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 cycli n-CDK complex Pho80-Pho85 phosphorylates the transcription factor Pho4 on f ive sites and inactivates it. Here, we describe the kinetic reaction betwee n Pho80-Pho85 and Pho4. Through experimentation and computer modeling we ha ve determined that Pho80-Pho85 phosphorylates Pho4 in a semi-processive fas hion that results from a balance between k(cat) and k(off). In addition, we show that Pho80-Pho85 phosphorylates certain sites preferentially. Phospho rylation of the site with the highest preference inhibits the transcription al 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. T his method of phosphorylation may allow Pho80-Pho85 to quickly inactivate P ho4 in the nucleus and efficiently phosphorylate Pho4 to completion. (C) 20 01 Academic Press.