In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.
Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.
Woodman
P G
PG
Department of Biochemistry and Molecular Biology, University of Manchester Medical School, United Kingdom.
Adamczewski
J P
JP
Hunt
T
T
Warren
G
G
D016203:CDC2 Protein Kinase / Q000494:pharmacology*; D002460:Cell Line; D016213:Cyclins / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D015388:Organelles / Q000187:drug effects / Q000378:metabolism*
D016213:Cyclins; D016203:CDC2 Protein Kinase