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首页> 外文期刊>American Journal of Physiology >Involvement of NH2 terminus of PKC-delta in binding to F-actin during activation of Calu-3 airway epithelial NKCC1.
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Involvement of NH2 terminus of PKC-delta in binding to F-actin during activation of Calu-3 airway epithelial NKCC1.

机译：在激活Calu-3气道上皮NKCC1期间，PKC-δ的NH2末端与F-肌动蛋白结合。

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摘要

Direct binding of nonmuscle F-actin and the C2-like domain of PKC-delta (deltaC2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid deltaC2-like domain. Truncations of the deltaC2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His6-tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His6 probe for expression of the His6 tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC-delta with nonmuscle F-actin. Peptides from the NH2-terminal region, but not the COOH-terminal region, of the deltaC2-like domain blocked binding of activated PKC-delta to F-actin. The deltaC2-like domain and three NH2-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC50 values ranging from 1.2 to 2.2 nmol (6-11 microM). NH2-terminal deltaC2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH2 terminus of the deltaC2-like domain encodes a beta1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the beta1-sheet region of the deltaC2-like domain in airway epithelial cells.
机译：非肌肉F-肌动蛋白与PKC-δ的C2样结构域（deltaC2样结构域）的直接结合参与Calu-3气道上皮中激素介导的上皮Na-K-2Cl共转运蛋白亚型1（NKCC1）的活化细胞系。这项研究的目的是确定肌动蛋白在123个氨基酸的deltaC2样结构域上的结合位点。通过限制性酶切消化deltaC2样结构域，并通过核苷酸测序证实。 His6-tagged肽在细菌中表达，纯化并用考马斯蓝染色分析预测大小，并用6xHis蛋白标签染色或INDIA His6探针分析His6标签的表达。测试了截短的肽竞争性抑制了活化的重组PKC-δ与非肌肉F-肌动蛋白的结合。来自ΔC2样结构域的NH 2末端区域而不是COOH末端区域的肽阻断了活化的PKCδ与F-肌动蛋白的结合。类似于deltaC2的结构域和17、83或108个氨基酸的三个NH2末端截短肽可阻止结合，IC50值范围为1.2至2.2 nmol（6-11 microM）。 NH2末端的deltaC2样肽还阻止了甲氧胺刺激的NKCC1活化，并从Calu-3细胞中提取了内源性肌动蛋白。 deltaC2样域的近端NH2末端编码一个beta1区域。肌动蛋白结合结构域的氨基酸序列不同于其他PKC同种型和肌动蛋白结合蛋白中的肌动蛋白结合结构域。我们的结果表明，F-肌动蛋白可能与气道上皮细胞中deltaC2样结构域的beta1区域结合。

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