Light-mediated remote control of signaling pathways

Cell signaling networks display an extraordinary range of temporal and spatial plasticity. Our programmatic approach focuses on the construction of intracellular probes, including sensors, inhibitors, and functionally unique proteins that can be temporally and spatially controlled by the investigator even after they have entered the cell. We have designed and evaluated protein kinase sensors that furnish a fluorescent readout upon phosphorylation. In addition, since the sensors are inert (i.e. cannot be phosphorylated) until activated by light, they can be carried through the various stages of any given cell-based behavior without being consumed. Using this strategy, we have shown that PKCβ is essential for nuclear envelope breakdown and thus the transition from prophase to metaphase in actively dividing cells. Photoactivatable proteins furnish the means to initiate cellular signaling pathways with a high degree of spatial and temporal control. We have used this approach to demonstrate that cofilin serves as a component of the steering apparatus of the cell. Finally, inhibitors are commonly used to assess the participation of specific enzymes in signaling pathways that control cellular behavior. We have constructed a photo-deactivatable inhibitor, an inhibitory species that can be switched off with light. In the absence of light, the target enzyme is inactive due to the presence of the potent inhibitory molecule. Upon photolysis, the inhibitory molecule is destroyed and enzymatic activity is released

Dual Wavelength Photoactivation of cAMP- and cGMP-Dependent Protein Kinase Signaling Pathways

The spatial and temporal organization of biological systems offers a level of complexity that is challenging to probe with conventional reagents. Photoactivatable (caged) compounds represent one strategy by which spatiotemporal organizational complexities can be addressed. However, since the vast majority of caged species are triggered by UV light, it is not feasible to orthogonally control two or more spatiotemporal elements of the phenomenon under investigation. For example, the cGMP- and cAMP-dependent protein kinases are highly homologous enzymes, separated in time and space, which mediate the phosphorylation of both distinct and common protein substrates. However, current technology is unable to discriminate, in a temporally or spatially selective fashion, between these enzymes and/or the pathways they influence. We describe herein the intracellular triggering of a cGMP-mediated pathway with 360 nm light and the corresponding cAMP-mediated pathway with 440 nm light. Dual wavelength photoactivation was assessed in A10 cells by monitoring the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a known substrate for both the cAMP- and cGMP-dependent protein kinases. Illumination at 440 nm elicits a cAMP-dependent phosphorylation of VASP at Ser157 whereas 360 nm exposure triggers the phosphorylation of both Ser157 and Ser239. This is the first example of wavelength-distinct activation of two separate nodes of a common signaling pathway

Light-Mediated Spatial Control via Photolabile Fluorescently Quenched Peptide Cassettes

Light-regulatable compounds are finding increasing utility as spatial and temporal probes of biological behavior. An independent measure of successful light-induced structural change is possible when alteration (e.g., activation, deactivation, etc.) of the bioprobe can be directly linked to a fluorescent readout. We have identified a series of photolabile fluorescently quenched cassettes that display extraordinarily large fluorescence enhancements upon photolysis. A pair of cassettes has been inserted into mitochondrial localization sequences to assess an organelle-targeted light-mediated release strategy for controlling biological activity. The peptide constructs are readily absorbed by mitochondria and subsequently can be cleaved in a light-dependent fashion as assessed by the predicted changes in absorbance and fluorescence

Merging of Confocal and Caging Technologies: Selective Three-Color Communication with Profluorescent Reporters

Falling apart, on cue: Signaling pathways often display a profound spatiotemporal component that is best studied using light-activatable reagents. Three separate photolabile moieties that can be distinguished based upon their response to three distinct wavelengths (360, 440, and 560 nm) have been synthesized and evaluated. This tri-color system is also applied to imaging in microwells and HeLa cells (see picture)

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

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

How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli

The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19) is essential for the biosynthesis of aromatic compounds in plants and microbes and is the unique target of the herbicide glyphosate. One of the first glyphosate-insensitive enzymes reported was a Gly96Ala mutant of EPSP synthase from Klebsiella pneumoniae. We have introduced this single-site mutation into the highly homologous EPSP synthase from Escherichia coli. The mutant enzyme is insensitive to glyphosate with unaltered affinity for its first substrate, shikimate-3-phosphate (S3P), but displays a 30-fold lower affinity for its second substrate, phosphoenolpyruvate (PEP). Using X-ray crystallography, we solved the structure of Gly96Ala-EPSP synthase liganded with S3P to 0.17 nm resolution. The crystal structure shows that the additional methyl group from Ala96 protrudes into the active site of the enzyme. While the interactions between enzyme and S3P remain unaffected, the accessible volume for glyphosate binding is substantially reduced. Exploiting the crystallographic results for molecular modeling, we demonstrate that PEP but not glyphosate can be docked in the Gly96Ala-modified binding site. The predicted PEP binding site satisfies the earlier proposed interaction pattern for PEP with EPSP synthase and corroborates the assumption that glyphosate and PEP target the same binding site

Multicolor Monitoring of the Proteasome’s Catalytic Signature

The proteasome, a validated anticancer target, participates in an array of biochemical activities, which range from the proteolysis of defective proteins to antigen presentation. We report the preparation of biochemically and photophysically distinct green, red, and far-red real-time sensors designed to simultaneously monitor the proteasome’s chymotrypsin-, trypsin-, and caspase-like activities, respectively. These sensors were employed to assess the effect of simultaneous multiple active site catalysis on the kinetic properties of the individual subunits. Furthermore, we have found that the catalytic signature of the proteasome varies depending on the source, cell type, and disease state. Trypsin-like activity is more pronounced in yeast than in mammals, whereas chymotrypsin-like activity is the only activity detectable in B-cells (unlike other mammalian cells). Furthermore, chymotrypsin-like activity is more prominent in transformed B cells relative to their counterparts from healthy donors

Dual Wavelength Photoactivation of cAMP- and cGMP-Dependent Protein Kinase Signaling Pathways

The spatial and temporal organization of biological systems offers a level of complexity that is challenging to probe with conventional reagents. Photoactivatable (caged) compounds represent one strategy by which spatiotemporal organizational complexities can be addressed. However, since the vast majority of caged species are triggered by UV light, it is not feasible to orthogonally control two or more spatiotemporal elements of the phenomenon under investigation. For example, the cGMP- and cAMP-dependent protein kinases are highly homologous enzymes, separated in time and space, which mediate the phosphorylation of both distinct and common protein substrates. However, current technology is unable to discriminate, in a temporally or spatially selective fashion, between these enzymes and/or the pathways they influence. We describe herein the intracellular triggering of a cGMP-mediated pathway with 360 nm light and the corresponding cAMP-mediated pathway with 440 nm light. Dual wavelength photoactivation was assessed in A10 cells by monitoring the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a known substrate for both the cAMP- and cGMP-dependent protein kinases. Illumination at 440 nm elicits a cAMP-dependent phosphorylation of VASP at Ser157 whereas 360 nm exposure triggers the phosphorylation of both Ser157 and Ser239. This is the first example of wavelength-distinct activation of two separate nodes of a common signaling pathway