK activation was evident when compared between MKP-2+/+ and MKP-2-/-. In further experiments in acute hippocampal slices, MKP-2 deletion leads to a reduction in ERK activity within the hippocampus. In contrast, increased ERK activity was observed in the heart and liver. Basal synaptic transmission was enhanced in MKP-2-/- mice at high stimulus intensities compared to MKP-2+/+ but PPF was unaltered at all in inter-stimulus times tested (10-500 ms). In summary, in this thesis I demonstrate that even though MKP-2 deletion reduced astrocyte growth and neurite length at 3 DIV, it doesn't affect their functional properties at early development. However, alterations of functional activity at 7 and 11 DIV and acute hippocampal culture suggest that MKP-2 deletion might play a role in functional activity when the neuron is fully developed. This data suggest a novel physiological role for MKP-2 in the brain and might reveal valuable insight for the drug development.
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Scotney, Pierre David. "The catalytic mechanism of Bacillus stearothermophilus pyruvate kinase." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266959.
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Veenstra, Cynthia. "The receptor tyrosine kinase Met and the protein tyrosine phosphatase PTPN2 in breast cancer." Doctoral thesis, Linköpings universitet, Avdelningen för kliniska vetenskaper, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-135047.
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Breast cancer is the most common form of cancer in women worldwide and the second leading cause of cancer death. It is a heterogeneous disease and is subdivided into different subtypes, all with different treatment responses and survival outcomes. Luminal breast cancers are characterised by the expression of oestrogen receptor and generally have a good prognosis. More aggressive tumours are marked by the presence of growth stimulating receptor tyrosine kinase HER2 (HER2-like breast cancer) or the absence of oestrogen receptor, progesterone receptor, and HER2 (triple-negative breast cancer,TNBC). The latter is the most aggressive form and is difficult to treat due to lack of treatment targets. This thesis aimed to explore possible prognostic and predictive biomarkers in different subtypes and study their role in breast cancer. To this aid, breast cancer tumours of pre- and post-menopausal patients enrolled in two cohorts were analysed for gene copy numbers and expression of proteins involved in cell proliferation. Gene copy numbers of receptor tyrosine kinases MET and EGFR, Met’s ligand HGF, and protein tyrosine phosphatase PTPN2 were determined by droplet digital PCR or quantitative PCR in both cohorts. Met, phosphorylated Met (pMet), HGF, and PTPN2 protein expression levels were analysed with immunohistochemical staining in the pre-menopausal cohort. Moreover,the role of the aforementioned proteins was investigated in breast cancer cell lines. Amplification of MET, HGF, and EGFR in breast tissues was found to be low (5-8%). These three genes, all located on chromosome 7, were found to be strongly correlated with eachother and to be associated with shortened distant recurrence-free survival. High protein expression of Met, pMet, and HGF was found in 33%, 53%, and 49% of the breast tumours. MET and EGFR were found to be more often amplified in TNBC disease, correlating with worse survival. Moreover, stromal expression of HGF was associated with shorter survival in TNBC. EGF stimulation in TNBC cell line MDA-MB-468 led to inhibited cell proliferation and migration. Partial knockdown of EGFR caused TNBC cells to proliferate and migrate more upon EGF treatment, mirroring EGFR inhibitor resistance. Knockdown of Met had in part the opposite effects, indicating that Met inhibitors might be useful in the treatment of TNBC. The increase in proliferation and migration upon EGFR depletion could be counteracted with simultaneous knockdown of EGFR and Met, indicating that dual inhibition of these proteins might be a future treatment option in TNBC. Copy loss of PTPN2 was reported in 15% of the cases in both pre- and post-menopausal cohorts. Low cytoplasmic PTPN2 protein expression was found in half of the cases. Loss of PTPN2 gene or protein was associated with a shorter distant recurrence-free survival in Luminal A and HER2-positive tumours, not in TNBC, suggesting a subtype-related prognostic value of PTPN2. Subtype relevance of PTPN2 was further implied by in vitro analyses. Whereas PTPN2 knockdown had no observed effect on TNBC cell lines, knockdown in the Luminal A cell line MCF7 inhibited Met phosphorylation and promoted phosphorylation of Akt, a key regulator of cellular proliferation and survival. The cell growth and survival regulating RAS/MAPK pathway remained unaffected. Knockdown in the HER2-positive cell line SKBR3 led to increased Met phosphorylation and decreased RAS/MAPK-related Erk phosphorylation as well as EGF-mediated transcription factor STAT3 phosphorylation. These results indicate that the role of PTPN2 in breast cancer is subtype-related and needs to be further investigated for future treatment options.
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Toman, Ondřej. "Stress response in Bacillus subtilis : signalization, adaptation and the role of protein L24." Paris 11, 2006. http://www.theses.fr/2006PA112175.
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Shakir, Salika Mehreen. "Characterization of a serine/threonine phosphatase-kinase pair in Bacillus anthracis." Oklahoma City : [s.n.], 2010.
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Lee, Jin Suk. "Role of phosphatases in controlling arabidopsis mapk signalling cascades." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/732.
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Plants possess integrated signalling networks that mediate the responses to various environmental conditions. Mitogen-activated protein kinases (MAPKs) constitute a highly conserved family of enzymes in eukaryotes, and in plants MAPK-based signal transduction modules regulate a large number of physiological processes, including responses to environmental stresses and phytohormones. Regulated dephosphorylation of active MAPKs is a key component of the control of MAPK signalling cascades, and in mammals, members of the MAPK phosphatase (MKP) sub-class of dual-specificity tyrosine phosphatases have been recognized as key players for inactivating MAPKs. Five MKP homologues are found in Arabidopsis thaliana, but only limited information is available concerning their properties and biological roles. Based on initial data derived from my reverse genetics and protein interaction studies of these five potential MKPs, as well as gene function information in the literature, I chose to focus on two putative Arabidopsis MKPs, AtMKP2 and Indole-3-Butyric Acid-response 5 (IBR5).
By using a combination of genetic and biochemical studies, I established that the previously uncharacterized MKP designated AtMKP2, participates in the regulation of cellular homeostasis in ozone-challenged tissue, and can influence the activation state of two MAPKs, MPK3 and MPK6. AtMKP2-suppressed plants displayed significantly prolonged MPK3 and MPK6 activation during ozone treatment, and recombinant AtMKP2 was able to dephosphorylate both phospho-MPK3 and phospho-MPK6 in vitro, providing direct evidence that AtMKP2 may target these oxidant-activated MAPKs.
A mutation in IBR5, one of the five potential AtMKPs, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis. My protein interaction studies demonstrated that IBR5 and MPK12 are physically coupled and that the C-terminus of MPK12 is essential for its interaction with IBR5. In vitro dephosphorylation assays indicated that recombinant phosphoMPK12 is efficiently dephosphorylated by IBR5. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, consistent with the lower auxin sensitivity reported for ibr5 mutants.
Taken together, my data demonstrate for the first time that both AtMKP2 and IBR5 are bona fide Arabidopsis MAPK phosphatases and that they serve as important regulators of oxidative stress and auxin signalling, respectively, in Arabidopsis.
