The cyclin-ubiquitin ligase activity of cyclosome/APC is jointly activated by protein kinases Cdk1-cyclin B and Plk.
The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, we have purified cyclosome from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, we found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylated the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases was not similar. Each protein kinase could restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity was observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases.
Golan
Amnon
A
Unit of Biochemistry, the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Yudkovsky
Yana
Y
Hershko
Avram
A
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D049872:Bivalvia; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002417:Cattle; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D019926:Cyclin B / Q000378:metabolism*; D016213:Cyclins / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D008938:Mitosis; D009865:Oocytes / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D025801:Ubiquitin / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes; D000097869:Polo-Like Kinase 1
D064173:Anaphase-Promoting Complex-Cyclosome; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D019926:Cyclin B; D016213:Cyclins; D008025:Ligases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D000097869:Polo-Like Kinase 1