Defining the Molecular Mechanisms that Regulate SHIP1 Activity and Membrane Localization in Immune Cells
Loading...
Date
2023-03-24
Authors
Waddell, Grace
Journal Title
Journal ISSN
Volume Title
Publisher
University of Oregon
Abstract
Spatial heterogeneity in many membrane proximal signaling reactions emerges from biochemical reactions involving phosphatidylinositol phosphate (PIP) lipids, kinases, phosphatases, and Rho-family GTPases. Interconnected positive and negative feedback loops control the communication between these distinct families of signaling molecules to regulate cell polarization, cortical oscillations, and transient spikes in biochemical activities. Together, these networks of biochemical reactions provide the molecular basis for signal adaptation modules that control cell polarity and migration. Of particular importance to these cellular processes is the hematopoietic-cell-specific lipid phosphatase, SHIP1, which regulates the dephosphorylation of PI(3,4,5)P3 lipids to generate PI(3,4)P2. We find that in non-migratory PLB-985 cells, SHIP1 colocalizes to cortical oscillations with Cdc42 GTPase and FBP17. In the presence of chemoattractant, SHIP1 polarizes to leading-edge membranes of migrating cells. Molecular dissection of SHIP1 domain organization and activities identified a minimal C-terminal motif that is necessary and sufficient for targeting to SHIP1 to cortical oscillations. Using supported lipid bilayers to biochemically characterize SHIP1 revealed that the full-length protein is autoinhibited. SHIP1 autoinhibition is predominantly regulated by its N-terminal SH2 domain and can be relived with the addition of immune receptor derived phosphotyrosine peptides. Finally, single molecule dwell time measurements in vitro and in vivo revealed that interactions between SHIP1 and various lipid species are surprisingly weak and likely serve a secondary role following membrane recruitment mediated by SHIP1-protein interactions. This dissertation includes unpublished co-authored material.