Somara Sita, Bitar Khalil N
Division of Pediatrics-Gastroenterology, University of Michigan Medical Center, Ann Arbor, MI 48109-5656, USA.
Am J Physiol Gastrointest Liver Physiol. 2008 Dec;295(6):G1246-54. doi: 10.1152/ajpgi.90461.2008. Epub 2008 Oct 23.
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-alpha in the membrane. In the present study, we have determined the domain of PKC-alpha involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-alpha. Calponin was found to bind directly to the full-length PKC-alpha. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-alpha. 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-alpha. Coimmunoprecipitation of calponin with His-tagged truncated forms of PKC-alpha showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-alpha 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-alpha and that the functional association of PKC-alpha with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells.
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Mg-ATP酶活性，参与平滑肌收缩的调节。先前的研究表明，收缩激动剂乙酰胆碱可诱导转位的钙调蛋白与膜中的PKC-α直接结合。在本研究中，我们确定了PKC-α中与钙调蛋白直接结合的结构域。通过将谷胱甘肽S-转移酶-钙调蛋白aa 92-229与PKC-α各个结构域以及不同结构域组合的His标签蛋白一起孵育，进行体外结合试验。发现钙调蛋白直接与全长PKC-α结合。钙调蛋白与PKC-α的C2和C4结构域结合，但不与C1和C3结构域结合。当与不同结构域组合的蛋白一起孵育时，钙调蛋白与C2-C3、C3-C4和C2-C3-C4结合，但不与C1-C2或C1-C2-C3结合。为了确定这些体外结合是否模拟体内结合，通过用PKC-α各个结构域以及不同结构域组合的His标签蛋白转染结肠平滑肌细胞进行体内结合试验。钙调蛋白与His标签的PKC-α截短形式的共免疫沉淀表明，C1-C2、C1-C2-C3、C2-C3和C3-C4不与钙调蛋白结合。在静息状态下，钙调蛋白仅与全长PKC-α以及C2-C3-C4在细胞中结合，并且这种结合在乙酰胆碱刺激后增加。这些数据表明，钙调蛋白与可能模拟PKC-α活性形式的片段结合，并且在结肠平滑肌细胞收缩过程中，PKC-α与钙调蛋白的功能结合需要C2和C4结构域。
