Binding of activated cyclosome to p13(suc1). Use for affinity purification.
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B by gel filtration chromatography; (b) cyclosome from interphase extracts, activated by a kinase in which cyclin B has been replaced by an N-terminally truncated derivative fused to glutathione S-transferase, binds well to p13(suc1)-Sepharose but not to glutathione-agarose.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.
Sudakin
V
V
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.
Shteinberg
M
M
Ganoth
D
D
Hershko
J
J
Hershko
A
A
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D016213:Cyclins / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism*; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism*; D008938:Mitosis; D008970:Molecular Weight; D009865:Oocytes / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus
D018797:Cell Cycle Proteins; D016213:Cyclins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C051655:Suc1 protein, S pombe; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D016203:CDC2 Protein Kinase; D008025:Ligases