ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43
ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43
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DOI：
10.1016/j.cellsig.2006.11.007
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发表时间：
2007-05-01
影响因子：
4.8
通讯作者：
Takemoto, Dolores J.
中科院分区：
文献类型：
--
作者：
Akoyev, Vladimir;Takemoto, Dolores J.
我们以前曾报道蛋白激酶C-γ(PKC-γ)被佛波醇-12-肉豆蔻酸酯-13-乙酸酯(TPA)激活，这导致PKC-γ易位到膜上，并导致缝隙连接蛋白43(Cx43)的磷酸化。Cx43的S368上的这种磷酸化导致Cx43从细胞连接斑块中分解出来，从而抑制染料的转移。本研究的目的是确定封闭带蛋白-1(ZO-1)在PKC-γ驱动的Cx43拆解中的特定作用。ZO-1是一种紧密连接蛋白，最近被证实对缝隙连接有影响。为此，使用特定的siRNA，培养的晶状体上皮细胞中ZO-1的水平降低了高达70%。ZO-1的下调导致了PKC-γ与Cx43的稳定相互作用，即使TPA没有正常的酶激活。然而，在TPA激活PKC-Gamma后，即使PKC-Gamma被激活，Cx43在S368上被磷酸化，Cx43也没有从斑块中解离出来。共聚焦显微镜显示，siRNA处理导致大的连接Cx43细胞到细胞斑块边界的ZO-1丢失，并导致Cx43聚集在细胞内。被ZO-1包围的大Cx43斑块的特殊“斑小体”排列的丢失伴随着功能性染料转移的完全丧失。这些结果表明，ZO-1是Cx43控制所必需的，无论是对于染料转移，还是对于PKC-伽马驱动的拆解反应。(C)2006 Elsevier Inc.保留所有权利。
We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma 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 PKC-gamma-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 PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma 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 PKC-gamma-driven disassembly response. (c) 2006 Elsevier Inc. All rights reserved.