Caged Catalytic Subunit of cAMP-Dependent Protein Kinase

Chang, Chih-Yuan; Fernández, Tania Miñes; Panchal, Rekha G.; Bayley, Hagan

doi:10.1021/ja981649v

Cited by 56 publications

(39 citation statements)

References 30 publications

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“…G-actin was the first protein to be caged using this 'shotgun' approach17. Proteins such as PKA and cofilin have also been caged using a similar strategy 19 , 47 , 48 . There are several specific points to consider when planning the synthesis of a caged enzyme: (i) use of caged proteins is complicated by issues of residual activity of the caged protein (it is very difficult to have 100% caging, so a few percent residual activity may give rise to ambiguous results), (ii) recovery of activity after uncaging is problematic, especially when 'shotgun' caging is used as multiple sites must be uncaged for activation, and (iii) caged proteins must be microinjected into cells 46 .…”

Section: Caging Peptides and Proteinsmentioning

confidence: 99%

Caged compounds: photorelease technology for control of cellular chemistry and physiology

2007

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Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group (Fig. 1). Illumination results in a concentration jump of the biologically active molecule that can bind to its cellular receptor, switching on (or off) the targeted process. Virtually every kind of signaling molecule or second messenger, of every size| [mdash]|from protons to proteins| [mdash]|has been caged 1 .Why are caged compounds so useful? A single component of cellular chemistry can control the function of a cell, and such cellular regulation can be temporally or spatially defined, intracellular or extracellular, and amplitude-or frequency-modulated 2 . Photomanipulation of cellular chemistry using caged compounds provides a uniquely powerful means to interact with such cellular dynamics, as it can touch upon any one of the above dimensions. Thus, since light passes through cell membranes, uncaging can rapidly release a biomolecule in an intracellular compartment. This space is not readily accessible to many second messengers (for example, inositol-1,4,5-trisphosphate (IP 3 ), ATP, Ca 2+ , cAMP, cGMP) when they are applied to cells externally, as their charge makes them impermeable to the plasma membrane. Furthermore, uniform illumination results in release throughout the cytosol, or the release can be localized by focusing the uncaging beam on one part of a cell. Likewise, extracellular uncaging of neurotransmitters and hormones is tunable, allowing stimulation of many neurons simultaneously or of single synapses| [mdash]|by global or focused illumination, respectively. Light cannot only be directed, but also modulated in time andCorrespondence should be addressed to Graham C R Ellis-Davies (cagedca@hotmail.com). (Fig. 2) has been widely used to study many Ca 2+ -controlled processes. In particular, secretory processes in neuronal and non-neuronal cells have been extensively studied with caged calcium 22 . Rapid uncaging of glutamate using two-photon photolysis is particularly useful for the rational stimulation of visually identified synapses in complex tissue preparations such as acutely isolated brain slices from the hippocampus 23 (Fig. 2b). Finally, uncaging of mRNA in vivo in zebrafish is an excellen...

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Section: Caging Peptides and Proteinsmentioning

confidence: 99%

Caged compounds: photorelease technology for control of cellular chemistry and physiology

2007

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“…[9] Several other proteins were reversibly inhibited by addition of a nitrobenzyl group to either a residue that is responsible for A C H T U N G T R E N N U N G inactivation or activation by phosphorylation. [10][11][12] Also, the activity of the well studied E. coli b-galactosidase was recovered after inhibition by a covalently attached nitrobenzyl derivate to a methionine residue in the active site. [13] Caging of proteins by conjugation with a photocleavable group is a powerful approach for reversibly blocking enzymatic activity.…”

Section: Introductionmentioning

confidence: 99%

Reversible Inactivation of the CG Specific SssI DNA (Cytosine‐C5)‐Methyltransferase with a Photocleavable Protecting Group

et al. 2007

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Caging of proteins by conjugation with a photocleavable group is a powerful approach for reversibly blocking enzymatic activity. Here we describe the covalent modification of the bacterial SssI DNA methyltransferase (M.SssI) with the cysteine-specific reagent 4,5-dimethoxy-2-nitrobenzylbromide (DMNBB). M.SssI contains two cysteine residues; replacement of the active-site Cys141 with Ser resulted in an approximately 100-fold loss of enzymatic activity; this indicates an important role for this residue in catalysis. However, replacement of Cys368 with Ala did not affect methyltransferase activity. Treatment of the Cys368Ala mutant enzyme with DMNBB led to an almost complete loss of activity. Irradiation of the inactivated enzyme with near-ultraviolet light (320-400 nm) restored 60 % of the catalytic activity. This indicates that caging by DMNBB can be used for the reversible inactivation of M.SssI.

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“…A variety of proteins have been created previously in a lightactivated form by caging critical functional groups, often to intervene within signal-transduction pathways, [21,27,46] to control rapid and transient processes such as ion channel gating [24] and cytoskeletal rearrangement, [47] or to control membrane permeability [20] (see refs. [48] and [49] for comprehensive reviews).…”

Section: Discussionmentioning

confidence: 99%

“…[20][21][22][23][24][25][26][27][28][29][30][31][32] In this approach, a photolabile protective group masks a critical functional element of the protein, yielding an inactive species. Photolysis of the masking group "uncages" the protein in its active composition.…”

Section: Introductionmentioning

confidence: 99%

Manipulating Cell Migration and Proliferation with a Light‐Activated Polypeptide

et al. 2009

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Remote control of cells: A polypeptide has been made that stimulates proliferation and migration of cells upon photochemical activation. This light-activated polypeptide enables spatially defined control of cell populations at the scale of tissue organization; this is accomplished without physically contacting the cells or modifying their substrate. Polypeptide growth and differentiation factors modulate a wide variety of cell behaviors and can be used to manipulate cells in vitro for tissue engineering and basic studies of cell biology. To emulate in vitro the spatial aspect of growth factor function, new methods are needed to generate defined spatial gradients of activity. Polypeptide factors that are engineered to be activated with light provide a method for creating concentration gradients with the fine precision in space and time with which light can be directed. As a first test of this approach, we have chemically synthesized a polypeptide with the sequence of epidermal growth factor in which a critical glutamate is "caged" with a photoremovable group. Photolysis of this polypeptide afforded maximal mitogenic and chemokinetic activity at concentrations at which the caged factor was inactive. Spatially resolved photolysis of the factor resulted in spatial patterning of fibroblasts. This system will be useful for ex vivo tissue engineering and for investigating the interactions of cells with their matrix and the role of chemical gradients in biological pattern formation.

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Caged Catalytic Subunit of cAMP-Dependent Protein Kinase

Cited by 56 publications

References 30 publications

Caged compounds: photorelease technology for control of cellular chemistry and physiology

Caged compounds: photorelease technology for control of cellular chemistry and physiology

Reversible Inactivation of the CG Specific SssI DNA (Cytosine‐C5)‐Methyltransferase with a Photocleavable Protecting Group

Manipulating Cell Migration and Proliferation with a Light‐Activated Polypeptide

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