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Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 16, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Kent S. Gates. Vita. Includes bibliographical references.

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Altered cytoskeletal regulation impacts numerous physiological phenomena: cell motility, apoptosis, oncogenic transformation and parasitic infection. The protein tyrosine phosphatase (PTP)-PEST contains multiple motifs mediating its recruitment to signalling components, and is required for actin filament organization. However, little is known regarding either the importance of PTP-PEST subcellular localization, or the role of PTP-PEST in either parasitic infection or apoptosis. My doctoral research was therefore focussed on elucidating the effect of subcellular distribution on PTP-PEST activity, specifically with respect to regulation of p130Cas (a PTP-PEST substrate), as well as on the involvement of PTP-PEST in both host-pathogen relations and apoptosis. First, PTP-PEST was found both within the cytosol and at the plasma membrane. Using PTP-PEST -/- rescued cell lines, I observed that tyrosine phosphorylation-dependent p130Cas interactions were controlled primarily by cytosolic PTP-PEST. Secondly, infection of fibroblasts with Leishmania major was observed to induce dramatic actin rearrangements, and to alter the phosphorylation state of numerous proteins. Importantly, both PTP-PEST and p130Cas were processed by the parasitic protease GP63 during infection. GP63 was also required for the cleavage of additional host proteins: cortactin, TC-PTP and caspase-3. Of note, Leishmania parasites mediated p38 inactivation, correlating with the proteolysis of its upstream activator TAB1, in a GP63dependent manner. These results indicate that GP63 plays a key role in a number of biochemical events, potentially contributing to Leishmania infectivity. Finally, PTP-PEST was found to relocalize to the edges of retracting membrane ruffles of apoptotic cells. Surprisingly, PTP-PEST was specifically cleaved by caspase-3 at the 549DSPD motif during apoptosis; leading to modification of catalytic activity and scaffolding properties, and sensitizing cells to Fas-mediated detachment. As this data demonstrated a potential role for caspase cleavage in PTP regulation, I also investigated the presence of conserved putative caspase-c1eavage sites in other family members. In summary, the data presented herein links PTP-PEST with various biological processes: oncogenic signalling, host-pathogen interactions, and apoptosis. In addition to demonstrating the involvement of PTP-PEST in diverse signalling pathways, these studies underscore the importance of subcellular localization and proteolysis in the regulation of this PTP.

SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, plays a critical role in regulation of cell signal transduction. SHP-1 is expressed not only in cells of hematopoietic lineages, but also in many non-hematopoietic cells under the control of a tissue-specific promoter, P1. In the first part of this thesis, the activity of the P1 promoter was analyzed in a region spanning 3.5 kb upstream of the major transcription start site in non-hematopoietic MCF-7 cells. An upstream Sp1 element (-126 to -118) positively regulated this TATA-box-lacking promoter. Two inverted CCAAT-elements (-332 to -328 and -66 to -62) played the important, but opposite roles, and transcription factor NF-Y predominantly bound to the two CCAAT-elements to control SHP-1 gene expression. Furthermore, incubation of MCF7 cells with 100 ng/ml trichostatin A (TSA), an inhibitor of histone deacetylase, significantly increased the activity of the P1 promoter. Mutation in the proximal CCAAT-element, however, eliminated the activating effect of trichostatin A on the promoter. In the second part of this thesis, the mechanism by which SHP-1 modulated TSA-induced MCF-7 cell apoptosis was elucidated. Analysis of cell survival signaling pathways revealed that overexpression of SHP-1 inactivated Akt ( eg. diminished phosphorylation resulted from modulation of PI3K expression) and increased caspase-9 and caspase-7 activities. Interestingly, a parallel decrease was observed in the phosphorylation of the pro-apoptosic Bcl-2 family member Bad at Ser112 as well as in the stress-activated MAP kinase JNK, both of which have been implicated in Akt- as well as ERK1/2-mediated functions. It was not surprising, therefore, to detect a diminished level of phosphorylated ERK1/2 in SHP-1-overexpressiog cells and that this effect was exacerbated by TSA treatment. Taken together, the data presented in this thesis suggest that SHP-1 expression is regulated by Sp1 and NF-Y factors, and SHP-1 sensitizes MCF-7 cells to TS

The aim of this work was to investigate the role of TGFbeta family members in the induction of cell growth arrest and apoptosis in immune cell types. The TGFbeta superfamily is a large group of evolutionary conserved polypeptide growth factors, involved in different physiological processes. Any deregulation of the different components of the TGFbeta signaling pathway, has been largely implicated in multiple human critical disorders including cancer. Activin, originally isolated from gonadal fluid, and more recently described as an antiproliferative and proapoptotic factor in different cell types has been implicated in different immune functions. In particular, activin and TGFbeta play an important role in the haematopoietic tissue. They are critical death inducers in the immune system contributing to the elimination of different activated immune cell types. Control of immune cell proliferation, activation and subsequent elimination of activated cell populations by cell growth arrest and apoptosis are critical events for controlling infections and preventing autoimmune disease. However, very limited information about the downstream target genes and their signaling mechanism that relay on the inhibitory effects on cell growth by activin and TGFbeta ligands. Using a screen for genes that are differentially regulated by activin and TGFbeta in haematopoietic cells, we found that the phospholipids phosphatase SHIP-1 was strongly upregulated by activin and TGFbeta. Thus, we hypothesized that TGFbeta and activin induce cell growth arrest in immune cells through up-regulation of SHIP-1 with a significant and subsequent decrease in PtdIns 3,4,5-P3 levels affecting cell survival. Furthermore, we attempted to characterize the different intracellular signalling pathways downstream of these serine/threonine kinase receptors that lead to SHIP-1 overexpression as well as the transcription factors involved in the mediation of transcriptional regulation of the SHIP-1 gene promoter. / Chapter 1 provides a broad introduction to the field of TGFbeta signaling focusing on TGFbeta-induced apoptosis in immune cell types and the biology of inositol phosphatases involved in phospholipide metabolism, mainly focused on SHIP-1. Chapter 2 contains data demonstrating that the activin/TGFbeta-induced cell growth arrests and apoptosis through expression of SHIP-1. Data in chapter 3 proved evidences about the cross-talk between the Smad and JNK MAP kinase signalling pathways and their role in the transcriptional regulation of the SHIP-1 gene promoter. Finally, chapter 4, is focused on the discussion and the propose model of how activin/TGFbeta-induced SHIP-1 expression blocks induction of cell survival signals. As a dynamic cellular and molecular process the induction of SHIP-1 by TGFbeta ligands might be in co-association with other apoptosis molecules leading to cell growth arrest and apoptosis in different cell populations of the immune system.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: c-Src-Associated PTPase. Includes bibliographical references (169-197). Also available online through Digital Dissertations.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 15, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Kent S. Gates. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Synaptic role of TYR-phosphatases. Includes bibliographical references (p. 135-172). Also available online through Digital Dissertations.