Multi-site phosphorylation of Pho4 by the cyclin-CDK Pho80-Pho85 is semi-processive with site preference

J Mol Biol. 2001 Mar 9;306(5):997-1010. doi: 10.1006/jmbi.2000.4417.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites*
  • Computer Simulation
  • Cyclin-Dependent Kinases / genetics
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclins / genetics
  • Cyclins / metabolism*
  • DNA-Binding Proteins*
  • Dipeptides / metabolism
  • Escherichia coli / enzymology
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Mutation
  • Phosphorylation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Repressor Proteins*
  • Saccharomyces cerevisiae Proteins*
  • Substrate Specificity
  • Transcription Factors*
  • Trypsin / metabolism

Substances

  • Cyclins
  • DNA-Binding Proteins
  • Dipeptides
  • Fungal Proteins
  • PHO4 protein, S cerevisiae
  • PHO80 protein, S cerevisiae
  • Recombinant Proteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Cyclin-Dependent Kinases
  • PHO85 protein, S cerevisiae
  • Trypsin