Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: a quantitative proteomic analysis.
The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosis and entry into S-phase. The activity of the APC is regulated by phosphorylation during mitosis. Because the phosphorylation pattern provides insights into the complexity of regulation of the APC, we studied in detail the phosphorylation patterns at a single mitotic state of arrest generated by various antimitotic drugs. We examined the phosphorylation patterns of the APC in HeLa S3 cells after they were arrested in prometaphase with taxol, nocodazole, vincristine, or monastrol. There were 71 phosphorylation sites on nine of the APC subunits. Despite the common state of arrest, the various antimitotic drug treatments resulted in differences in the phosphorylation patterns and phosphorylation stoichiometries. The relative phosphorylation stoichiometries were determined by using a method adapted from the isotope-free quantitation of the extent of modification (iQEM). We could show that during drug arrest the phosphorylation state of the APC changes, indicating that the mitotic arrest is not a static condition. We discuss these findings in terms of the variable efficacy of antimitotic drugs in cancer chemotherapy.
Steen
Judith A J
JA
Departments of Neurobiology and Pathology, Harvard Medical School and Children's Hospital Boston, Boston, MA 02115, USA.
Steen
Hanno
H
Georgi
Ann
A
Parker
Kenneth
K
Springer
Michael
M
Kirchner
Marc
M
Hamprecht
Fred
F
Kirschner
Marc W
MW
D064173:Anaphase-Promoting Complex-Cyclosome; D050256:Antimitotic Agents / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation; D049468:Prometaphase / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011743:Pyrimidines / Q000494:pharmacology; D008941:Spindle Apparatus / Q000187:drug effects; D013871:Thiones / Q000494:pharmacology; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D014750:Vincristine / Q000494:pharmacology
D050256:Antimitotic Agents; D021122:Protein Subunits; D011743:Pyrimidines; D013871:Thiones; D014750:Vincristine; C400223:monastrol; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D017239:Paclitaxel; D015739:Nocodazole