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.
Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94143, USA.
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DA
Jeffery
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Springer
D S
DS
King
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EK
O'Shea
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