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Claywell, Ja. "CHARACTERIZATION AND STUDY OF THE PHYSIOLOGICAL ROLE OF CTL0511, A CHLAMYDIAL PROTEIN PHOSPHATASE TYPE 2C." OpenSIUC, 2019. https://opensiuc.lib.siu.edu/dissertations/1679.
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Chlamydia are obligate intracellular bacterial pathogens that are responsible for infectious blindness, sexually transmitted infections, and acute respiratory disease in humans. These pathogens undergo an essential biphasic developmental cycle differentiating between two functionally distinct forms known as the infectious elementary body (EB) and the replicative reticulate body (RB). Identifying the signals and regulatory mechanisms that enable Chlamydia to establish infection, differentiate between the two developmental forms, and survive within the host cell is critical to understanding chlamydial pathogenesis and developing future therapeutic strategies. In pathogenic bacteria, serine, threonine, and tyrosine (Ser/Thr/Tyr) protein kinases and phosphatases are critical for development, metabolism, and virulence. Chlamydia encode two validated protein kinases (pkn1 and pknd), a putative protein phosphatase (ctl0511; CppA), and appear capable of global phosphorylation that differs between the developmental forms. While these findings support a role for protein phosphorylation in chlamydial pathogenesis, a validated cognate protein phosphatase for Pkn1 and PknD mediating reversible phosphorylation was lacking. We hypothesized that CppA is the partner phosphatase for the chlamydial protein kinases, and in this study we validated and characterized CppA as a broad specificity protein phosphatase type 2C. Using in vivo and in vitro approaches we demonstrated that CppA acts on P-Ser/Thr/Tyr residues and can dephosphorylate multiple chlamydial protein substrates including PknD and the FHA 2 domain of CdsD, a component of the type 3 secretion apparatus. The importance of CppA for chlamydial growth and development was determined using a chemical “knock-out” approach and study of CppA missense mutations identified in slow growing C. trachomatis L2 chemical mutants. Treatment of C. trachomatis L2, C. trachomatis D, and C. muridarum with CppA inhibitors significantly reduced progeny levels and inclusion size in a time dependent manner with more significant growth inhibition in the first 12 hours post infection. Collectively, our findings support that CppA works in conjunction with PknD, and likely Pkn1, to mediate reversible phosphorylation of multiple protein substrates leading to changes in chlamydial physiology that appear to be key for early steps in development.
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Briedis, Kristine Mary. "The distribution and evolution of protein kinase and phosphatase families in the three superkingdoms of life." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3307357.
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Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed July 22, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 171-195).
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Back, Steven. "TULA-2: A Novel Protein Tyrosine Phosphatase That Regulates Osteoclast Differentiation and Function." Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/254886.
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Cell BiologyPh.D.
The human skeleton is a dynamic organ that serves multiple functions to maintain normal physiology and health. It protects vital organs, provides support for movement, houses marrow and maintains calcium homeostasis. The skeleton is maintained by the work of two cells with opposing functions: osteoblasts, cells that synthesize organic bone matrix and osteoclasts that degrade and resorb it. These cells interact with one another in a tightly regulated process known as the bone remodeling cycle. This cycle maintains the health of bone by removing and replacing weak or damaged bone and responding to stress loads by remodeling portions of the skeleton that require reinforcement. Osteoblasts differentiate from mesenchymal stem cells and respond to hormonal stimuli by synthesizing and secreting cytokines necessary for osteoclast differentiation. Osteoblasts may become embedded within mineralized matrix, becoming osteocytes, cells that can sense changes in mechanical loading and facilitate localization of the remodeling cycle. Osteoclasts differentiate from hematopoietic stem cells (HSC) when the cell surface receptors, c-FMS and RANK, are activated by ligands produced by osteoblasts, M-CSF and RANKL respectively. In addition to c-FMS and RANK stimulation, another calcium-mediated, co-stimulatory pathway must be activated to ensure proper osteoclast differentiation. This pathway is activated by two immunoreceptors, OSCAR and TREM-2 that interact with adaptor proteins termed FcRγ and DAP12 respectively. These adaptor proteins harbor immunoreceptor tyrosine-based activation motifs (ITAM), which exist on their cytoplasmic tail. Once the immunoreceptors are triggered, specific tyrosines within the ITAM motifs become phosphorylated and act as docking points for the tyrosine kinase, Syk. Once bound, Syk autophosphorylates and acts on its downstream targets. Syk dephosphorylation is, therefore, necessary to attenuate this signal to prevent over activation of osteoclasts. Recently, a novel tyrosine phosphatase, T-cell Ubiquitin ligand -2 (TULA-2) has been shown to dephosphorylate specific phosphotyrosine residues on Syk in various systems and has shown an increased specificity to dephosphorylate tyrosine 352. The goal of this project is to determine how TULA-2 mediated dephosphorylation of Syk regulates osteoclast differentiation and function. TULA-2 is a member of the TULA family of proteins, TULA and TULA-2. In spite of a significant homology and similar domain organization between TULA and TULA-2, only TULA-2 has significant phosphatase activity. Furthermore, whereas TULA is expressed only in lymphocytes, TULA-2 is expressed in most tissues albeit a higher level of expression is seen in cells of hematopoietic origin. In vivo analysis including Micro-computed tomography (Micro CT) and histomorphometry indicated that mice that lack both TULA and TULA-2 (DKO) have decreased bone mass compared to wild-type (WT) counterparts. An in vitro cell differentiation assay revealed that a larger population of osteoclast-like cells (OCL) could be cultivated from bone marrow isolated from DKO mice compared to OCL derived from WT bone marrow. An in vitro resorption pit assay revealed that DKO osteoclasts could resorb bone at a faster rate than WT counterparts. Additionally, over-expression of phosphatase-dead TULA-2 in WT osteoclasts increased the ability of the cells to resorb bone. At the molecular level, activation of the co-stimulatory pathway revealed increased tyrosine phosphorylation of Syk 352 in DKO pre-osteoclasts when compared to phosphorylation of Syk isolated from WT pre-osteoclasts. Cumulatively, the above data indicates that the absence of TULA-2 results in an increased signaling response leading to a larger population of hyperactive osteoclasts, which contributes to decreased bone mass in mice. These data suggest that the phosphatase activity of TULA-2 is required for negative regulation of bone resorption.
Temple University--Theses
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Campbell, Sean Thomas. "Protein Engineering for Biochemical Interrogation and System Design." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/560940.
