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

Stefan, Eduard; Aquin, S.; Berger, Nathalie; Landry, Christian R.; Nyfeler, Beat; Bouvier, Michel; Michnick, Stephen W.

doi:10.1073/pnas.0704257104

Cited by 174 publications

(184 citation statements)

References 47 publications

Supporting

Mentioning

179

Contrasting

Order By: Relevance

“…To substantiate our results, we performed in vivo split-luciferase complementation assays, which allow the analysis of dynamic protein-protein interactions (Stefan et al, 2007). As expected, the wild-type U-box domains were able to interact, as evidenced by the luminescence due to luciferase reconstitution ( Figure 5F; Supplemental Figure 8A).…”

Section: Phosphorylation Of Thr-62 Regulates Dimerizationmentioning

confidence: 99%

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

et al. 2017

View full text Add to dashboard Cite

Crosstalk between posttranslational modifications, such as ubiquitination and phosphorylation, play key roles in controlling the duration and intensity of signaling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here, we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signaling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and, thus, autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo.

show abstract

Section: Phosphorylation Of Thr-62 Regulates Dimerizationmentioning

confidence: 99%

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

et al. 2017

View full text Add to dashboard Cite

show abstract

“…The Renilla luciferase-based PCA assay has been described in detail previously (33). PCA assays, SPOT synthesis and overlay experiments, cAMP-agarose precipitations and phosphorylation assays have been described (18) and are in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Reciprocal regulation of PKA and Rac signaling

Bachmann

Riml

Huber

et al. 2013

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

View full text Add to dashboard Cite

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac-bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTPRac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA-boosted activation of Raf-Mek-Erk. Consequently GTPRac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac-mediated activation of nuclear Erk1/2 signaling. signal transduction | cross-talk S ignal transduction cascades coordinate the plethora of extracellular stimuli into biological responses within cells. The specificity of receptor-initiated signaling responses is encoded by spatial and temporal dynamics of downstream signaling networks (1). These networks, initiating from e.g., the G protein-coupled receptor (GPCR) superfamily and receptor tyrosine kinases (RTK), tightly regulate signaling pathways at several critical points via feedback loops and cross-talk among other pathways (2-5). A large number of GPCR signaling cascades uses cAMP as an intracellular second messenger (3, 6). In response to hormone binding to distinct GPCRs, cAMP is produced and binds to its canonical effector, the cAMP-dependent protein kinase A (PKA). cAMP binding to the PKA regulatory subunits (R) induces dissociation of the tetrameric PKA holoenzyme, resulting in active PKA catalytic subunits (PKAc; Fig. 1C) (7, 8). To ensure substrate specificity, PKA is tethered to distinct subcellular compartments through physical interaction with A-kinase anchoring proteins (AKAPs; refs. 9 and 10). It has been long regarded that the...

show abstract

“…Rluc bioluminescence signals were integrated for 10 s following addition of the Rluc substrate benzylcoelenterazine to intact cells (5 μM; NanoLight). For immunoblot analyses, anti-Renilla luciferase antibodies to detect either F[1]-fused (Millipore; MAB4410) or F[2]-fused (Millipore; MAB4400) hybrid proteins were used (23,24).…”

Section: Methodsmentioning

confidence: 99%

“…Rluc-based PCA sentinels have been designed and used to study the dynamics of PPIs in vivo (23,24). An advantage of this assay is that it reports absolute values of protein-complex formation in real time.…”

Section: A Library Of Pyridine Compounds Yields Effective Inhibitors mentioning

confidence: 99%

Inhibitor of MYC identified in a Kröhnke pyridine library

Hart¹,

Garner

et al. 2014

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

115

131

View full text Add to dashboard Cite

In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The K d of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.M YC is a transcriptional regulator that occupies an apex position in the organizational hierarchy of the cell (1-3). It belongs to a family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins that dimerize with the small bHLH-LZ protein MAX to become functional (4). The MYC-MAX heterodimer preferentially binds to the palindromic DNA sequence CACGTG, referred to as the E-box motif. As a transcription factor, MYC can bind to the promoters of target genes to stimulate or repress transcriptional activity (5-7). The human genome contains three MYC genes, c-MYC, N-MYC, and L-MYC. Throughout this paper, we will use "MYC" to indicate the protein product of the c-MYC gene.MYC is involved in almost all cancers (8, 9). It is rarely mutated, but achieves gain of function through overexpression or amplification. Because of this broad pathogenic significance, MYC is an important cancer target. However, both conceptual and practical difficulties have stood in the way of identifying potent and effective small-molecule inhibitors of MYC. The conceptual obstacles reflect concern about inhibiting a gene that controls essential cellular activities. Because MYC plays an important role in cell proliferation (10, 11), it is often argued that inhibition of this function would lead to broad and unacceptable side effects in vivo. However, studies with the dominant-negative MYC construct Omomyc have shown that inhibiting MYC has only mild and rapidly reversible effects on normal, fast-proliferating tissues (8,12,13). The main practical difficulty in targeting MYC is the absence of pockets or grooves that could serve as binding sites for small molecules (14).The preferred strategy for the identification of potential MYC inhibitors has been interference with MYC-MAX dimerization (15-18). The formation of the MYC-MAX heterodimer involves the bHLH-LZ domains of the two partner molecules with a protein-protein interaction (PPI) surface of ∼3,200 Å 2 . This surface lacks well-defined binding sites for small molecules and therefore is widely considered as "undruggable." However, despite the large interaction surface, a single-amino acid substitution can completely disrupt the dimerization of MYC with MAX (14...

show abstract

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

Cited by 174 publications

References 47 publications

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

Reciprocal regulation of PKA and Rac signaling

Inhibitor of MYC identified in a Kröhnke pyridine library

Contact Info

Product

Resources

About