Intramolecular Conformational Changes Optimize Protein Kinase C Signaling

Antal, Corina E.; Violin, Jonathan D.; Kunkel, Maya T.; Skovsø, Søs; Newton, Alexandra C.

doi:10.1016/j.chembiol.2014.02.008

Cited by 55 publications

(84 citation statements)

References 72 publications

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“…These phosphorylations -unlike those found in many other kinases -are constitutive and not agonist-evoked. The 'matured' PKC remains in the cytosol in its inactive, autoinhibited form [29,36]. Agonist-triggered, receptor-mediated activation of phospholipase C (PLC), typically via coupling with the G protein Gq, results in the generation of DG, the key allosteric activator of PKC [37].…”

Section: Regulation Of Pkc Activitymentioning

confidence: 99%

“…All PKC isozymes are processed by a series of ordered phosphorylations and ordered conformational transitions to yield a signaling-competent enzyme that is maintained in an autoinhibited conformation until the correct second messengers are present [28][29][30]. Specifically, binding of a pseudosubstrate segment in the substrate-binding cavity of the kinase domain prevents activation in the absence of agonist.…”

Section: Regulation Of Pkc Activitymentioning

confidence: 99%

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Conventional protein kinase C in the brain: 40 years later

Callender

Newton

2017

Neuronal Signaling

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Protein kinase C (PKC) is a family of enzymes whose members transduce a large variety of cellular signals instigated by the receptor-mediated hydrolysis of membrane phospholipids. While PKC has been widely implicated in the pathology of diseases affecting all areas of physiology including cancer, diabetes, and heart disease -it was discovered, and initially characterized, in the brain. PKC plays a key role in controlling the balance between cell survival and cell death. Its loss of function is generally associated with cancer, whereas its enhanced activity is associated with neurodegeneration. This review presents an overview of signaling by diacylglycerol (DG)-dependent PKC isozymes in the brain, and focuses on the role of the Ca 2 + -sensitive conventional PKC isozymes in neurodegeneration.

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Section: Regulation Of Pkc Activitymentioning

confidence: 99%

Section: Regulation Of Pkc Activitymentioning

confidence: 99%

Conventional protein kinase C in the brain: 40 years later

Callender

Newton

2017

Neuronal Signaling

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“…6B, panel iii). Fully localized plasma membrane staining is evident by the spindle-like appearance of MARK2 in these cells along with the absence of visible nuclei, as decreases in nuclear visibility correlate with increases in plasma membrane localization (47). Curiously, coexpression of the Par6 PB1 domain did not change MARK2 localization relative to the plasma membrane (Fig.…”

Section: Scaffold Proteins Differentially Regulate the Phosphorylatiomentioning

confidence: 85%

Protein Scaffolds Control Localized Protein Kinase Cζ Activity

Tobias

Newton²

2016

Journal of Biological Chemistry

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Atypical protein kinase C (aPKC) isozymes modulate insulin signaling and cell polarity, but how their activity is controlled in cells is not well understood. These enzymes are constitutively phosphorylated, insensitive to second messengers, and have relatively low activity. Here we show that protein scaffolds not only localize but also differentially control the catalytic activity of the aPKC PKC, thus promoting activity toward localized substrates and restricting activity toward global substrates. Using cellular substrate readouts and scaffolded activity reporters in live cell imaging, we show that PKC has highly localized and differentially controlled activity on the scaffolds p62 and Par6. Both scaffolds tether aPKC in an active conformation as assessed through pharmacological inhibition of basal activity, monitored using a genetically encoded reporter for PKC activity. However, binding to Par6 is of higher affinity and is more effective in locking PKC in an active conformation. FRETbased translocation assays reveal that insulin promotes the association of both p62 and aPKC with the insulin-regulated scaffold IRS-1. Using the aPKC substrate MARK2 as another readout for activity, we show that overexpression of IRS-1 reduces the phosphorylation of MARK2 and enhances its plasma membrane localization, indicating sequestration of aPKC by IRS-1 away from MARK2. These results are consistent with scaffolds serving as allosteric activators of aPKCs, tethering them in an active conformation near specific substrates. Thus, signaling of these intrinsically low activity kinases is kept at a minimum in the absence of scaffolding interactions, which position the enzymes for stoichiometric phosphorylation of substrates co-localized on the same protein scaffold.

