Browsing by Subject "cell signaling"
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Item Identification and Characterization of Effectors/Binding Molecules for the Small GTPase Rab15(2005-03-18) David Jay Strick; Lisa A. Elferink, Ph.D.; Pomila Singh, Ph.D.; Ping Wu, M.D. Ph.D.; Nancy K. Wills, Ph.D.; Mary L. Thomas, Ph.D.; Gregg T. Nagle, Ph.D.; Brian J. Knoll, Ph.D.Endocytic trafficking is a key mechanism for regulating receptor availability on\r\nthe plasma membrane as well as receptor degradation. Clathrin-dependent endocytosis\r\ninvolves receptor internalization into early endosomes. Here internalized receptors are\r\nsorted for degradation in lysosomes, direct recycling back to the cell surface or indirect\r\nrecycling via a second recycling compartment called the pericentriolar recycling\r\nendosome. Rab GTPases regulate specific membrane trafficking steps including vesicle\r\nbudding, vesicle transport and fusion with downstream target compartments. Rab\r\nfunction is mediated by the cyclical binding and hydrolysis of GTP, which in turn\r\nregulates the recruitment of downstream effector molecules directly involved in\r\nmembrane transport steps. This dissertation focuses on the endocytic GTPase Rab15.\r\nRab15 localizes to early and pericentriolar recycling endosomes, and differentially\r\nregulates receptor transport at these distinct organelles. For example, over expression of\r\nGTP-bound Rab15 inhibits internalization of the Transferrin Receptor and inhibits\r\nhomotypic endosome fusion in vitro. Conversely, over expression of Rab15-GDP\r\ndifferentially stimulates Transferrin receptor recycling from the early endosome and\r\npericentriolar recycling endosome respectively. Rab15 may differentially regulate\r\nreceptor trafficking through these distinct endocytic compartments by binding\r\ncompartment specific effectors. To test this hypothesis, I performed yeast two-hybrid\r\nscreens to identify and characterize Rab15 binding partners. This dissertation is the\r\nfunctional characterization of three Rab15 binding proteins; Mammalian Suppressor of\r\nSec4, Rab15 Effector Protein and Rab15 Binding Protein. Using molecular, biochemical\r\nand imaging approaches, I demonstrated that interactions between Rab15 and Mss4\r\nmodulate the inhibitory effect of Rab15-GTP on receptor entry into early endosomes.\r\nThe second binding partner, Rab15 Effector Protein, localized specifically to the\r\npericentriolar recycling endosome where it regulated Transferrin receptor recycling back\r\nto the cell surface. Finally, Rab15 Binding Protein is a neural specific protein of\r\nunknown function, suggesting an important regulatory function for Rab15 in neural\r\nreceptor trafficking. These results confirm that Rab15 is a bi-functional GTPase, which\r\ndifferentially regulates receptor trafficking through early and pericentriolar recycling\r\nendosomes, by binding specific effector proteins. Moreover, identification of putative\r\nRab15 effector molecules further defines the endocytic pathway, thus providing valuable\r\ninformation for the characterization of trafficking-related diseases and potential drug\r\ntargets in the future.Item A mechanism of activation of c-MET receptor tyrosine kinase(2006-06-23) Payal Sheth; Lisa A. Elferink; Vincent J. Hilser; Stanley J. Watowich; Rolf Konig; James C. Lee; Bing Suc-MET receptor tyrosine kinase-mediated signaling governs numerous important cellular responses including cellular proliferation, differentiation, migration and apoptosis. Deregulation of these signals result in malignant behaviors, often leading to cancers. While the identity of the many signaling molecules that are activated following hepatocyte-growth factor (HGF)-induced activation of c-MET had been established, little was known about the mechanism of activation of c-MET. From a therapeutic perspective, it is necessary to understand the detailed molecular mechanisms regulating c-MET activation to selectively target these molecules. c-MET, in presence of its cognate ligand, is oligomerized, and is autophosphorylated on specific tyrosines on its cytoplasmic domain. The phosphorylated tyrosines in specific sub-domains of c-MET cytoplasmic region perform specific functions including increase in catalytic activity and recruitment of effector molecules. Classically, it has been believed that the sole role of ligand-induced oligomerization was to autophosphorylate the receptor, thereby switching the receptor’s kinase activity on. However, in light of a recent body of evidence suggesting that certain RTKs are kinase active on cell surface in absence of ligand-induced oligomerization, we hypothesized that oligomerization could be important for other aspects of RTK activation. Using c-MET as our model system, we investigated the role of oligomerization, irrespective of its role in autophosphorylation, in regulating c-MET activation. Previous studies from our laboratory have conclusively shown that oligomerization increases c-MET’s substrate binding affinity and substrate phosphorylation kcat. The work presented here addresses the role of oligomerization in regulating c-MET’s susceptibility to dephosphorylation, another important regulator of c-MET activation. The biochemical parameters measured for c-MET are used to build a unified kinetic model for c-MET activation. The model building and its subsequent validation using cell culture experiments are described here. Furthermore, the model is probed using parameter sensitivity analyses to understand how oligomerization-induced changes in the kinetic, thermodynamic and dephosphorylation properties of c-MET work synergistically to selectively induce specific signaling from the dimeric and not the monomeric receptor. Using