Signaling Mechanisms On Membrane Surfaces: Studies of Membrane-Bound Master Kinases

Buckles, Thomas

Graduate Thesis Or Dissertation

Signaling Mechanisms On Membrane Surfaces: Studies of Membrane-Bound Master Kinases 公开 Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/1n79h540w

Abstract

The leading edge pseudopod of polarized chemotaxing eukaryotic cells is a membrane associated signaling platform integral to movement, gene expression, growth and proliferation. In spontaneously polarized leukocytes such as macrophages, for example, the leading edge pseudopod guides cell movement up attractant gradients during the innate immune response. To accomplish this, a complex network of kinases and other proteins, including PI3K, AKT1, Ras superfamily G-proteins and RTKs assemble at the leading edge membrane where they control pseudopod stability and the direction of migration. More broadly, many of the same proteins assemble on membrane surfaces in growing cells where they control normal cell growth and survival, as well as oncogenesis in many human cancers. Despite their importance to practical applications and human health, the protein-protein interactions and regulatory mechanisms of these proteins’ interactions remain largely unresolved. The first part of this research focuses on elucidating the mechanism by which Ras and soluble phosphopeptides synergistically activate PI3K. Specifically, the small G-protein Ras is known to bind and activate the lipid kinase PI3K, which phosphorylates the constitutive plasma membrane lipid phosphatidyl-inositol-(4,5)-bisphosphate (PIP₂) thereby generating the crucial signaling lipid phosphatidylinositol-(3,4,5)-bisphosphate (PIP₃). This Ras-PI3K-PIP₃ signal regulates cell migration through cytoskeletal and membrane reorganization. In cell growth and survival pathways, the same Ras-PI3K-PIP₃ signal stimulates normal cell growth, while excessive PIP₃generation can trigger or support oncogenesis leading to many human cancers. Consequently, Ras and PI3K are important oncogenes, such that about one quarter of all human tumors show mutations in a member of the Ras family, and over 200 oncogenic mutations linked to human cancers have been identified in PI3Kα. Two contrasting mechanisms of Ras activation of PI3K have been discussed in the literature: one proposes that Ras recruits additional PI3K to the membrane surface, while the other proposes that Ras binding allosterically activates the PI3K catalytic site. In vitro single molecule TIRF microscopy was employed to resolve these hypotheses by measuring the lipid kinase turnover rate of PI3K and its membrane surface binding density in the presence and absence of Ras. The findings directly reveal the mechanism of synergistic PI3K activation by Ras and soluble phosphopeptides: Ras is found to dramatically increase the membrane recruitment of PI3K rather than allosterically activating its catalytic site. The second part of this work focuses on the effects of common therapeutics on the leading edge pseudopod of polarized macrophages and illustrates simple cell morphology and PIP₃signaling assays employing live cell fluorescence and DIC microscopy to quantify these effects, both at a single-cell and population level. The findings reveal that certain drugs do not detectably perturb the leading edge, while rapid addition of other drugs causes temporary leading edge pseudopod collapse followed by slow adaptation and recovery. Specifically, both ibuprofen and acetaminophen, over the counter pain therapeutics, cause leading edge collapse when added rapidly to polarized macrophages, followed by a full recovery within three hours. Live cell fluorescent and DIC microscopy was used to address this effect. The results suggest the need to identify drugs that perturb the leading edge pseudopod of macrophages and other leukocytes, and to administer those drugs sufficiently slowly to avoid pseudopod collapse and likely inhibition of the innate immune response.

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