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Proteins are intimately involved in almost every cellular phenomenon, from life to death. Understanding the interactions of proteins with each other and other macromolecules and the ability to rationally redesign them to improve their activities or control their function are of considerable current interest. Split-protein methodologies provide an avenue for achieving many of these goals. Since the original discovery of conditionally activated split-ubiquitin, the field has grown exponentially to include the activities of over a dozen different proteins. The flexibility of the systems has resulted in their use across a wide spectrum, both literally and figuratively, to primarily screen, visualize and quantitate macromolecular interactions in a variety of biological systems. In another arena, there is significant interest the apoptosis-regulating proteins: the Bcl-2 family. These proteins are found in many cell types and control, through expression levels as well as other mechanisms, the apoptotic state of a protein as governed by intrinsic death signals generated from such sources as DNA damage and viral infection. The apoptotic function of these proteins are mainly governed by a single type of interaction: the helix:receptor binding of the BH3-Only helices to the anti-apoptotic receptor proteins. While this often promiscuous helix:receptor interaction has received much scrutiny, the nature of the anti-apoptotic binding pocket, especially with regard to the specific residues that govern the interaction, has been lacking. With the high sensitivity and rapid analysis platform afforded by the cell-free split-luciferase analysis methodology, we devised and carried out the first systematic and large scale alanine mutagenesis of all five major anti-apoptotic members of the Bcl-2 family, validated these results both with biophysical methods as well as correlation with previous studies. Our results help explain how different receptors can bind a wide range of helices and also uncovered details regarding binding that are not possible with structural or computational analysis alone. In a second area of research, we have utilized the interaction of BH3 helices and their receptors for designing small molecule controlled protein kinases and phosphatases. In this protein design area, BH3-Only helices were inserted using a knowledge based approach into particular loops within both a protein kinase and a protein phosphatase. The BH3-Only helix interaction with added receptors, such as Bcl-xL provided an allosteric switch for turning-off the activity of the helix-inserted enzymes. The activity of the enzymes could then be turned-on by the addition of a cell-permeable small molecule that is known to bind the receptor. This plug-and-play design was demonstrated to be successful for two very different enzyme classes and likely provides a general and tunable biological element for controlling the activity of one or more proteins and enzymes in a biochemical networks.
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Dong, Yu. "Ca2+/Calmodulin Dependent Protein Kinase II Subcellular Re-distribution and Activation of Protein Phosphatase After a Brief Pentylenetertrazol Seizure: Potential Role in Kindling." University of Toledo Health Science Campus / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=mco1082463968.
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Orton, Christopher R. "Analysis of Protein Adduction Kinetics and the Effects of Protein Adduction on C-Jun N-Terminal Kinase Signaling." Diss., The University of Arizona, 2006. http://hdl.handle.net/10150/194247.
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Defining the mechanics and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. In this dissertation I describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein using quantitative mass spectrometry. Adducts are formed by electrophiles at Cys-14 and Cys-47 on the metabolic enzyme glutathione-S-transferase P1-1 and accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples. This method was used to measure rate constants for adduction at both positions with two different model electrophiles, IAB and BMCC. The results indicate that Cys-47 was approximately 2-3-fold more reactive toward both electrophiles than was Cys-14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system. Quantitative analyses of protein modifications provide a means of determining the reactivity and selectivity of damaging protein modifications in chemical toxicity.Another area of study explored in this dissertation is looking at the effects of protein alkylation on activating cellular signaling pathways, specifically the JNK signaling pathway. Protein adduction has been shown to be selective between different alkylating agents. It would then be reasonable to think this selectivity of adduction translates to selectivity of downstream consequences or cellular events directly tied to specific adductions. My work will show how treatment of HEK293 cells with either IAB or BMCC leads to differences in activation of JNK signaling. In addition, I've been able to show a difference in selectivity of a number of adducted targets by each alkylating agent, which are directly involved in regulation of the JNK signaling pathway. These studies illustrate not only the significance of protein adduction, but the importance for continual research to better understand their behavior in living systems.
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Heriche, Jean-Karim. "Protéine kinase CK2 et prolifération cellulaire." Grenoble 1, 1996. http://www.theses.fr/1996GRE10174.
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La proteine kinase ck2 est une serine/threonine kinase dont la sous-unite catalytique (ck2a) est souvent associee a une sous-unite regulatrice (ck2b) au sein d'un tetramere a#2b#2. Bien que sa fonction et sa regulation ne soient pas connues, on suppose qu'elle participe au controle de la proliferation cellulaire. Un signal mitogene majeur dans de nombreux types cellulaires est forme par l'activation sequentielle des trois kinases raf/mek1/mapk. Nous nous sommes donc demandes si la ck2 pouvait etre connectee a cette voie de transduction du signal proliferatif. Pour cela, nous avons suivi une demarche a base de transfections et d'experiences in vitro en utilisant des mutants des proteines impliquees. Nous avons ainsi pu montrer qu'une population de ck2a independante de ck2b presente les caracteristiques d'un nouveau substrat pour raf. En effet, ck2a est phosphorylee et inhibee en presence de raf actif in vitro et in vivo et la presence de ck2b empeche cette phosphorylation. Nous avons egalement montre que ck2a peut s'associer a mek1 et inhiber son activation par le serum. Cet effet de ck2a sur mek1 passe par l'association de ck2a avec la phosphatase pp2a car un mutant de ck2a incapable de s'associer a la phosphatase n'a plus d'effet sur mek1. L'inhibition de mek1 par ck2a correle avec une forte inhibition de la croissance clonale des cellules en presence de ck2a. De plus, le complexe ck2a/pp2a est dissocie en reponse au pdgf in vivo et par raf in vitro, suggerant que ce complexe est bien un regulateur physiologique de mek1 et de la proliferation cellulaire. Enfin, en exploitant les analogies entre ck2a et la serine/threonine kinase apparentee gsk-3, nous avons egalement trouve que ck2a pouvait etre un substrat associe de la tyrosine kinase abl. La signification biologique de cette association est discutee
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Neamatallah, Thikryat A. "The role of mitogen-activated protein kinase phosphatase-2 (MKP-2) in macrophage development and gene expression." Thesis, University of Strathclyde, 2014. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=23627.
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Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP4) and an important immune regulator. It specifically dephosphorylates the MAP kinases ERK and JNK to influence pro- and antiinflammatory cytokine production. MKP-2 has recently been shown to play a significant role in controlling Leishmania mexicana infection (Al-Mutairi et al., 2010) primarily by influencing macrophage activity. However, information on the effect of MKP-2 deletion at the molecular level on macrophage development and function is limited. This project utilised a novel DUSP4 gene knockout mouse and investigated the effects of MKP-2 deletion on M-CSF induced MAPK signalling and macrophage development as well as macrophage gene expression. Experiments in bone marrow derived macrophages demonstrated that in response to M-CSF macrophage, proliferation was reduced following to MKP-2 deletion. This was correlated with ERK phosphorylation, the expression of CD115 and CD34 on macrophage progenitors as well as the induction of genes related to macrophage differentiation and proliferation, colony stimulating factor-2 (Csf2) and monocyte to macrophage differentiation associated (Mmd) genes. In addition a comparative microarray gene expression analysis was conducted on MKP-2-/- and MKP-2+/+ macrophages following (LPS) or (IL-4) activation. As demonstrated previously, and associated with a role for MKP-2 in antimicrobial activity, arginase-1 expression was up-regulated in MKP-2-/- compared with MKP- 2+/+ macrophages. Surprisingly, and in contrast, we found that other alternative activation markers Ym1 (Chi3l3) and Fizz1/Retnla (Relm-a) were significantly reduced in MKP-2-/- macrophages when compared with their wild-type counterparts. As both Ym1 and Fizz1 have been implicated to play a major role in extracellular matrix disposition this suggests a significant role for MKP-2 in wound healing. Collectively, the findings in the current study have established that MKP-2 plays an important role in macrophage development and immune function.
