G(1) CYCLIN-DEPENDENT ACTIVATION OF P34(CDC28) (CDC28P) IN-VITRO

In Saccharomyces cerevisiae, transient accumulation of G(1) cyclin/p34(CDC28) (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division. To investigate posttranslational controls that modulate Cdc28p activity during the G(1) phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G(1) extract. A glutathione S-transferase-G(1) cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase. Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase. This assay may be useful for distinguishing genes that promote directly the posttranslational assembly of active Cln2p/Cdc28p kinase complexes from those that stimulate the accumulation of active complexes via a positive-feedback loop that governs synthesis of G(1) cyclins.