Activating phosphorylation of the Saccharomyces cerevisiae cyclin-dependent kinase, cdc28p, precedes cyclin binding.
Eukaryotic cell cycle progression is controlled by a family of protein kinases known as cyclin-dependent kinases (Cdks). Two steps are essential for Cdk activation: binding of a cyclin and phosphorylation on a conserved threonine residue by the Cdk-activating kinase (CAK). We have studied the interplay 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 cell cycle. The extent of Thr-169 phosphorylation was similar for monomeric Cdc28p and Cdc28p bound to cyclin. By varying the order of the addition of cyclin and Cak1p, we determined that Cdc28p was activated most efficiently when it was phosphorylated before cyclin binding. Furthermore, we found that a Cdc28p(T169A) mutant, which cannot be phosphorylated, bound cyclin less well than wild-type Cdc28p in vivo. These results suggest that unphosphorylated 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 phosphorylation.
Ross
K E
KE
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-2114, USA.
Kaldis
P
P
Solomon
M J
MJ
D000918:Antibody Specificity; D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002453:Cell Cycle; D019925:Cyclin A / Q000378:metabolism; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism*; D004789:Enzyme Activation; D000939:Epitopes; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine / Q000276:immunology / Q000378:metabolism; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C093749:CLB2 protein, S cerevisiae; D019925:Cyclin A; D019926:Cyclin B; D016213:Cyclins; D000939:Epitopes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D017489:CDC28 Protein Kinase, S cerevisiae; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase