This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The cell division cycle of the budding yeast Saccharomyces cerevisiae is mainly driven by one Cdk (Cyclin-Dependent Kinase), which becomes active when bound to one of nine cyclin subunits. The majority of the specific associates and substrates for these cyclin-Cdk complexes remain elusive, though their elucidation is essential for a full understanding of the cell cycle. Here we report the results of a targeted proteomics study that identified numerous proteins associated with particular cyclin-Cdk complexes. These included phosphorylation substrates, proteins involved in the ubiquitination-degradation pathway, adaptor proteins and inhibitors. We investigated and confirmed the specificity and biological relevance of some of these interactions. We demonstrate that this approach for studying protein associations within a biological module allowed the detection of many new associations. Our data includes many associations that were missed in previous proteome-wide studies, and shows that even transient and dynamic interactions can be detected by mass spectrometry-based targeted proteomic approaches. This work was presented in Archambault V, Chang EJ, Drapkin BJ, Cross FR, Chait BT, Rout MP, Targeted proteomic study of the cyclin-Cdk module Mol Cell. 2004, 14, 699-711. We are currently revisiting this experiment under conditions that have been highly optimized over the past several years, phosphorylation enrichment procedures, as well as using time-resolved proteomics, optimized mass spectrometric readout, together with heavy isotope labeling strategies. Under these new conditions, we have isolated additional putative substrates, which we are now testing biologically.
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