Taulés M, Rius E, Talaya D, López-Girona A, Bachs O, Agell N
Department of Cell Biology, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Faculty of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain.
J Biol Chem. 1998 Dec 11;273(50):33279-86. doi: 10.1074/jbc.273.50.33279.
Although it is known that calmodulin is involved in G1 progression, the calmodulin-dependent G1 events are not well understood. We have analyzed here the role of calmodulin in the activity, the expression, and the intracellular location of proteins involved in G1 progression. The addition of anti-calmodulin drugs to normal rat kidney cells in early G1 inhibited cyclin-dependent kinase 4 (Cdk4) and Cdk2 activities, as well as retinoblastoma protein phosphorylation. Protein levels of cdk4, cyclin D1, cyclin D2, cyclin E, p21, and p27 were not affected after CaM inhibition, whereas decreases in the amount of cyclin A and Cdc2 were observed. The decrease of Cdk4 activity was due neither to changes in its association to cyclin D1 nor to changes in the amount of p21 or p27 bound to cyclin D1-Cdk4 complexes. Calmodulin inhibition also produced a translocation of nuclear cyclin D1 and Cdk4 to the cytoplasm. This translocation could be responsible for the decreased Cdk4 activity upon calmodulin inhibition. Immunoprecipitation, calmodulin affinity chromatography, and direct binding experiments indicated that calmodulin associates with Cdk4 and cyclin D1 through a calmodulin-binding protein. The facts that Hsp90 interacts with Cdk4 and that its inhibition induced Cdk4 and cyclin D1 translocation to the cytoplasm point to Hsp90 as a good candidate for being the calmodulin-binding protein involved in the nuclear accumulation of Cdk4 and cyclin D1.
尽管已知钙调蛋白参与G1期进程，但对钙调蛋白依赖性的G1期事件了解尚少。我们在此分析了钙调蛋白在参与G1期进程的蛋白质的活性、表达及细胞内定位方面的作用。在G1早期向正常大鼠肾细胞中添加抗钙调蛋白药物可抑制细胞周期蛋白依赖性激酶4（Cdk4）和Cdk2的活性，以及视网膜母细胞瘤蛋白的磷酸化。钙调蛋白受到抑制后，cdk4、细胞周期蛋白D1、细胞周期蛋白D2、细胞周期蛋白E、p21和p27的蛋白质水平未受影响，而细胞周期蛋白A和Cdc2的量有所减少。Cdk4活性的降低既不是由于其与细胞周期蛋白D1的结合变化，也不是由于与细胞周期蛋白D1-Cdk4复合物结合的p21或p27量的变化。钙调蛋白抑制还导致核内细胞周期蛋白D1和Cdk4转位至细胞质。这种转位可能是钙调蛋白抑制后Cdk4活性降低的原因。免疫沉淀、钙调蛋白亲和层析及直接结合实验表明，钙调蛋白通过一种钙调蛋白结合蛋白与Cdk4和细胞周期蛋白D1结合。热休克蛋白90（Hsp90）与Cdk4相互作用且其抑制可诱导Cdk4和细胞周期蛋白D1转位至细胞质，这些事实表明Hsp90是参与Cdk4和细胞周期蛋白D1核内积累的钙调蛋白结合蛋白的良好候选者。
