cDNA Phage Display Assay for the identification of Tyrosine Kinase Substrates

Courtney Stritar
Yale University

Tyrosine kinases play a central role in the activation and mediation of cellular signal transduction. One component of tyrosine kinase activation, particularly for the Src-family tyrosine kinases, involves the phosphorylation of protein substrates, leading to activation of signal transduction cascades. A major obstacle in elucidating signal transduction cascades is the identification of kinase substrates. We are currently developing a cDNA phage display to identify kinase substrates through an affinity-based selection assay. Phage displayed peptides and protein fragments are treated with Src overexpressing 527 cell lysate and captured with a phosphotyrosine-specific assay. Rescued phage are captured, amplified and reselected. Analysis of substrate peptide sequence is accomplished by DNA sequence analysis of amplified phage. By identifying the cellular substrates of tyrosine kinases, novel members of signal transduction pathways can be identified .