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A more recent version of this article appeared on November 1, 2006. Originally published In Press as doi:10.1074/mcp.M600238-MCP200 on July 6, 2006.
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Submitted on June 27, 2006
Revised on January 1, 1998
Accepted on July 6, 2006

A quantitative results-driven approach to analyzing multi-site protein phosphorylation: The phosphate-dependent phosphorylation profileof the transcription factor pho4

Francesca Zappacosta, Therese S. Collingwood, Michael J. Huddleston, and Roland S. Annan

Computational, Analytical and Structural Sciences, GlaxoSmithKline Pharm, King of Prussia, PA 19406

Corresponding Author: roland_s_annan{at}gsk.com

Multi-site protein phosphorylation appears to be quite common. Nevertheless our understanding of how multiple phosphorylation events regulate a protein's function is limited in many cases. The ability to measure temporal changes in the site specific phosphorylation profile of a protein in response to a given stimulus or cellular activity would provide an immediate indication of the functional significance of any phosphorylation site to a given process. Here we describe a mass spectrometry-based method to identify functionally relevant phosphorylation sites on a protein. It combines stable isotope labelling with a highly selective mass spectrometry analysis to detect and quantitate phosphorylation sites in response to a cellular signal. This approach requires no a priori knowledge of the phosphorylation state of the protein, does not require purification of phosphopeptides and reliably detects sub-stoichiometric levels of phosphorylation. Following a review of the quantitative results, only those phosphorylation sites which show a change in relative abundance are selected for identification and further study. We used this results-driven approach to study phosphorylation of the budding yeast transcription factor Pho4 in response to phosphate starvation. Phosphorylation of Pho4 on five cyclin-dependent kinase (Cdk) consensus sites has been shown to regulate Pho4's transcriptional activity in response to changes in environmental phosphate levels. Here we show that in phosphate-rich media Pho4 is phosphorylated on at least 15 distinct sites including the five Cdk sites previously described. In excellent agreement with the known mechanism for regulation of Pho4 we found that phosphorylation at all five of the Cdk sites were repressed in phosphate-depleted media. In addition to these five sites, we identified four novel phosphorylation sites which were also responsive to changes in phosphate availability. Selecting a limited number of Pho4 phos phorylation sites we performed a more detailed kinetic analysis using an isotope-free strategy. We used LC-SRM to greatly improve the accuracy, sensitivity and dynamic range of the subsequent experiments. A detailed analysis of the cell-based phosphorylation at the selected Pho4 sites confirmed an apparent site preference for the Pho80/Pho85 cyclin-cyclin dependent kinase complex.


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