Abstract Adhesive interactions between cells and extracellular matrix proteins play a vital role in biological processes such as cell proliferation, differentiation and survival. Integrins comprise a major family of cell surface receptors that mediate these interactions. Integrin engagement triggers adhesion-dependent intracellular signalling cascades that include the phosphorylation of tyrosines in intracellular signalling proteins. Integrin-dependent signals act in concert with signals from growth factors and other signalling receptors. The objective of this thesis was to study how cell adhesion and growth factors interact with intracellular components to regulate cell behavior in normal and transformed cells. One of the main proteins phosphorylated following integrin ligation in several different cell types is the docking protein p130Cas (Cas), which is tyrosine phosphorylated after stimulation of cells with low concentrations of epidermal growth factor (EGF). Tyrosine-phosphorylated Cas associates with an adapter protein c-Crk, the main binding protein for Cas, suggesting a novel role for EGF in Cas signalling. The interaction of cells with a variety of agonists such as growth factors and integrin ligation results in stimulation of mitogen-activated protein kinases (MAPKs), which control the expression of genes important for many cell functions. Expression of Cas and Crk induces activation of C-Jun N-terminal kinases (JNKs), which are members of MAPK family. JNK activation induced by integrin ligand binding is blocked by the expression of a dominant-negative mutant of Cas or Crk demonstrating an important role for the Cas-Crk complex in integrin-mediated JNK activation. The proto-oncogene product p120Cbl (Cbl) was identified as the main tyrosine-phosphorylated protein following integrin ligation in hematopoietic cells of myeloid lineage. Tyrosine-phosphorylated Cbl interacts with and activates other signalling proteins, such as Src tyrosine kinase and phosphatidylinositol 3"-kinase (PI 3-kinase), thereby mediating adhesion-dependent signals in hematopoietic cells. Unlike the cellular Cbl, the transforming mutants of Cbl were tyrosine-phosphorylated in an adhesion-independent manner and interacted with and activated signalling molecules both in suspended and in adherent cells. Further, the oncogenic forms of Cbl induced anchorage-independent but serum-dependent proliferation of cells. These results support the view that transformation by Cbl results from constitutive activation of integrin-dependent rather than growth factor-dependent signalling events.

ABSTRCT In mammals protein tyrosine phosphorylation plays an important role in the activation of programmed cell death. However, tyrosine phosphorylation involved in cell death has not been examined in plants. These studies demonstrated that D-mannose induced cell death and DNA fragmentation in rice suspension cells. In the presence of mannose for 24 hours, tyrosine phosphorylation of two proteins, 20 kDa and 43 kDa markedly increased. After incubating 3 days, the level of phosphotyrosine accumulation declined in bands of 16 and 20 kDa. In addition, the occurrence of DNA fragmentation and two tyrosine-phosphorylated proteins, 26 kDa and 40 kDa, were detected in aged suspension-cultured cells. The expression of genes that encode mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK signalling pathway, OsMEK and OsMAPK2, are up regulated during D-mannose treatment. The results provide evidence that protein tyrosine phosphorylation as well as MEK/MAPK signalling pathway is associated with cell death in rice.

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Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 111-129).

Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 155-180). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

No / We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.

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<p>Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Although the temporal manner in which effectors are secreted is important for the proper manipulation of host cell functions, the mechanism remains a mystery. In this study, we provide several lines of evidence that T3S chaperones may impart coherence to effector secretion. In addition, we identified a new early T3S effector in Chlamydia. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. By defining proteins that associate with the three most abundant T3S chaperones, Slc1, Scc2 and Mcsc in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry, we identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form stable complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses and lack of C. trachomatis-induced morphological changes. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.</p> / Dissertation