Activating phosphorylation of the Saccharomyces cerevisiae cyclin-dependent kinase, Cdc28p, precedes cyclin binding

Eukaryotic cell cycle progression is controlled by a family of protein kina ses known as cyclin-dependent kinases (Cdks). Two steps are essential for C dk activation: binding of a cyclin and phosphorylation on a conserved threo nine residue by the Cdk-activating kinase (CAK). We have studied the interp lay between these regulatory mechanisms during the activation of the major Saccharomyces cerevisiae Cdk, Cdc28p. We found that the majority of Cdc28p was phosphorylated on its activating threonine (Thr-169) throughout the cel l cycle. The extent of Thr-169 phosphorylation was similar for monomeric Cd c28p and Cdc28p bound to cyclin. By varying the order of the addition of cy clin and Cak1p, we determined that Cdc28p was activated most efficiently wh en it was phosphorylated before cyclin binding. Furthermore, we found that a Cdc28p(T169A) mutant, which cannot be phosphorylated, bound cyclin less w ell than wild-type Cdc28p in vivo. These results suggest that unphosphoryla ted Cdc28p may be unable to bind tightly to cyclin. We propose that Cdc28p is normally phosphorylated by Cak1p before it binds cyclin. This activation pathway contrasts with that in higher eukaryotes, in which cyclin binding appears to precede activating phosyhorylation.