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“…Low-and high-resolution mass spectra were recorded on a Bruker Daltonics micrOTOF-2focus. For RP-HPLC, Cosmosil 5C 18 AR-II column (4.6×250 mm for analytical runs, 20×250 mm for preparative runs, Nacalai Tesque, Inc., Kyoto, Japan) was employed with a linear gradient of CH 3 combined organic layer was washed with brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. To a solution of the crude aldol product in CH 2 Cl 2 were added MsCl (62 µL, 0.8 mmol) and Et 3 N (223 µL, 1.6 mmol) at 0°C.…”

Section: Methodsmentioning

confidence: 99%

“…FRET efficiency and sensitivity depend on the distance between the donor and the acceptor. The FRET phenomenon can be applied as a spectroscopic measure and a probe of conformational change of macro-biomolecules [1][2][3] and a FRET-mediated competitive assay can be used for studies of the binding between two molecules. 4,5) Radioisotopebased methods are extremely sensitive and are widely used for ligand binding assays, but fluorescent-based assays, which are suitable for high-throughput screening are free of hazards associated with radioactivity.…”

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confidence: 99%

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

Ohashi

Nomura

Minato

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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Protein kinase C (PKC) is correlated with cell signaling pathways and also receives attention as a therapeutic target for cancer and Alzheimer-type dementia. The application of Förster/fluorescence resonance energy transfer (FRET) phenomena to detect binding between proteins and small molecules, for example, PKC and its ligands, underlies a fluorescence-based assay method suitable for high-throughput screening. To accelerate studies on PKC functions in processing signals using small molecules and the development of drugs that target PKC, novel methods for the assessment of the PKC binding affinity of compounds are necessary. We previously developed solvatochromic fluorophore-based methods for that assessment. In this study, a novel method for a FRET-based PKC binding assay was developed and is expected to overcome the limitations of solvatochromic fluorophores.Key words protein kinase C; protein kinase C ligand; ligand screening; Förster resonance energy transfer; fluorescence resonance energy transfer Förster/fluorescence resonance energy transfer (FRET) is the transfer of excitation energy from a fluorescence donor to an acceptor. FRET efficiency and sensitivity depend on the distance between the donor and the acceptor. The FRET phenomenon can be applied as a spectroscopic measure and a probe of conformational change of macro-biomolecules [1][2][3] and a FRET-mediated competitive assay can be used for studies of the binding between two molecules. 4,5) Radioisotopebased methods are extremely sensitive and are widely used for ligand binding assays, but fluorescent-based assays, which are suitable for high-throughput screening are free of hazards associated with radioactivity. Previously, we described fluorescence-based ligand screening assays as an alternative to radioisotope assays. 6,7)Protein kinase C (PKC) isozymes, which are classified as Ser/Thr kinases, play a critical role in cellular signaling pathways related to proliferation, 8,9) differentiation, 10) and apoptosis.11,12) PKC has 11 isozymes which are classified into three subtypes: conventional PKC (cPKC; α, β I/II , γ), novel PKC (nPKC; δ, ε, η, τ) and atypical PKC (aPKC; ζ, λ, ι). The binding of diacylglycerols (DAG) to the C1 domain of PKC is an important step in an activation process except in the case of aPKC which contains an atypical C1 domain which fails to bind to DAG.13) Various synthetic PKC ligands targeting the C1 domain have been developed as chemical probes or drug candidates by radioisotope assays.14-17) We have previously developed a fluorescent assay for PKC-ligand binding affinity using a synthetic PKC δC1b domain labeled with a solvatochromic fluorophore on the edge of its ligand-binding pocket. This can detect ligand binding through changes in the surrounding environment.18) However, this method would not be suitable for high-throughput screening. The reason might be due to several possibilities: For instance, the change of fluorescence intensity is affected by properties of test compounds. It would be concerned that a fluorescen...

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Intramolecular Conformational Changes Optimize Protein Kinase C Signaling

Cited by 55 publications

References 72 publications

Conventional protein kinase C in the brain: 40 years later

Conventional protein kinase C in the brain: 40 years later

Protein Scaffolds Control Localized Protein Kinase Cζ Activity

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

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