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Castillo-Montoya, Javier, and Javier Castillo-Montoya. "Development of Orthogonal Split-Kinase and Split-Phosphatase Systems for Interrogating and Rewiring Signal Transduction." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/623179.
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The function of most proteins is regulated by post-translational modifications, of which phosphorylation in particular has been shown to be ubiquitous and of paramount importance to cell signaling. Two enzyme families, protein kinases and phosphatases, regulate phosphorylation, and aberrant activities of family members have been implicated in many diseases such as cancer and neurological disorders. Thus, understanding the function of these enzymes in living cells is important for understanding their biology and for designing new therapies, but a challenging task due to their highly conserved architecture. The major focus of the dissertation is on the development of a new approach to selectively turn-on multiple specific kinases and/or phosphatases using orthogonal ligands as chemical inducers of dimerization (CIDs). Specific kinases or phosphatases were dissected at particular sites into two inactive fragments or split-proteins. The split fragments are attached to interacting protein pairs of CID systems, such that upon addition of the specific ligand they heterodimerize with subsequent reassembly of the split-protein and concomitant activity. We demonstrated the in vitro and in cellulo feasibility of this approach using three orthogonal CIDs, rapamycin, abscisic acid, and gibberellic acid, to turn-on members of the tyrosine kinase group such as Lyn and Src, and of the tyrosine phosphatase group such as PTP1B and SHP1. We have also developed a new synthetic photocleavable di-trimethoprim CID that allows for ligand-gated turn-on of desired kinases in live cells. The new CID can be cleaved or turned-off by UV irradiation which results in a turn-off of kinase activity. Small molecule controlled split-proteins allow for developing logic gates and we demonstrate that the systems we have developed can be used to construct 7 out of the 10 basic, circuit-type Boolean phosphorylation-based logic gates in living cells. These post-translational logic gates may have interesting applications in synthetic biology. Finally, we present an initial approach to use redesigned kinases and redesigned ligands as potential scaffolds for developing new CIDs. Thus, we provide and extend new methodologies that potentially allow for posttranslational control over the activity of user defined split-kinases and split-phosphatases for interrogating and redesigning signaling pathways. The last section of this work focuses on understanding small-molecule selectivity toward protein kinases. We systematically analyzed different reported kinase screens to further understand the reliability of large scale data in the kinome field as the design of selective inhibitors is one the most useful approaches for understanding the function of enzymes or the development of drugs in a natural setting such as a primary cell or an organism.
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Ananieva-Stoyanova, Elitsa Antonova. "Identification and Functional Role of Myo-Inositol Polyphosphate 5-Phosphatase Protein Complexes." Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/28028.
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To survive, an organism must constantly adjust its internal state to changes in the environment from which it receives signals. The signals set off a chain of events referred to signal transduction. Signal transduction systems are especially important in multicellular organisms, such as plants and animals, because of the need to coordinate the activities of hundreds to trillions of cells. Plants, in particular, have a special need for perceiving signals from their environment because of their static nature. As in the animal cell, the first steps in perception of a signal include signal interaction with a receptor, signal amplification through second messenger production, and signal termination through second messenger hydrolysis. Myo-inositol polyphosphate 5-phosphatases (5PTases) (EC 3.1.3.56) have unique signal terminating abilities toward the second messenger inositol trisphosphate (Ins (1,4,5)P3, InsP3). In Arabidopsis thaliana there are 15 members of the 5PTase family, the majority of which contain a single 5PTase catalytic domain. Four members of the Arabidopsis 5PTase family, however, have a unique protein domain structure, with additional N-terminal WD40 repeats that are implicated in protein-protein interactions. The research presented here focused on the identification of 5PTase interacting proteins and the characterization of their functional role in Arabidopsis. To accomplish this goal, I examined a 5PTase13-interacting protein, the sucrose (Suc) nonfermenting-1-related kinase, SnRK1.1, an important energy sensor that is highly conserved among eukaryotes. My identification of a 5PTase13:SnRK1.1 complex points to the novel interaction of this metabolic modulator and inositol signaling/metabolism. 5PTase13 , however, plays a regulatory role in other plant specific processes as well, since I also identified the Arabidopsis homolog (Atp80) of the human WDR48 (HsWDR48, Hsp80) as a novel protein interactor of 5PTase13. My results indicate that Atp80 is important for leaf emergence, development and senescence likely via a regulatory interaction with 5PTase13 and PINOID â binding protein (PBP1).Ph. D.
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Puhl, Aaron A., and University of Lethbridge Faculty of Arts and Science. "Expanding our knowledge of protein tyrosine phosphatase-like phytases : mechanism, substrate specificity and pathways of myo-inositol hexakisphosphate dephosphorylation." Thesis, Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2006, 2006. http://hdl.handle.net/10133/526.
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A novel bacterial protein tyrosine phosphatase (PTP)-like enzyme has recently been isolated that has a PTP-like active site and fold and the ability to dephosphorylate myo-inositol hexakisphosphate. In order to expand our knowledge of this novel class of enzyme, four new representative genes were cloned from 3 different anaerobic bacteria related to clostridia and the recombinant gene products were examined. A combination of site-directed mutagenesis, kinetic, and high-performance ion-pair chromatography studies were used to elucidate the mechanism of hydrolysis, substrate specificity, and pathways of Ins P6 dephosphorylation. The data indicate that these enzymes follow a classical PTP mechanism of hydrolysis and have a general specificity for polyphosphorylated myo-inositol substrates. These enzymes dephosphorylate Ins P6 in a distributive manner, and have the most highly ordered pathways of sequential dephosphorylation of InsP6 characterized to date. Bioinformatic analyses have indicated homologues that are involved in the regulation of cellular function.x, 138 leaves ; 29 cm.
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Selner, Nicholas. "PROFILING THE INTRINSIC SEQUENCE SPECIFICITY OF PROTEIN TYROSINE PHOSPHATASES." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1384269733.
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Son, Sunghun. "Systematic analysis of phosphatase genes in aspergillus nidulans and a role of FCP1 in cell cycle regulation." The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1196228508.
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Dong, Yu. "Ca²⁺/calmodulin dependent protein kinase II subcellular re-distribution and activation of protein phosphatase after a brief pentylenetetrazol seizure potential role in kindling /." Connect to full-text via OhioLink ETD Center, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1082463968.
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Thesis (Ph. D.)--Medical College of Ohio, 2003."In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Howard Rosenberg. Document formatted into pages: iv, 144 p. Title from title page of PDF document. Includes bibliographical references (p. 104-132).
