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 r equirement for anaphase onset and exit from mitosis. To facilitate ide ntification of specific molecules that regulate this event in mammalia n cells, I have developed a cell-free assay in which cdc2 kinase assoc iated with a chromosomal fraction from metaphase tissue culture cells is inactivated by a cell-cycle-regulated cytosolic system. In vitro ki nase 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 compo nents that are both required for inactivation and are differentially r egulated during mitosis.