Abstract
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B by gel filtration chromatography; (b) cyclosome from interphase extracts, activated by a kinase in which cyclin B has been replaced by an N-terminally truncated derivative fused to glutathione S-transferase, binds well to p13(suc1)-Sepharose but not to glutathione-agarose.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.
Highlights
Previous studies have indicated that a ;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 following evidence indicates that the binding of the cyclosome to p13suc1 is not mediated via the Cdc2-cyclin B complex: (a) activated cyclosome binds to p13suc1-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B by gel filtration chromatography; (b) cyclosome from interphase extracts, activated by a kinase in which cyclin B has been replaced by an N-terminally truncated derivative fused to glutathione S-transferase, binds well to p13suc1-Sepharose but not to glutathione-agarose
We found that the active form of the cyclosome2 bound tightly to p13suc1-Sepharose
Summary
Vol 272, No 29, Issue of July 18, pp. 18051–18059, 1997 Printed in U.S.A. Valery Sudakin‡, Michal Shteinberg‡, Dvorah Ganoth, Judith Hershko, and Avram Hershko§¶. Activation of the cyclosome by protein kinase Cdc2-cyclin B includes a time lag [1, 9], which may serve to prevent the premature inactivation of the kinase in the cell cycle Based on these findings, we suggested the cyclosome has a regulated cyclin-ubiquitin ligase activity, which targets cyclin B for destruction at the end of mitosis [1]. Recent biochemical studies with immunodepleted extracts of Xenopus eggs further indicated that Suc1/Cks is required in at least two stages of the embryonic cell cycle: in the activation of the Cdc2-cyclin B complex by tyrosine dephosphorylation of Cdc, and in exit from mitosis due to cyclin B degradation [26]. This information was utilized to develop an affinity procedure for the purification of the cyclosome
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