Genetic analysis of the relationship between activation loop phosphorylation and cyclin binding in the activation of the Saccharomyces cerevisiae Cdc28p cyclin-dependent kinase.
We showed recently that a screen for mutant CDC28 with improved binding to a defective Cln2p G1 cyclin yielded a spectrum of mutations similar to those yielded by a screen for intragenic suppressors of the requirement for activation loop phosphorylation (T169E suppressors). Recombination among these mutations yielded CDC28 mutants that bypassed the G1 cyclin requirement. Here we analyze further the interrelationship between T169E suppression, interaction with defective cyclin, and G1 cyclin bypass. DNA shuffling of mutations from the various screens and recombination onto a T169E-encoding 3' end yielded CDC28 mutants with strong T169E suppression. Some of the strongest T169E suppressors could suppress the defective Cln2p G1 cyclin even while retaining T169E. The strong T169E suppressors did not exhibit bypass of the G1 cyclin requirement but did so when T169E was reverted to T. These results suggested that for these mutants, activation loop phosphorylation and cyclin binding might be alternative means of activation rather than independent requirements for activation (as with wild type). These results suggest mechanistic overlap between the conformational shift induced by cyclin binding and that induced by activation loop phosphorylation. This conclusion was supported by analysis of suppressors of a mutation in the Cdk phosphothreonine-binding pocket created by cyclin binding.
Cross
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The Rockefeller University, New York, NY 10021, USA. fcross@rockvax.rockefeller.edu
Levine
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D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics / Q000378:metabolism*; D056746:Cyclin G; D016213:Cyclins / Q000378:metabolism*; D004789:Enzyme Activation; D009154:Mutation; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
C082721:CLN2 protein, S cerevisiae; D056746:Cyclin G; D016213:Cyclins; D029701:Saccharomyces cerevisiae Proteins; D010769:Phosphothreonine; D017489:CDC28 Protein Kinase, S cerevisiae