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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Feb 14;92(4):1182–1186. doi: 10.1073/pnas.92.4.1182

G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro.

R J Deshaies 1, M Kirschner 1
PMCID: PMC42662  PMID: 7862657

Abstract

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.

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Selected References

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