Inactivation of cdc2 kinase during mitosis requires regulated and constitutive proteins in a cell-free system.
Inactivation of the cyclin-p34cdc2 protein kinase complex is a major requirement for anaphase onset and exit from mitosis. To facilitate identification of specific molecules that regulate this event in mammalian cells, I have developed a cell-free assay in which cdc2 kinase associated with a chromosomal fraction from metaphase tissue culture cells is inactivated by a cell-cycle-regulated cytosolic system. In vitro kinase inactivation requires ATP, Mg2+ and the dephosphorylation of one or more sites in the chromosomal fraction by protein phosphatase 1 and/or 2A. Cyclin B is destroyed during inactivation, while the level of p34cdc2 remains constant. Ammonium sulfate fractionation resolves the cytosolic inactivating system into at least two distinct protein components that are both required for inactivation and are differentially regulated during mitosis.
Suprynowicz
F A
FA
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors*; D016466:CHO Cells; D002474:Cell-Free System; D006224:Cricetinae; D003600:Cytosol / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008938:Mitosis / Q000502:physiology*; D011506:Proteins / Q000378:metabolism*
D011506:Proteins; D016203:CDC2 Protein Kinase