An Optogenetic Toolkit for Spatial and Temporal Control of the cAMP Dependent Protein Kinase Public Deposited

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  • February 22, 2019
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  • Hughes, Robert M.
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
  • Lawrence, David
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
  • Whicker, Alex R.
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
  • O'Banion, Colin Padraic
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
  • Priestman, Melanie A.
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
Abstract
  • Cellular signaling is highly compartmentalized in both time and space as exemplified by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. PKA and associated signaling proteins are sequestered to specific subcellular compartments by A-kinase anchoring proteins to generate distinct signaling microenvironments. These signaling nodes provide spatial specificity to PKA so that this otherwise ubiquitous signaling pathway is only activated in the right location and at the right time. Although many tools are available to manipulate cellular signaling on a global scale, it is difficult to control intracellular signaling with any degree of spatiotemporal resolution. Here, we present a set of optogenetic tools to control PKA signaling at the plasma membrane, cytoskeleton, and outer mitochondrial membrane. We used molecular engineering approaches in conjunction with biochemical and cell biology assays such as western blotting and fluorescent microscopy to show that activation of our optogenetic toolset in cells results in compartment specific PKA phosphorylation events upon stimulation with light, and that activity is localized to discrete locations within the cell using a PKA reporter system generated by our group. Abbreviations: Photoactivated adenylate cyclase (PAC, AC), Nucleus (Nu), Plasma Membrane (PM), Outer Mitochondrial Membrane (OMM), Vasodilator Stimulated Phosphoprotein (VASP)
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  • In Copyright
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  • American Society for Biochemistry and Molecular Biology. Annual Meeting. (2016: San Diego, CA)
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