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Dries, Daniel Robert. "Kinetics and mechanism of the interaction of the C1 domain of protein kinase C with lipid membranes." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3244326.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed February 23, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 114-120).
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Abourjeily, Nay. "Involvement of tyrosine phosphorylation during Leishmania donovani differentiation." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=32742.
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Dimorphic Leishmania donovani parasites exist as promastigotes in the sandfly vector and differentiate into amastigotes once injected into the skin of human hosts during a blood meal. The mechanisms and signals that are involved in triggering differentiation are not well understood in Leishmania. We have investigated whether tyrosine phosphorylation is a possible signalling component. Differential levels of tyrosine-phosphorylated proteins were observed in extracts from in vitro promastigote and amastigote cultures, with an overall reduction in the latter stage. Following this observation, the inhibition of tyrosine phosphorylation was examined in promastigotes using Tyrphostin AG1433, a broad-spectrum tyrosine phosphorylation inhibitor. AG1433 treated in vitro promastigote cultures differentiate into amastigote-like morphology, have reduced tyrosine phosphorylation level, and express the amastigote-specific marker A2 proteins. Our studies demonstrate that signal transduction mechanisms involving tyrosine phosphorylation/dephosphorylation events are involved in controlling L. donovani promastigote differentiation into amastigote forms.
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Robert, Perle. "Caractérisation fonctionnelle des modifications post traductionnelles de la protéine Arpp19, un inhibiteur de la phosphatase PP2A." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT085/document.
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La phosphorylation/déphosphorylation des protéines est une modification clé dans les mécanismes qui contrôlent les évènements mitotiques.Classiquement, l’entrée en mitose requiert l’activation de Cdk1. Pour se faire, les phosphorylations inhibitrices sur Cdk1 par Myt1 et Wee1 doivent être éliminées par Cdc25. Le complexe Cdk1-Cycline B (MPF) est ainsi actif, les kinases inhibitrices inactivées.Dernièrement, une nouvelle protéine kinase clé pour l’entrée en mitose a été mise en évidence : Greatwall (Gwl). Les récents résultats publiés par notre équipe montrent que Gwl permet l’entrée et le maintien en mitose en inhibant l’activité de la phosphatase PP2A, la phosphatase responsable de la déphosphorylation des substrats de la protéine kinase Cdk1-Cycline B, via son substrat Arpp19. Gwl phosphoryle Arpp19 sur la sérine 71 lui conférant ainsi la capacité d’inhiber l’activité de la phosphatase PP2A.Une étude sur les modifications post traductionnelles d’Arpp19 a été initiée dans l’équipe et met en évidence plusieurs sites de phosphorylation : • La sérine 71, site de phosphorylation par Gwl • La sérine 28, dont la phosphorylation est attribuée à Cdk1 (vérifié in vitro) • La sérine 113, site de phosphorylation par pKA Ce projet de thèse s’inscrit dans la suite logique du travail déjà effectué dans l’équipe et a pour objectif de caractériser les modifications post traductionnelles d’Arpp19, leurs rôles dans la progression mitotique, leurs incidences sur la liaison et l’inhibition de la cible d’Arpp19, PP2A.Cette partie du projet repose sur la synthèse de mutants d’Arpp19Xe, mutants phosphomimétiques d’une part (sérine transformée en acide aspartique par mutagenèse dirigée) ou mutants dont la phosphorylation est impossible (sérine en alanine). Ces mutants nous ont permis de travailler sur l’impact de ces différentes phosphorylations dans l’extrait d’œufs de Xénope.Ce projet s’attache également à mettre en lumière l’ensemble de la voie de signalisation aboutissant aux différentes modifications post traductionnelles d’Arpp19, leurs chronologies au cours du cycle et ainsi identifier les protéines effectrices de ces phosphorylations sur Arpp19 qui sont autant de leviers potentiels sur lesquels les thérapies anti-tumorales pourraient s’appuyerProteins phosphorylation and dephosphorylation are key post translational modifications controlling mitotic events.Traditionally, mitotic entry requires Cdk1 activation. To allow this to occur, inhibitory phosphorylations on Cdk1 by Myt1 and Wee1 kinases must be removed by phosphatase Cdc25. Thus, the Cdk1-Cyclin B complex, also called MPF (Mitotic Promoting Factor), is active and inhibitory kinases inactivated.Along this canonic scheme, another key kinase has been shown to play a critical role: the Greatwall (Gwl) kinase also called MAST-L for MAST like. Results published by our team show that in Xenopus laevis, Gwl allows entry and maintains mitosis by inhibiting the activity of the phosphatase responsible for dephosphorylation of Cdk1/Cycline B substrates: PP2A. This activity is driven by Gwl target: Arpp19. Gwl phosphorylates Arpp19 on its 71st residue turning it into a potent inhibitor of PP2A.A study of Arpp19 post translational modifications of Arpp19 has been initiated in the team which will allow the further study of several phosphosites: • Serine 71, Gwl phosphosite, the best documented site. • Serine 28, shown in vitro to be a Cdk1-CycB phosphosite. • Serine 113, assigned to PKA. This thesis project joins logically after the work already made in the team and has for objective to characterize the post translational modifications of Arpp19, their roles in mitotic progress, their incidences on binding and inhibition of Arpp19’s target, PP2A.This part of the project relies on mutants' synthesis of Arpp19Xe, phosphomimetics’ mutants on one hand (serine transformed into aspartic acid by mutagenesis) or mutants unable to be phosphorylated (serine into alanine). These mutants allowed us to work on the impact of these various phosphorylations in Xenopus eggs extracts.This project also attempts to highlight the whole signalization pathway ending in the various post translational modifications of Arpp19, their timelines during the cycle and thus to identify effector proteins of these phosphorylations on Arpp19 which are as much as potential levers on which can serve as targets for cancer therapy
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Semiz, Sabina. "Effects of diabetes, insulin, and vanadium on regulation of glycogen synthesis : roles of glycogen synthase kinase-3 and protein phosphatase-1." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ61172.pdf.
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Chen, Chun-Ti. "Regulation of the Cdc14-like Phosphatase CLP1 in Schizosaccharomyces pombe and Identification of SID2 Kinase Substrates: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/449.
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Coordination of mitosis and cytokinesis is crucial to generate healthy daughter cells with equal amounts of genetic and cytoplasmic materials. In the fission yeast Schizosaccharomyces pombe, an evolutionarily conserved Cdc14-like phosphatase (Clp1) functions to couple mitosis and cytokinesis by antagonizing CDK activity. The activity of Clp1 is thought to be regulated in part by its subcellular localization. It is sequestered in the nucleolus and the spindle pole body (SPB) during interphase. Upon mitotic entry, it is released into the cytoplasm and localized to the kinetochores, the actomyosin ring, and the mitotic spindle to carry out distinct functions. It is not clear how Clp1 is released from the nucleolus, however, once released, a conserved signaling pathway termed Septation Initiation Network (SIN) functions to retain Clp1 in the cytoplasm until completion of cytokinesis. The SIN and Clp1 function together in a positive feedback loop to promote each other’s activity. That is, the SIN promotes cytoplasmic retention of Clp1, and cytoplasmic Clp1 antagonizes CDK activity and reverses CDK inhibition on the SIN pathway to promote its function and activity. However, at the start of this thesis, the mechanism by which the SIN regulated Clp1 was unknown. The SIN pathway is also required to promote constriction of the actomyosin ring, and the septum formation. However, its downstream targets were still uncharacterized. In two separate studies, we studied how Clp1 is released from the nucleolus at mitotic entry and how the SIN kinase Sid2 acts to retain Clp1 in the cytoplasm. We identified several Sid2 candidate substrates, and revealed other functions of the SIN pathway in coordinating mitotic events.