these data, we propose an alternative feed-forward model for c-MET activation mechanism differs from the traditional view of the RTK activation.Item Regulation of hepatocyte growth factor receptor endocytic trafficking(2006-03-21) Ning Li; Stan Watowich, Ph.D.; Sarita Sastry, Ph.D.; Lisa A. Elferink, Ph.D.; Cornelis Elferink, Ph.D.; Andrew Bean, Ph.D.The Hepatocyte Growth Factor Receptor (HGFR/cMet) is a receptor tyrosine kinase that is essential for multiple cell responses, including cell proliferation, survival, motility and branching morphogenesis. Normal HGFR signaling regulates embryonic development, organ regeneration and wound healing, whereas deregulated HGFR signaling is linked to tumor progression, metastasis and angiogenesis. Ligand activation of HGFR leads to receptor downregulation via endocytosis and lysosomal degradation, the major mechanism for terminating HGFR signaling. Perturbation of HGFR trafficking, either at the level of internalization or during sorting at the early endosome for degradation, leads to oncogenic activation of HGFR. Impaired HGFR trafficking is emerging as a key mechanism for HGFR-induced cancer progression and metastasis. \r\nA major goal of my dissertation was to lay the foundation for future studies examining different mechanisms leading to altered HGFR trafficking in human cancers, by determining and characterizing the mechanisms which normally function to regulate HGFR internalization and degradation. Two ligands have been identified for HGFR, the physiological ligand HGF and the Listeria surface protein Internalin B (InlB). I characterized the mechanisms for InlB and HGF induced HGFR trafficking using a combination of imaging, molecular biological and biochemical approaches. First, I demonstrated that InlB and HGF were mechanistically equivalent in triggering HGFR internalization primarily through clathrin-coated pits. Then, I determined that the Y1349 and Y1356 docking sites and tyrosine kinase activity of HGFR were required for receptor internalization. Recruitment of the adaptor protein Grb2, but not Gab1, was essential for ligand induced HGFR internalization. I then showed Cbl, an E3 ubiquitin ligase recruited by Grb2 to HGFR, played an essential role in receptor internalization. Furthermore, the E3 ligase activity of Cbl and ubiquitination machinery was involved in HGFR internalization. Finally, I demonstrated that ligand induced HGFR degradation occurred through the lysosomal pathway, involving the function of Hrs and PI3K. My studies represent the first detailed characterization of the trafficking events that normally function to inactivate HGFR signaling. My findings contribute to better understanding of how HGFR-induced tumorigenesis and tumor metastasis may result from impaired HGFR trafficking, and identify novel mechanisms that may function as therapeutic targets for treatment of human cancers due to impaired HGFR trafficking.\r\nItem Signaling pathways regulating self-renewal, differentiation, and multipotency of CD133+ umbilical cord blood stem cells(2008-12-17) Margaret Corbett Howe; Larry Denner; Ronald Tilton; Randall Urban; Ping Wu; Mark Evers; Chunming Liu; Austin CooneyOur goal is to increase the number of immature umbilical cord blood stem cells (UCBSCs) for hematopoietic transplantation. Towards this goal, our lab adapted a culture technique to grow immature CD133+ UCBSCs (CD133+ cells). Although CD133+ cells proliferate rapidly in culture, a minority self-renew and remain CD133+, while a majority differentiate and become CD133-. Therefore, new strategies to identify and grow immature UCBSCs are important. Since little is known about signally mechanisms regulating self-renewal and differentiation of UCBSCs, we sought insight from embryonic stem cell (ESC) literature to guide our studies. \r\nTo identify a population of UCBSCs that grow without differentiating, we focused on Oct-4, a transcription factor essential for self-renewal in ESCs that we previously reported expression in UCBSCs. During our studies, new challenges in the field arose. Two isomers of Oct-4 were discovered, Oct-4A and Oct-4B 3, in which only Oct-4A conferred the ability of ESCs to self-renew 4. We redesigned our experiments to detect Oct-4A and discovered that freshly isolated CD133+ cells expressed Oct-4A mRNA and protein. Since these cells proliferated in culture, they lost expression of adult stem cell markers including CD133, and gained markers of hematopoietic differentiation. However, Oct-4A mRNA and protein were expressed regardless of the differentiation status. Therefore, Oct-4A, despite its essential roles in ESCs, neither defined nor conferred self-renewal of CD133+ cells.\r\nTo discover strategies to grow immature CD133+ cells without differentiation, we focused on the Wnt pathway which is essential for self-renewal in ESCs. Differentiation of CD133+ cells to CD133- cells corresponded to down-regulation of Wnt signaling. Pharmacological activation of the Wnt pathway by (2’Z,3’E)-6-Bromoindirubin-3’-oxime (BIO) inhibition of GSK-3beta resulted in accelerated differentiation, instead of decreased differentiation, of CD133+ cells. BIO-treated CD133- cells that were differentiated maintained multipotency while proliferating at similar rates to vehicle-treated CD133+ cells that self-renewed. Therefore, inhibition of GSK-3beta could be a strategy for differentiating CD133+ cells into hematopoietic progenitor cells while maintaining their proliferation capacity. \r\nIn conclusion, this project demonstrated that pathways regulating UCBSC properties are not similar to pathways regulating ESCs properties. Our findings are the first studies that derive UCBSCs properties of self-renewal, differentiation, and multipotency.\r\n