General Methods for Identifying G1-phase Substrates of Cdk Protein Kinases

摘要

We have used two-dimensional gels in combination with genetics and biochemistry to identify G1-phase substrates of cyclin-Cdk complexes. We have optimized protocols for labeling proteins with P-32 for two/two-dimensional gel electrophoresis. So far, we have identified and characterized one important in vivo substrate, Sic 1. Other workers have shown that an analogous substrate, p27, is important in human cells. More recently, we have identified and started to characterize three more substrates, Orc2, Mcm3, and Mcm4 (= Cdc54). In a second line of work, we have made peptide antigens for the purpose of making antibodies against phosphorylated Cdk substrates. Monoclonal antibodies reactive against phospho-Thr-Pro have been obtained, and monoclonal antibodies reactive against phospho-Thr-Pro may also have been obtained, though these are not yet fully characterized. These antibodies, which will be specific for phosphorylated Cdk sites, will be extremely useful in characterizing Cdk substrates and their phosphorylation.