Direct association of calponin with specific domains of PKC-alpha.
Direct association of calponin with specific domains of PKC-alpha.
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钙调蛋白与 PKC-α 特定结构域的直接关联。
DOI：
10.1152/ajpgi.90461.2008
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发表时间：
2008
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：
Somara,Sita;Bitar,KhalilN
钙调蛋白通过与F-肌动蛋白相互作用和抑制磷酸化肌球蛋白的肌动蛋白激活的Mg-ATP酶活性来调节平滑肌收缩。以前的研究表明，收缩激动剂乙酰胆碱诱导膜上易位的calponin和PKC-α直接结合。在本研究中，我们已经确定了PKC-α的结构域参与直接与calponin。体外结合试验通过孵育谷胱甘肽S-转移酶-钙调蛋白aa 92-229与PKC-α的单个结构域和不同结构域组合的His标记蛋白进行。发现Calponin直接与全长PKC-α结合。Calponin与PKC-α的C2和C4结构域结合，但不与PKC-α的C1和C3结构域结合。当与不同结构域组合的蛋白质孵育时，钙调蛋白结合C2-C3、C3-C4和C2-C3-C4，但不结合C1-C2或C1-C2-C3。为了确定这些体外结合是否模拟体内结合，通过用PKC-α的单个结构域和不同结构域组合的His标记的蛋白质转染结肠平滑肌细胞来进行体内结合测定。Calponin与His标记的PKC-α截短形式的免疫共沉淀显示C1-C2、C1-C2-C3、C2-C3和C3-C4不与Calponin结合。在静息状态下，Calponin仅与全长PKC-α和C2-C3-C4相关，并且这种相关性在乙酰胆碱刺激后增加。这些数据表明，钙调蛋白结合片段，可能模拟的活性形式的PKC-α和PKC-α与钙调蛋白的功能协会需要在结肠平滑肌细胞收缩的C2和C4域。
Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC-α in the membrane. In the present study, we have determined the domain of PKC-α involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathioneS-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-α. Calponin was found to bind directly to the full-length PKC-α. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-α. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with His-tagged proteins of individual domains and different combinations of domains of PKC-α. Coimmunoprecipitation of calponin with His-tagged truncated forms of PKC-α showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-α and with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKC-α and that the functional association of PKC-α with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells.