Agonist-induced association of tropomyosin with protein kinase Calpha in colonic smooth muscle.
Agonist-induced association of tropomyosin with protein kinase Calpha in colonic smooth muscle.
复制标题
激动剂诱导结肠平滑肌中原肌球蛋白与蛋白激酶 Cα 的关联。
DOI：
10.1152/ajpgi.00330.2004
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Bitar,KhalilN
中科院分区：
文献类型：
--
作者：
Somara,Sita;Pang,Haiyan;Bitar,KhalilN
由肌球蛋白轻链磷酸化调节的平滑肌收缩也在细丝水平调节。原肌球蛋白是一种细丝调节蛋白，通过调节肌动蛋白-肌球蛋白相互作用来调节收缩。目前的研究表明，乙酰胆碱诱导结肠平滑肌细胞原肌球蛋白磷酸化的PKC介导的和钙依赖性。我们的数据还表明，乙酰胆碱诱导结肠平滑肌细胞颗粒部分中PKC介导的原肌球蛋白与PKCα的结合显著且持续增加。免疫印迹研究显示，在结肠平滑肌细胞中，颗粒组分中的原肌球蛋白或肌动蛋白的量对乙酰胆碱的反应没有显著变化，表明颗粒组分中原肌球蛋白与PKCα的结合增加可能是由于乙酰胆碱诱导PKCα移位到颗粒组分。为了研究PKCα与原肌球蛋白的结合是否是由于直接的相互作用，我们进行了体外直接结合试验。从结肠平滑肌mRNA中扩增原肌球蛋白cDNA，表达GST-原肌球蛋白融合蛋白。GST-原肌球蛋白和重组PKCα的体外结合实验表明原肌球蛋白与PKCα直接相互作用。PKC介导的原肌球蛋白磷酸化以及PKCα与原肌球蛋白的直接相互作用表明原肌球蛋白可能是PKC的底物。原肌球蛋白的磷酸化可能有助于保持滑动的原肌球蛋白远离肌动蛋白上的肌球蛋白结合位点，导致肌动球蛋白相互作用和持续收缩。
Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCα in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCα in the particulate fraction may be due to acetylcholine-induced translocation of PKCα to the particulate fraction. To investigate whether the association of PKCα with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCα indicated direct interaction of tropomyosin with PKCα. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCα with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.