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Li, Dailin. "Regulation of Na⁺, K⁺-ATPase activity by protein phosphatase-1 and protein kinase C : studies in infant and adult renal tissue and on vascular smooth muscle cells /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3563-7/.
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Guo, Dongni Lily Centre for Vascular Research Faculty of Medicine UNSW. "Protein kinase A and related pathways in the regulation of apolipoprotein E secretion and catalase activity." Awarded By:University of New South Wales. Centre for Vascular Research, 2009. http://handle.unsw.edu.au/1959.4/41512.
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Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.
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Al-Nasser, Sulaiman M. A. "The effect of mitogen-activated protein kinase phosphatase-2 (MKP-2) overexpression in prostate cancer cell function and its clinical prostate cancer progression." Thesis, University of Strathclyde, 2013. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=22729.
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MAP kinase signals have been reported as a crucial cancer chemopreventive and chemotherapeutic target due to their involvement in tumour cell growth, proliferation, apoptosis and survival. They consist principally of extracellular signal-regulated kinase (ERK), c-jun NH2- terminal kinase (JNK) and p38 MAP kinase, which has been correlated with a more malignant phenotype in several tumour models and in vivo. A key regulatory off switch for the MAP kinases is the dual specificity phosphatase, DUSP-4, also known as MAP kinase phosphatase-2 (MKP-2). This phosphatase is a type one DUSP, located in the nucleus, induced in cells in response to a number of extracellular stimuli and selective for ERK and JNK in vitro. This study was designed to examine the role of MKP-2 in the prostate cancer cell lines LNCaP Androgen sensitive (AS) and Androgen Insensitive (AI) in proliferation and apoptosis in vitro and cancer development in vivo. MKP-2 was found to be expressed endogenously in both (AS ) and (AI) cells. Adv.MKP-2 was then used as a tool to study the effect of MKP-2 overexpression in LNCaP (AS) and (AI). Immunofluorescent staining revealed strictly nuclear expression of the MKP-2 adenovirus, with more than 90% of the cells infected. In LNCaP (AS), ERK phosphorylation in response to EGF was transient whilst in LNCaP (AI) cells it was sustained. In contrast, JNK phosphorylation was sustained in both LNCaP (AS) and (AI) in response to Ultraviolet (UV) light C (60 j/m2). Infection of both LNCaP (AS) and (AI) cells with Adv.MKP-2 significantly inhibited the phosphorylation of ERK and JNK. The kinetics of cell cycle progression was established. Stimulation with FCS over 48 h in LNCaP (AS) and 24 h in LNCaP (AI) caused a marked increase in cells both in S phase and G2/M phase. Following infection with Adv.MKP-2 progression inhibited cell numbers in both S phase and G2/M phase where they were reduced by over 50%. This correlated with a reduction in the expression of both cyclin B and D1. In contrast, Adv.MKP-2 did not modify apoptosis in both LNCaP cell lines stimulated by several agents including; UV-C, Doxorubicin and X-ray, These results indicate the potential for MKP-2 to influence cancer cell function proliferation but not apoptosis. To specify the affects are mediated via dephosphorylation of ERK and/or JNK, we used a form of MAP kinase which has a triple substitution of arginine within positions R74, R75 and R76 to alanine and is unable to bind to ERK. Using this mutation further specifies the role of ERK or, in particular, JNK in regulating cellular proliferation, Adv.MKP-2-NLS1 caused inhibition of either EGF or UV-C ERK and JNK phosphorylation respectively in both LNCaP cells, whilst Adv.MKP-2-CI was not effective, suggesting that Adv.MKP-2-NLS1 can still bind and deactivate ERK. However, both NLS1 and CI MKP-2 adenoviruses inhibited cell cycle progression. The effect of either WT-MKP-2 or CI-MKP-2 on histone H3 phosphorylation was also ass essed and both were found to cause substantial inhibition, suggesting the potential of a phosphataseindependent function for MKP-2 in the regulation of cell cycle progression. Finally, work using human prostate tissue confirmed that MKP-2 is expressed in invasive prostate tumours and localised in the nucleus, and staining intensity was positively correlated with PSA below 10 ng/ml levels and in early grade of Gleason score. These findings clearly suggest the potential for MKP-2 to be used as a possible gene tool in prostate cancer, which requires not only the inhibition of LNCaP cells proliferation in vitro, but PSA below 10 ng/ml levels and in early stage of Gleason score survival in vivo.
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Beck, Eichler-Jonsson Claudia. "Aspects of mitogen-activated protein kinase cascade activation by epidermal growth factor (EGF): kinetics and crosstalk mechanism with tumor necrosis factor Alpha (TNFalpha) /." [S.l. : s.n.], 2002. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10252924.
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Dickey, Audrey Sarah. "Role of pp2a/bβ2 and pka/akap1 in brain development and function via dynamin-related protein 1 (drp1) control of mitochondria shape and bioenergetics." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/3444.
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Mitochondria are critical for energy production and Ca2+ homeostasis and undergo fission and fusion reactions, perturbation of which can contribute to neuronal injury and disease. Mitochondrial fission is catalyzed by Drp1 (dynamin-related protein 1), a large GTPase tightly controlled by various posttranslational modifications, including phosphorylation. Bβ2 is a neuron-specific postnatally induced protein phosphatase 2A (PP2A) regulatory subunit that mediates PP2A translocation to the outer mitochondrial membrane (OMM) to promote mitochondrial fragmentation and sensitize neurons to various injuries. Opposing PP2A/Bβ2's effect on mitochondrial morphology and cell death is protein kinase A (PKA) anchored to the OMM via A kinase anchoring protein 1 (AKAP1). This dissertation describes how reversible phosphorylation of Drp1 at a conserved Serine residue by an outer mitochondrial kinase (PKA/AKAP1) and phosphatase complex (PP2A/Bβ2) affects dendrite and synapse development in hippocampal neurons and synaptic plasticity and learning and memory in vivo.
Inducing mitochondria fragmentation decreases dendritic arbor complexity, but increases spine and synapse number. Mitochondrial elongation induces opposite effects. L-carnitine increases mitochondria membrane potential and recapitulates the dendritic and synaptic effects of mitochondrial elongation. Epistasis experiments substantiate our hypothesis that PP2A/Bβ2 dephosphorylates and PKA/AKAP1 phosphorylates Drp1 to change mitochondrial shape and regulate mitochondria localization, dendrite outgrowth, and synapse development.
