Kaneko T, Amano M, Maeda A, Goto H, Takahashi K, Ito M, Kaibuchi K
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101, Japan.
Biochem Biophys Res Commun. 2000 Jun 24;273(1):110-6. doi: 10.1006/bbrc.2000.2901.
Calponin, an F-actin-associated protein implicated in the regulation of smooth muscle contraction, is known to be phosphorylated in vitro by protein kinase C (PKC) and Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II). Unphosphorylated calponin binds to F-actin and inhibits the actin-activated myosin ATPase activity; these properties are lost on phosphorylation. In the present study, we found that Rho-kinase phosphorylated basic calponin stoichiometrically in vitro. We identified the sites of phosphorylation of calponin by Rho-kinase as Thr-170, Ser-175, Thr-180, Thr-184, and Thr-259, and prepared antibodies that specifically recognized calponin phosphorylated at Thr-170 and Thr-184. We showed that the phosphorylation of calponin by Rho-kinase inhibited the binding of calponin to F-actin. Taken together, these results suggest that calponin is a substrate of Rho-kinase and that Rho-kinase regulates the interaction of calponin with F-actin.
钙调蛋白是一种与F-肌动蛋白相关的蛋白质，参与平滑肌收缩的调节，已知它在体外可被蛋白激酶C（PKC）和钙（2+）/钙调蛋白依赖性蛋白激酶II（CaM激酶II）磷酸化。未磷酸化的钙调蛋白与F-肌动蛋白结合并抑制肌动蛋白激活的肌球蛋白ATP酶活性；这些特性在磷酸化后丧失。在本研究中，我们发现Rho激酶在体外能使碱性钙调蛋白发生化学计量的磷酸化。我们确定了Rho激酶使钙调蛋白磷酸化的位点为苏氨酸-170、丝氨酸-175、苏氨酸-180、苏氨酸-184和苏氨酸-259，并制备了能特异性识别在苏氨酸-170和苏氨酸-184磷酸化的钙调蛋白的抗体。我们表明，Rho激酶使钙调蛋白磷酸化会抑制钙调蛋白与F-肌动蛋白的结合。综上所述，这些结果表明钙调蛋白是Rho激酶的底物，且Rho激酶调节钙调蛋白与F-肌动蛋白的相互作用。
