General Methods for Identifying Gl-phase Substrates of Cdk Protein Kinases.
Abstract
We are developing methods to identify Gi-phase substrates of cyclin-Cdk: complexes. In earlier years of this project, we optimized two-dimensional gels for visualization of phosphorylation of Cdc28 substrates, and we developed monoclonal antibodies with reactivity to phospho-Ser-Pro or phospho-Thr- Pro. We identified one important in vivo substrate, Sic 1. However, it appears that these methods by themselves are insufficiently powerful to identify many substrates, since most substrates seem to be very rare. We have therefore supplemented these methods with three other approaches: (1) purification of affinity tagged cyclins, to look for co-purifying proteins; (2) complete genome two-hybrid screens with Cdc28 and with Gi cyclins, to look for interacting proteins; and (3) genome-wide analysis of cell cycle regulated transcription, on the basis that some important substrates might be regulated at the transcriptional level as well as at the activity level. All three approaches are going very well, and we have excellent candidates for new substrates, which will be characterized in part using the 2D gel system.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 1998
- Accession Number
- ADA366953
Entities
People
- A. B. Futcher