A distal, high-affinity binding site on the cyclin-CDK substrate Pho4 is important for its phosphorylation and regulation.
Cyclins and cyclin-dependent kinases (CDKs) are key components of signaling pathways essential for cell growth and survival. The cyclin-CDK Pho80-Pho85 inactivates the transcription factor Pho4 in budding yeast by phosphorylating it on five sites. We isolated seven single amino acid substitutions outside of the phosphorylation sites that cause Pho4 to be constitutively active. The substitutions decrease the amount of Pho4 phosphorylation in vivo, and they increase the apparent K(M) of the in vitro phosphorylation reaction by an order of magnitude but do not alter k(cat) substantially. These data suggest that the substituted residues are part of a cyclin-CDK-binding site that is distal to the phosphorylation sites. Further analysis revealed that all of Pho4 variants were phosphorylated by Pho80-Pho85 in a more distributive manner than the wild-type protein, further supporting the idea that binding at a distal, high-affinity binding site is important in determining the processivity of Pho4 phosphorylation. In addition, computational modeling of the Pho4 phosphorylation reactions shows that the K(D) of binding between the Pho4 mutants and Pho80-Pho85 increases, confirming that the mutations are located in a relatively high-affinity "docking site" for the kinase. Interestingly, the K(D) derived from the in vitro data correlates well with the strength of the in vivo phenotypes, demonstrating that the in vitro data are relevant to the in vivo regulation of Pho4.
Byrne
Meghan
M
Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, 600 16th Street, Genentech Hall Room S472D, San Francisco, CA 94143-2240, USA.
Miller
Nicole
N
Springer
Michael
M
O'Shea
Erin K
EK
D019943:Amino Acid Substitution; D001665:Binding Sites; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012097:Repressor Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D016213:Cyclins; D004268:DNA-Binding Proteins; C060292:PHO4 protein, S cerevisiae; C066824:PHO80 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae