Chartier L, Rankin L L, Allen R E, Kato Y, Fusetani N, Karaki H, Watabe S, Hartshorne D J
Department of Animal Sciences, University of Arizona, Tucson 85721.
Cell Motil Cytoskeleton. 1991;18(1):26-40. doi: 10.1002/cm.970180104.
Calyculin-A, an inhibitor of type 1 and 2A phosphatases, was applied extracellularly to 3T3 fibroblasts. At 0.1 microM, calyculin-A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phosphorylation following treatment with calyculin-A was observed in vimentin, the 20-kD myosin light chain, and an unidentified 440-kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells. Calyculin-A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin-A (0.1 microM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin-A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin-A an unusual "ball-like" structure was observed with transmission electron microscopy. This unique structure was 2-3 microM in diameter and was located close to the nucleus. The use of calyculin-A adds further support to the idea that cell shape is controlled, at least in part, by concerted actions of a kinase-phosphatase couple.
将1型和2A型磷酸酶抑制剂花萼海绵诱癌素A（Calyculin-A）施加于3T3成纤维细胞的细胞外环境。在0.1微摩尔浓度下，花萼海绵诱癌素A使细胞溶质和不溶性细胞组分中的蛋白质磷酸化显著增加。这种效应与细胞外的钙离子无关。用肌球蛋白抗体形成的免疫沉淀物含有几种细胞骨架成分。在用花萼海绵诱癌素A处理后，波形蛋白、20kD肌球蛋白轻链和一种未鉴定的440kD成分中观察到磷酸化增加。在处理过的细胞的中间丝制剂中发现波形蛋白磷酸化水平增强。花萼海绵诱癌素A还引起3T3细胞明显的形态变化。添加花萼海绵诱癌素A（0.1微摩尔）后几分钟内，细胞变圆并失去与基质的附着。在附着的对照细胞中突出的应力纤维、中间丝和微管，在变圆的细胞中不明显。形态变化是可逆的，去除花萼海绵诱癌素A后，变圆的细胞附着于基质，恢复扁平形状，并进行活跃的有丝分裂。在用花萼海绵诱癌素A处理的细胞中，透射电子显微镜观察到一种不寻常的“球状”结构。这种独特的结构直径为2 - 3微米，位于靠近细胞核的位置。花萼海绵诱癌素A的使用进一步支持了细胞形状至少部分由激酶 - 磷酸酶对的协同作用控制的观点。
