Enzyme Specificity: Adventures in protein-peptide selectivity (or research in the Witucki Lab)

Enzymes called protein tyrosine kinases (PTKs) are important in regulating many cellular functions through signal transduction including cell growth, proliferation, survival, and gene expression. These enzymes are responsible for phosphorylating the phenol oxygen of the amino acid tyrosine as shown.

One particular kinase, Focal Adhesion Kinase (FAK), has been implicated in a number of cancers, including skin, larynx, colon, prostate and breast cancer.  As such, FAK has become an important anti-cancer target enzyme. Our work focuses on developing specific peptide substrates for FAK and the conversion of these substrates into inhibitors of the enzyme. This research involves organic synthesis via solid phase peptide synthesis (SPPS, 1984 Nobel Prize winning methodology, B. Merrifield), chemical purification & characterization methods, and enzyme assays for both activity and inhibition of the kinase.

RESEARCH: Undergraduate students in my lab have used two main methods to identify novel FAK peptide binding partners:

1. Synthesized combinatorial peptide libraries: We design and synthesize libraries and then use them to screen or “pan” for peptide substrates, while the peptide is tethered to a solid support or “bead”. We have used several different colorimetric methods to label the phosphorylated beads; the dye called BM purple can be used to color the peptide substrate beads purple once phosphorylated, as shown here:

2. Using Nature: We also identify peptides from known or proposed protein substrates of FAK, these peptides are then synthesized in varying length and assayed to determine activity. One of the protein substrates we investigate in our lab is p¹³⁰CAS. Using CAS we have identified a very efficient peptide for FAK and are currently converting it into potential inhibitors via organic synthesis using an instrument called a peptide synthesizer, which is shown here:

Some of the tyrosine modifications we have made on our synthesized inhibitors are shown here: