Multi-site phosphorylation of Pho4 by the cyclin-CDK Pho80-Pho85 is semi-processive with site preference.
As part of a nutrient-responsive signaling pathway, the budding yeast cyclin-CDK complex Pho80-Pho85 phosphorylates the transcription factor Pho4 on five sites and inactivates it. Here, we describe the kinetic reaction between Pho80-Pho85 and Pho4. Through experimentation and computer modeling we have determined that Pho80-Pho85 phosphorylates Pho4 in a semi-processive fashion that results from a balance between kcat and k(off). In addition, we show that Pho80-Pho85 phosphorylates certain sites preferentially. Phosphorylation of the site with the highest preference inhibits the transcriptional activity of Pho4 when it is in the nucleus, while phosphorylation of the lowest-preference sites is required for export of Pho4 from the nucleus. This method of phosphorylation may allow Pho80-Pho85 to quickly inactivate Pho4 in the nucleus and efficiently phosphorylate Pho4 to completion.
Jeffery
D A
DA
Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94143, USA.
Springer
M
M
King
D S
DS
O'Shea
E K
EK
D001665:Binding Sites; D003198:Computer Simulation; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004151:Dipeptides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014157:Transcription Factors; D014357:Trypsin / Q000378:metabolism
D016213:Cyclins; D004268:DNA-Binding Proteins; D004151:Dipeptides; D005656:Fungal Proteins; C060292:PHO4 protein, S cerevisiae; C066824:PHO80 protein, S cerevisiae; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae; D014357:Trypsin