Sudakin V, Shteinberg M, Ganoth D, Hershko J, Hershko A
Unit of Biochemistry, the B. Rappaport Faculty of Medicine and the Rappaport Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.
J Biol Chem. 1997 Jul 18;272(29):18051-9. doi: 10.1074/jbc.272.29.18051.
Previous studies have indicated that a approximately 1,500-kDa complex, designated the cyclosome or anaphase-promoting complex, has a regulated cyclin-ubiquitin ligase activity that targets cyclin B for degradation at the end of mitosis. The cyclosome is inactive in the interphase of the embryonic cell cycle and is converted to the active form in late mitosis in a phosphorylation-dependent process initiated by protein kinase Cdc2-cyclin B. We show here that the active, phosphorylated form of the cyclosome from clam oocytes binds to p13(suc1), a protein known to associate with Cdc2. The following evidence indicates that the binding of the cyclosome to p13(suc1) is not mediated via the Cdc2-cyclin B complex: (a) activated cyclosome binds to p13(suc1)-Sepharose following its separation from Cdc2-cyclin B by gel filtration chromatography; (b) cyclosome from interphase extracts, activated by a kinase in which cyclin B has been replaced by an N-terminally truncated derivative fused to glutathione S-transferase, binds well to p13(suc1)-Sepharose but not to glutathione-agarose. An alternative possibility, that the phosphorylated cyclosome binds directly to a phosphate-binding site of p13(suc1), is supported by the observation that the cyclosome is efficiently eluted from p13(suc1)-Sepharose by phosphate-containing compounds. This information was utilized to develop a procedure for the affinity purification of the cyclosome. A factor abundant in the fraction not adsorbed to p13(suc1)-Sepharose stimulates the activity of purified cyclosome. It is suggested that binding of Suc1 may have a role in the regulation of cyclosome activity.
先前的研究表明，一种大约1500 kDa的复合物，被称为周期体或后期促进复合物，具有受调控的细胞周期蛋白 - 泛素连接酶活性，该活性在有丝分裂末期将细胞周期蛋白B作为降解靶点。周期体在胚胎细胞周期的间期无活性，并在有丝分裂后期通过蛋白激酶Cdc2 - 细胞周期蛋白B启动的磷酸化依赖性过程转化为活性形式。我们在此表明，来自蛤卵母细胞的活性磷酸化形式的周期体与p13(suc1)结合，p13(suc1)是一种已知与Cdc2相关的蛋白质。以下证据表明周期体与p13(suc1)的结合不是通过Cdc2 - 细胞周期蛋白B复合物介导的：(a) 活化的周期体在通过凝胶过滤色谱与Cdc2 - 细胞周期蛋白B分离后，与p13(suc1) - 琼脂糖凝胶结合；(b) 间期提取物中的周期体，被一种激酶激活，其中细胞周期蛋白B已被与谷胱甘肽S - 转移酶融合的N端截短衍生物取代，能很好地与p13(suc1) - 琼脂糖凝胶结合，但不与谷胱甘肽 - 琼脂糖结合。另一种可能性是，磷酸化的周期体直接与p13(suc1)的磷酸结合位点结合，这一观察结果得到了支持，即周期体可被含磷酸盐的化合物从p13(suc1) - 琼脂糖凝胶上有效洗脱。利用这些信息开发了一种亲和纯化周期体的方法。在未吸附到p13(suc1) - 琼脂糖凝胶的部分中大量存在的一种因子可刺激纯化的周期体的活性。有人提出Suc1的结合可能在周期体活性的调节中起作用。
