G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro.
In Saccharomyces cerevisiae, transient accumulation of G1 cyclin/p34CDC28 (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 G1 phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G1 extract. A glutathione S-transferase-G1 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 G1 cyclins.
Deshaies
R J
RJ
Department of Biochemistry and Biophysics, University of California Medical Center, San Francisco 94143.
Kirschner
M
M
D000595:Amino Acid Sequence; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004789:Enzyme Activation; D016193:G1 Phase; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D016213:Cyclins; D017489:CDC28 Protein Kinase, S cerevisiae