Bβ2 null mice are viable and fertile, without obvious abnormalities. Bβ2 null mice demonstrate significantly larger cortical and hippocampal neuronal mitochondria than in wildtype. Bβ2 deletion decreases spine number on apical and basal cortical dendrites and hippocampal dendrites. Bβ2 null mice display significantly decreased input/output relationship in the hippocampus, consistent with a decrease in synapse number. In a combined context and cued fear-conditioning protocol, the hippocampal-dependent context recall trial revealed significant deficits in Bβ2 null and heterozygous mice. This deficit is also seen in hippocampal-dependent Barnes maze performance. These results are consistent with the reduced hippocampal long-term potentiation (LTP) found in Bβ2 null mice and demonstrate the importance of Bβ2 in hippocampal synaptic plasticity and memory. In conclusion, PP2A/Bβ2 and PKA/AKAP1 have important roles in mitochondria regulation and dendritic and synaptic development as seen in our results in vitro with rat hippocampal cultures and in vivo with Bβ2 null mice.
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Persson, Camilla. "Protein Tyrosine Phosphatases as Regulators of Receptor Ryrosine Kinases." Doctoral thesis, Uppsala University, Ludwig Institute for Cancer Research, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3345.
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Tyrosine phosphorylation is a crucial mechanism in cellular signaling and regulates proliferation, differentiation, migration and adhesion. The phosphorylation reaction is reversible and is governed by two families of enzymes: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). This thesis investigates the role of PTPs in regulating receptor protein tyrosine kinases (RTKs), and explores a mechanism for regulation of phosphatase activity.
Most receptor tyrosine kinases are activated by ligand induced dimerization, which results in an increase in receptor phosphorylation. Preparations of ligand-stimulated dimeric PDGF β-receptors were shown to be less susceptible to dephosphorylation compared with unstimulated receptors. This revealed that reduced receptor dephosphorylation contributes to ligand-induced increase in RTK phosphorylation.
The receptor-like phosphatase DEP-1 site-selectively dephosphorylates the PDGF β-receptor. One of the most preferred sites is the PLC-γ binding phosphotyrosine pY1021, and the autoregulatory pY857 is one of the least preferred sites. By using chimeric phospho-peptides derived from these two sites as substrate for DEP-1, it was shown that a lysine residue at position +3 acts as a negative determinant for DEP-1 and that an aspartic acid residue at position –1 is a positive determinant.
The modulatory effect of TC-PTP on PDGF β-receptor signaling was explored by using mouse embryonic fibroblasts derived from TC-PTP knockout mice. PDGF β-receptors derived from knockout cells exhibited a higher level of ligand-induced phosphorylation compared to receptors from wildtype cells. The increase was unevenly distributed between different autophosphorylation sites. The PLC-γ binding site, previously implicated in chemotactic response, displayed the largest increase. Consistently, a cell migration assay revealed hyper-responsiveness to PDGF of TC-PTP knockout cells as compared to wildtype cells.
Reversible oxidation of the active site cysteine in PTPs is a mechanism, which have been postulated to regulate phosphatase specific activity. An antibody-based generic method for detection of oxidized PTPs was developed. Using this method it was revealed for the first time that UV-induced inactivation of PTPs involves oxidation of the active site cysteine.
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Bignon, Jérôme. "Rôle de la protéine tyrosine phosphatase, PTP1C, dans la régularité de l'activité catalytique de la protéine tyrosine kinase pp60c-src et au cours du développement des cellules hématopoietiques." Paris 5, 1995. http://www.theses.fr/1995PA05CD08.
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Pour étudier le rôle de la tyrosine, PTP1C, dans la régulation de l'activité kinase de pp60c-src, nous avons utilisé un modèle murin (la souris motheaten) présentant un déficit congénitale en PTPIC. Nous avons montré que l'activité kinase de pp60c-src était fortement diminuée dans les thymocytes (Th-) de cette souris. De plus, après immunoprécipitation de pp60c-src à partir de thymocytes (Th-), l'addition d'une forme recombinante de PTPIC restaure son activité kinase. Enfin, nous avons observé que PTPIC déphosphorylait spécifiquement le résidu tyrosine, localisé dans la partie C-terminale de pp60-src, dans la forme hosphorylée participe au maintien de cette protéine dans une conformation inactive. D'autre part, une association physique entre PTPIC et pp60-src a été détectée, in vitro et in vivo, dans des thymocytes murins ainsi que dans des plaquettes et des lymphoblastes humains. Cette association est uniquement observée en présence d'orthovanadate , ce qui suggère qu'elle serait dépendante du degré de phosphorylation de ces protéines sur des résidus tyrosine. Nous avons donc identifié, dans des conditions physiologiques, la première phosphatase impliquée dans la régulation de l'activité pp60c-src et nous avons montré que PTPIC participait à la régulation positive de certaines molécules impliquées dans des processus intracellulaires. La souris motheaten est caractérisée par des anomalies au niveau des cellules hématopoïétiques. Pour étudier le rôle joué par PTPIC au cours du développement de ces cellules, nous avons choisi d'exprimer cette protéine de façon ectopique, dans les cellules souche hématopoïétiques de la moelle osseuse de cette souris. Nous avons construit deux vecteurs rétroviraux capables d'exprimer PTPIC dans des cellules cibles infectées et nous avons préparé des cellules d'empaquetage qui produisent ces virions avec une efficacité permettant l'infection des cellules progénitrices de la moelle osseuse (titre viral = 10 exposant 7). L'obtention de ces vecteurs rétroviraux recombinante nous permettre d'étudier, in vitro et in vivo, le rôle de PTPIC au cours du développement et du fonctionnement des cellules hématopoïétiques.
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Gnangnon, Bénédicte. "Caractérisation moléculaire et fonctionnelle de la pseudo-tyrosine kinase-like (pTKL) de plasmodium." Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S003/document.
