UVM Theses and Dissertations
Svec, Kathryn Victoria
Cellular, Molecular, and Biomedical Sciences Graduate Program
Cellular migration is a cornerstone of many biological processes, from the creation and repair of complex tissues and organs to the metastatic spread of cancer cells. This complex process involves the coordination of diverse cellular pathways and structures. While many molecular players have been identified, much remains unknown about how these structures and pathways work in concert to promote cellular motility. Previous work has revealed the pleiotropic serine/threonine kinase PKA (cAMP dependent protein kinase) as an important regulator of cell migration. PKA is dynamically regulated in protrusive structures of migrating cells and its activity is upstream of numerous GTPases and effectors involved in processes such as membrane protrusion/retraction, actin organization, and adhesion dynamics. The importance of PKA activity in migration extends to many different cell types. Although the canonical regulation of PKA occurs downstream of G protein coupled receptors, G proteins, and adenylyl cyclases, no unifying mode of PKA regulation in migration has been identified. Here we seek to understand the regulation of leading edge PKA activity in cellular migration. In this work, we show that leading edge PKA activity is dependent upon mechanical inputs. Disruption of actomyosin contractility leads to rapid ablation of leading edge (LE) PKA activity. Further, regions of high PKA activity spatially coincide with regions of high contractility. PKA is rapidly and locally activated by mechanical stretch of the substrate underlying cells, further defining a spatiotemporal relationship between PKA and contractile forces. Finally, inhibiting PKA activity blocks durotaxis, a mode of cell motility in which migration is directed by increases in matrix stiffness, further underscoring the importance of PKA as an important facet of mechanotransduction. However, the mechanism through which PKA activity is linked to mechanical inputs during cell migration remains unclear. Investigation into this link led to the discovery of a novel relationship between PKA and Focal Adhesion Kinase (FAK), a cytosolic tyrosine kinase required for migration and mechanosensation canonically distinct from cAMP and PKA signaling. Surprisingly, treatment of migrating cells with a specific FAK inhibitor leads to a rapid decrease in leading edge PKA activity. Also, cells genetically lacking FAK exhibit decreased LE PKA activity which is rescued by re-expression of WT, but not kinase-dead, FAK. Further, treatment with a myosin II inhibitor does not lead to immediate decreases in global active FAK but does have a rapid, temporary effect on active FAK in focal adhesions, suggesting that smaller pools of FAK may be dynamically regulated downstream of contractile forces. Lastly, preliminary studies find that Src family kinase (SFK) inhibitors similarly diminish PKA activity in migrating cells and that pre-treatment with FAK or SFK inhibitors decrease PKA response to a general [Beta] adrenergic receptor agonist. Together, these findings identify novel connections between mechanical signaling, tyrosine kinase activity, and the cAMP/PKA pathway critical for cell migration and, likely, other essential cellular processes regulated by the pleiotropic cAMP dependent protein kinase.
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