EFFECTS OF PHOSPHORYLATION BY CAK ON CYCLIN BINDING BY CDC2 AND CDK2

The cyclin-dependent protein kinases (CDKs) are activated by associati on,vith cyclins and by phosphorylation at a conserved threonine residu e by the CDK-activating kinase (CAK). We have studied the binding of v arious human CDK and cyclin subunits in vitro, using purified proteins derived from baculovirus-infected insect cells. We find that most CDK -cyclin complexes known tb exist in human cells (CDC2-cyclin B, CDK2-c yclin A, and CDK2-cyclin E) form with high affinity in the absence of phosphorylation or other cellular components. One complex (CDC2-cyclin A) forms with high affinity only after CAK-mediated phosphorylation o f CDC2 at the activating threonine residue. CDC2 does not bind with hi gh affinity to cyclin E in vitro, even after phosphorylation of the CD C2 subunit. Thus, phosphorylation is of varying importance in the form ation of high-affinity CDK-cyclin complexes.