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Le paludisme, première endémie parasitaire mondiale ayant engendré près d’un demi-million de morts en 2017 (d’après l’OMS), est due à une infection par un parasite du genre Plasmodium. Cet apicomplexe infecte, au cours de son cycle de vie, un hôte définitif, un moustique femelle du genre Anopheles, et un hôte intermédiaire homéotherme (l’Homme pour au moins 6 espèces). Chez ce dernier, après une phase de développement hépatique, le parasite envahit puis lyse les érythrocytes. L’accroissement exponentiel de la parasitémie engendre les symptômes du paludisme et permet la production de formes sexuées (gamétocytes) qui seront transmises au vecteur arthropode, permettant ainsi la complétion du cycle de vie du parasite.Plasmodium a co-évolué avec ses hôtes et mis en place divers modes de régulation de l’expression de ses gènes. La phosphorylation est l’une des modifications post-traductionnelles majeures et rapides qu’il utilise pour répondre aux changements environnementaux auxquels il est confronté au cours de son cycle de vie. Nombre de ses kinases et phosphatases jouent un rôle essentiel dans l’invasion de cellules hôtes, la croissance et la division cellulaires, ainsi que la motilité de certains stades. En revanche, le rôle des cinq pseudokinases de Plasmodium dans son développement n’a jusqu’ici pas été exploré.Durant ma thèse, j’ai caractérisé l’unique pseudo-Tyrosine Kinase-like (pTKL) de Plasmodium et étudié son rôle au cours du cycle intra-érythrocytaire du parasite.L’annotation de la pTKL de P. falciparum (PfpTKL) m’a permis d’identifier différents domaines et motifs, et notamment un domaine SAM (Sterile Alpha Motif), deux motifs RVxF (connus pour leur capacité d’interaction avec la Protéine Phosphatase de type 1, PP1) et un pseudo-domaine kinase appartenant à la famille des Tyrosine Kinases-like (TKL). Nous avons montré que ce pseudo-domaine kinase est capable de lier l’ATP de manière cation-indépendante, mais est dépourvu d’activité enzymatique. Des études d’interaction in vitro couplées à l’utilisation de modèles hétérologues (Levure, ovocytes de Xénope) m’ont permis d’identifier deux protéines parasitaires partenaires de PfpTKL : le domaine SAM de PfpTKL interagit directement avec la pseudo-protéase PfSERA5 (SErine Repeat Antigen 5), alors que les deux régions de la protéine contenant les motifs RVxF de PfpTKL interagissent avec PfPP1c (phosphatase majeure de Plasmodium). De façon intéressante, le deuxième motif RVxF est directement impliqué dans l’interaction avec PP1c et serait capable de moduler l’activité de cette dernière de manière allostérique.La localisation de la pTKL de P. berghei (PbpTKL) a ensuite été étudiée par immunofluorescence et confirmée par des expériences de fractionnement cellulaire. Nous avons ainsi observé que PbpTKL est exportée dans l’érythrocyte infecté au stade trophozoïte, puis retenue dans le parasite et la vacuole parasitophore au stade schizonte. L’étude de l’interactome de PbpTKL par IP/MS au stade trophozoïte a montré que PbpTKL s’associe à diverses protéines impliquées dans l’organisation du cytosquelette de l’érythrocyte, ainsi que dans l’érythropoïèse et l’homéostasie cellulaire. Ces observations suggèrent que pTKL joue un rôle, direct ou via ses partenaires, à l’interface entre le parasite et sa cellule hôte.Enfin, afin d’approcher la fonction de pTKL chez le parasite, nous avons généré différentes lignées génétiquement modifiées. L’étude phénotypique des souches de P. berghei KO et iKD pour pTKL a montré qu’elle était dispensable pour la complétion du cycle intra-érythrocytaire, l’expression des gamétocytes ainsi que l’activation des gamétocytes mâles. Ces données suggèrent que pTKL est dispensable pour ces stades de développement ou que l’expression de gènes redondants compense son absence. Quoi qu’il en soit, il est important de poursuivre les recherches sur le rôle de cette protéine aux autres stades de développement du parasite, notamment du zygote aux stades hépatiquesMalaria is the first endemic parasitic disease in the world with nearly half million deaths in 2017 according to the WHO. This disease is the result of an infection by an agent belonging to the Plasmodium genus. This apicomplexan parasite infects two hosts over its complex life cycle: a definitive one – a mosquito belonging to the Anopheles genus – and a homoeothermic intermediate host. At least six Plasmodium species can infect humans. In its intermediate host, Plasmodium first replicates in hepatocytes before releasing erythrocyte-infectious stages in the bloodstream. Once there, parasites invade and replicate within erythrocytes, before lysing them to release other infectious stages. This triggers an exponential rise in the parasitemia, as well as malaria symptoms. Sexual stages, called gametocytes, are produced over this intra-erythrocytic cycle to be transmitted to the arthropod vector, thus allowing the completion of the parasite life cycle.Plasmodium co-evolved with its hosts and set up diverse gene expression regulation pathways accordingly. Phosphorylation is one of the major and fastest post-translational modifications used by the parasite to respond to environmental changes. Many of its kinases and phosphatases play key roles in host cell invasion, cellular growth and division, as well as motility of specific developmental stages. However, the role of the five pseudo-kinases expressed by Plasmodium has not been explored yet.During my PhD project, I have performed the characterization of the unique Plasmodium pseudo-Tyrosine Kinase-like (pTKL) and explored its role over the parasite intra-erythrocytic cycle.P. falciparum pTKL (PfpTKL) in silico annotation allowed the delineation of the protein domains. Notably, a SAM (Sterile Alpha Motif) domain, two RVxF motifs (known for their binding potential with the major protein phosphatase type 1, PP1) and a pseudo-kinase domain belonging to Tyrosine Kinase-like (TKL) family were found. This pseudo-kinase domain was found to be able to bind ATP in a cation-independent way although devoid of kinase activity. Two parasite protein partners of PfpTKL have been identified using in vitro protein-protein interaction studies together with heterologous models (yeast, Xenopus ovocytes). First, PfSERA5 (SErine Repeat Antigen 5) specifically and strongly interacts with PfpTKL SAM domain and second, PfPP1c binds the two RVxF-containing regions of PfpTKL. Interestingly, the second RVxF motif, which is located within the pseudo-kinase domain, directly binds PfPP1c and seems to be involved in the allosteric regulation of the phosphatase activity. The subcellular localization of P. berghei pTKL (PbpTKL) was studied by IFA as well as sequential lysis of erythrocytes followed by immunoprecipitation assays. PbpTKL was shown to be exported to the host cell cytosol at the trophozoite stage, but retained in the parasitophorous vacuole and the parasite cytosol at the schizont stage. Furthermore, our interactome analysis conducted at the trophozoite stage by IP/MS showed that PbpTKL binds many host cell proteins involved in erythrocyte cytoskeleton organization, as well as erythropoiesis and cell homeostasis. These data suggest that pTKL plays a role at the parasite/host interface, either directly or via its protein partners.Finally, in an attempt to understand the role of pTKL for the parasite development, we generated genetically modified P. berghei strains. The phenotypic study of PbpTKL KO and iKD strains did not show any difference between the defective parasites and the parental wild type ones during the intra-erythrocytic cycle, gametocyte expression and male gametocyte activation. These data suggest the dispensability of pTKL or the expression of redundant gene(s) with similar functions in these parasite stages. Whatever the explanation, it is still important to follow up this investigation in other parasite stages, from zygotes to hepatic stages
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Lowe, Julie. "Identification of NF-kappa B and DNA-dependent protein kinase (DNA-PK) as new players in the regulation and signaling of the oncogenic phosphatase Wip1." Connect to Electronic Thesis (CONTENTdm), 2010. http://worldcat.org/oclc/642326246/viewonline.
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Beck, Eichler-Jonsson Claudia. "Aspects of mitogen activated protein kinase cascade activation by epidermal growth factor (EGF) kinetics and crosstalk mechanism with tumor necrosis factor [alpha] (TNF[alpha]) /." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=965959937.
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