LEDGF activation of PKC γ and gap junction disassembly in lens epithelial cells

Nguyen, Thu Annelise; Boyle, Daniel L.; Wagner, Lynn M.; Shinohara, Toshimichi; Takemoto, Dolores J.

doi:10.1016/s0014-4835(03)00049-6

Cited by 25 publications

(22 citation statements)

References 19 publications

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“…In contrast, activation of PKC␣ and ␥ in N/N1003A cells by treatment with TPA leads to a decrease in the number of gap junctional plaques without changes in Cx43 levels, suggesting redistribution of Cx43 (167). Similar results are obtained after treatment of N/N1003A cells with diacylglycerol (DAG), insulin-like growth factor 1 (IGF-1), or lens epithelium-derived growth factor (LEDGF), suggesting that IGF-1 and LEDGF may correspond to the endogenous agonists that trigger activation of PKC (78,107). PKC involvement may also explain the decreased intercellular communication of lens epithelial cells elicited by extracellular ATP (84); this effect is sensitive to PKC and phospholipase C inhibitors and requires a serine…”

Section: Effects Of Pkc Activationmentioning

confidence: 52%

Oxidative Stress, Lens Gap Junctions, and Cataracts

Berthoud

Beyer

2009

Antioxidants & Redox Signaling

213

141

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The eye lens is constantly subjected to oxidative stress from radiation and other sources. The lens has several mechanisms to protect its components from oxidative stress and to maintain its redox state, including enzymatic pathways and high concentrations of ascorbate and reduced glutathione. With aging, accumulation of oxidized lens components and decreased efficiency of repair mechanisms can contribute to the development of lens opacities or cataracts. Maintenance of transparency and homeostasis of the avascular lens depend on an extensive network of gap junctions. Communication through gap junction channels allows intercellular passage of molecules (up to 1 kDa) including antioxidants. Lens gap junctions and their constituent proteins, connexins (Cx43, Cx46, and Cx50), are also subject to the effects of oxidative stress. These observations suggest that oxidative stress-induced damage to connexins (and consequent altered intercellular communication) may contribute to cataract formation. Antioxid. Redox Signal. 11, 339-353. 339The Eye Lens

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Section: Effects Of Pkc Activationmentioning

confidence: 52%

Oxidative Stress, Lens Gap Junctions, and Cataracts

Berthoud

Beyer

2009

Antioxidants & Redox Signaling

213

141

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“…Additionally, the activation of PKC␥ by LEDGF induced increases in DAG can cause a decrease in gap junction activity through the phosphorylation of Cx43 (1). A schematic diagram of PKC␥ activation is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It appears that both the C1A and C1B domains are exposed and that this PKC isoform can be activated without increased Ca 2ϩ (23). Growth factors such as LEDGF or IGF-I can cause the translocation of PKC␥ in cells (1,2). LEDGF, a lens and retina growth factor (36), can increase the endogenous DAG level, PKC enzyme activity, and phosphorylation level of PKC␥ (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Previously in our laboratory, we have shown that IGF-I or LEDGF decreases gap junction activity through the activation of PKC␥, causing a PKC-mediated phosphorylation of connexin 43 (Cx43), and then a decrease in gap junction activity (1,2). PKC-mediated phosphorylation of Cx43 and subsequent decrease of gap junction activity could be regulated through the release of PKC␥ from 14-3-3, followed by the activation and translocation of PKC to the membrane, and then by subsequent PKC-catalyzed phosphorylation of Cx43.…”

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confidence: 99%

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Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Nguyen¹,

Takemoto

Takemoto³

2004

Journal of Biological Chemistry

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Gap junction activity in lens epithelial cells is regulated by PKC␥-mediated phosphorylation of Cx43. PKC␥ activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKC␥-C1B domain that are responsible for its interaction with 14-3-3⑀. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKC␥. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3⑀, resulting in the release of endogenous cellular PKC␥ from 14-3-3⑀, in vivo or in vitro, in activation of PKC␥ enzyme activity, phosphorylation of PKC␥, in the subsequent translocation of PKC␥ to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 M of C1B1 or C1B5 peptides also caused a 10-or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKC␥ are regulated by its binding to 14-3-3⑀ at the C1B domain of PKC␥. Synthetic peptides corresponding to these regions of PKC␥ successfully competed for the binding of 14-3-3⑀ to endogenous PKC␥, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.

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“…To study the gap junction activities in cells the scrape-loading/dye-transfer (SL/DT) assay with lucifer yellow and rhodamine dextran was carried out as described before [21][22][23]. Gap junction activity was expressed as the number of cells transferring lucifer yellow minus cells with rhodamine dextran per total number of DAPI stained cells.…”

Section: Dye Transfer -Gap Junction Activity Assaymentioning

confidence: 99%

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

Akoyev

Takemoto

2007

Cellular Signalling

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We have previously reported that protein kinase C gamma (PKCγ) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKCγ translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKCγ-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKCγ with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKCγ, the Cx43 did not disassemble out of plaques even though the PKCγ enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKCγ-driven disassembly response.

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LEDGF activation of PKC γ and gap junction disassembly in lens epithelial cells

Cited by 25 publications

References 19 publications

Oxidative Stress, Lens Gap Junctions, and Cataracts

Oxidative Stress, Lens Gap Junctions, and Cataracts

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

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