Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET)

Prinz, Anke; Diskar, Mandy; Erlbruch, Andrea; Herberg, Friedrich W.

doi:10.1016/j.cellsig.2006.01.013

Cited by 62 publications

(72 citation statements)

References 46 publications

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“…COS-7 cells (ATCC CRL-1651) were cultivated in DMEM including 5% FBS Gold (PAA) as described before (27) and transfected in white 96-well microplates (Nunc), using 1 μL PEI (1 mg/mL; Polysciences) and 0.2 μg DNA per plasmid and well. About 1 h after transfection, cells were rinsed with PBS and cultivated for another ∼6 h in medium without FBS and subjected to treatments as indicated in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…To further complement in vitro studies of DARPin/ERK interaction, we designed bioluminescence resonance energy transfer (BRET) sensors to verify specific binding of selected DARPins to ERK and pERK directly within COS-7 cells. Several chimeras of selected DARPins E40, pE59, EpE82, the unselected library member E3_5, and ERK2 fused to the N and C termini of either Renilla luciferase (Rluc) (27) or a variant of green fluorescent protein (termed GFP 2 ) (27) were created. Successful expression of the combination of fusion proteins in COS-7 cells, to be used for BRET 2 assays (the superscript denoting the use of GFP 2 ; see Materials and Methods), was confirmed by Western blot analysis and immunofluorescence microscopy.…”

Section: Affinity Precipitation With Selected Erk Phosphorylation-spementioning

confidence: 99%

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Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

Kummer

Parizek

Rube

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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126

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We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state-specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.intrabodies | X-ray crystallography

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Section: Methodsmentioning

confidence: 99%

Section: Affinity Precipitation With Selected Erk Phosphorylation-spementioning

confidence: 99%

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

Kummer

Parizek

Rube

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

126

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“…Several cell-based assays have been developed to detect specific activation of PKA, including fluorescence and bioluminescent resonance energy transfer assays for detecting catalytic activity (10) or cAMP-induced PKA subunit dissociation (11)(12)(13). These methods have been invaluable to the study of protein complex dynamics and particularly to the integration of compartmentalized GPCR signaling pathways (14)(15)(16).…”

mentioning

confidence: 99%

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Stefan

Aquin²,

Berger³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

173

179

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The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-tobackground ratio to identify endogenously expressed G␣s proteincoupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive ␤-2 adrenergic-but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.G protein-coupled receptor ͉ complementation assays ͉ protein-protein interactions ͉ protein fragment G protein-coupled receptors (GPCRs) represent the largest family of cell-surface molecules involved in signal transmission. GPCRs play roles in a broad range of biological processes through regulating the majority of cell-to-cell and cell-toenvironment communication, and, consequently, their dysfunction manifests in numerous diseases (1, 2). The GPCR family has enormous pharmacological importance, as demonstrated by the fact that Ͼ30% of approved drugs elicit their therapeutic effect by selectively acting on known members of this family (3). The human genome harbors Ͼ800 putative GPCRs including a considerable number with unknown physiological function or ligands. GPCR cascades hence remain a major focus of molecular pharmacology (4, 5).Signal transduction by GPCRs is mediated by activation of protein kinases (4), among which the most intensively studied is the cAMP-dependent protein kinase A (PKA) (6). Various extracellular signals converge on the cAMP/PKA pathway through ligand binding to GPCRs. The adenylyl cyclase then converts ATP to the ubiquitous second messenger cAMP. Intrac...

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“…To date, only cyclic nucleotide analogs have been reported to bind to these sites. However, as the charged cyclic phosphate group is a strict requirement, these compounds exhibit poor in vivo efficacy 14 due to cell permeability and sensitivity to phosphodiesterases.…”

Section: Targeting Pka Allosteric Sitesmentioning

confidence: 99%

“…To prove probe specificity and demonstrate that this assay is not reporting artifacts such as protein or peptide aggregation, a truncated version of IP20, PKI (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), was shown to compete with FAM-IP20 for binding to C-subunit in a dose dependent manner, with an IC 50 of 2.7 µM (Figure 2c). …”

Section: Interaction Of Fam-ip20 With the Catalytic Subunitmentioning

confidence: 99%

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

et al. 2006

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Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named Ligand-Regulated Competition (LiReC), in an effort to find non-ATP competitive small molecule regulators for type Iα cAMP-dependent Protein Kinase A (PKA-Iα), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Iα complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.

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Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET)

Cited by 62 publications

References 46 publications

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

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