Direct association and translocation of PKC-alpha with calponin.
Direct association and translocation of PKC-alpha with calponin.
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PKC-α 与钙调蛋白的直接关联和易位。
DOI：
10.1152/ajpgi.00477.2003
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Bitar,KhalilN
中科院分区：
文献类型：
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作者：
Patil,SureshB;Pawar,MercyD;Bitar,KhalilN
钙调蛋白通过与 F-肌动蛋白相互作用并抑制磷酸化肌球蛋白的肌动蛋白激活的 MgATP 酶活性，参与平滑肌收缩的调节。 Calponin 也已被证明与 PKC 相互作用。我们研究了钙调蛋白与 PKC-α 以及低分子量热休克蛋白 (HSP)27 在结肠平滑肌细胞收缩中的相互作用。使用抗钙调蛋白抗体对分离的平滑肌细胞的颗粒级分进行免疫沉淀，并使用抗 HSP27 和抗 PKC-α 的抗体进行蛋白质印迹分析。乙酰胆碱诱导颗粒部分中钙调蛋白与 HSP27 以及钙调蛋白与 PKC-α 的免疫复合物持续增加，表明膜中易位蛋白的关联。为了检查观察到的体内相互作用是否是由于钙调蛋白与 PKC-α 的直接相互作用所致，对人钙调蛋白基因的 1.3 kb cDNA 进行了 PCR 扩增。编码 622 nt 钙调蛋白 cDNA（nt 351-972 对应于氨基酸 92-229）的 PCR 产物在载体 pGEX-KT 中表达为融合谷胱甘肽 S-转移酶 (GST) 蛋白。我们在体外研究了 GST-calponin 融合蛋白与重组 PKC-α 的直接关联。用还原型谷胱甘肽缓冲液（pH 8.0）洗脱后收集的级分进行蛋白质印迹分析，显示 GST-钙调蛋白与 PKC-α 共洗脱，表明 GST-钙调蛋白与 PKC-α 直接关联。这些数据表明，膜中易位的钙调蛋白和 PKC-α 存在直接关联，并且复杂的钙调蛋白-PKC-α-HSP27 在结肠平滑肌细胞的收缩中发挥作用。
Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-α and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-α. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-α in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-α, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351–972 corresponding to amino acids 92–229) was expressed as fusion glutathioneS-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-α in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-α, indicating a direct association of GST-calponin with PKC-α. These data suggest that there is a direct association of translocated calponin and PKC-α in the membrane and a role for the complex calponin-PKC-α-HSP27, in contraction of colonic smooth muscle cells.