Isozyme-specific Interaction of Protein Kinase Cδ with Mitochondria Dissected Using Live Cell Fluorescence Imaging

Wu-Zhang, Alyssa Xia; Murphy, Anne N.; Bachman, Mackenzie; Newton, Alexandra C.

doi:10.1074/jbc.m112.412635

Cited by 24 publications

(36 citation statements)

References 64 publications

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“…17. We cloned Cx43 into the pcDNA3.0 ␦CKAR using a BamHI restriction site and incorporated a 9-amino acid linker consisting of GSAAASFAT between the end of Cx43 and the beginning of the CFP.…”

Section: Methodsmentioning

confidence: 99%

“…Stimulation-The CKAR reporters (nonspecific and ␦CKAR version) have previously been appended to amino acid sequences that target to specific organelles such as mitochondria, the Golgi apparatus, nuclei, and the plasma membrane (15,17,24) as well as to specific proteins such as AKAP79 (25) and PSD95. 3 Here, we appended ␦CKAR to the C terminus of Cx43.…”

Section: The Cx43-␦ckar Biosensor Was Phosphorylated At Ser-368 and Mmentioning

confidence: 99%

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Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Cone

Cavin

Ambrosi

et al. 2014

Journal of Biological Chemistry

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Background: Connexin43, a ubiquitous gap junction protein, is phosphorylated by protein kinase C on serine 368. Results: After PKC␦ activation, phospho-Ser-368 Connexin43 channels segregated into the gap junction center and were subsequently internalized and degraded. Conclusion: PKC␦ phosphorylation triggered internalization and degradation of Connexin43 channels without dephosphorylation. Significance: Differential phosphorylation events are used to sort and traffic Connexin43 channels within gap junctions and into the cytoplasm.

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Section: Methodsmentioning

confidence: 99%

Section: The Cx43-␦ckar Biosensor Was Phosphorylated At Ser-368 and Mmentioning

confidence: 99%

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Cone

Cavin

Ambrosi

et al. 2014

Journal of Biological Chemistry

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“…For instance, Newton and colleagues detected a relatively small but significant PKCδ translocation to the outer mitochondrial membrane (OMM) after the application of a phorbol ester using fluorescence resonance energy (FRET) by generating an OMM-targeted [31] CFP (mt-CFP) and PKCδ-GFP [32]. They also confirmed that PKCδ activity was increased at the OMM upon phorbol-ester stimulation, using the OMM-targeted FRET-based PKCδ kinase activity reporter.…”

Section: Discussionmentioning

confidence: 97%

“…In addition, genetic models showed that PKCβ expression is not necessary for the development of cardiac hypertrophy nor does its absence attenuate the hypertrophic response [30]. PKCε has been suggested to have a protective role in myocardial ischemia-reperfusion injury [31] and arrhythmias [32]. Moreover, the use of specific PKC isoenzyme blockers may lead to novel treatments for heart failure [35; 36] and also for cancers [33] and Alzheimer’s disease [34].…”

Section: Discussionmentioning

confidence: 99%

Isoform-specific dynamic translocation of PKC by α 1 -adrenoceptor stimulation in live cells

Sorenson

Jhun

Mishra

et al. 2015

Biochemical and Biophysical Research Communications

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Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated at downstream of Gq-protein coupled receptor (GqPCR) signaling and translocated to the various cellular compartments including plasma membrane (PM). Recent reports suggested that a different type of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is the one of the GqPCR highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (< 30s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (<30s), with sustained membrane localization. One of novel PKCs (nPKCs), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, β and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.

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“…For experiments with forskolin treatment, cells were treated ϳ24 h prior to imaging and transfected the day before. Kinase activity was monitored via intramolecular FRET of the activity reporters (CKAR or CKAR-PB1), and protein translocation was monitored via intermolecular FRET between the YFP-tagged protein and the CFP-tagged target using methods previously described (6,36).…”

Section: Methodsmentioning

confidence: 99%

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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Isozyme-specific Interaction of Protein Kinase Cδ with Mitochondria Dissected Using Live Cell Fluorescence Imaging

Cited by 24 publications

References 64 publications

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Isoform-specific dynamic translocation of PKC by α 1 -adrenoceptor stimulation in live cells

Protein Scaffolds Control Localized Protein Kinase Cζ Activity

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