The longstanding goal of our research is to elucidate the molecular regulation of protein kinases controlled by protein posttranslational modifications (PTMs) and seek novel therapeutic opportunities for cancer treatment. We have developed and applied protein chemical approaches for site-specific installations of PTMs (i.e. phosphorylation, ubiquitination, etc. …) into target protein kinases, and then employ biochemical and cellular assays to define their posttranslational regulation mechanisms. Currently, we are investigating three key Ser/Thr kinases S6K1 (mTOR-S6K1 signaling), Cdk5, and LKB1.
Approach 1: Expressed protein ligation (EPL)
Overview of expressed protein ligation for making protein kinases with site-specific PTMs. On the one hand, the N-terminal fragment of protein of interest (POI) C-terminally fused with intein and CBD is recombinantly expressed in insect cells and purified by chitin resin affinity chromatography. The N-terminal protein fragment will be cleaved off from the intein by adding MESNA and resulting in a C-terminal thioester. On the other hand, the C-terminal peptide fragment of POI with an N-ter Cysteine is synthesized using solid-phase peptide synthesis with Fmoc strategy, chemical modifications such as phosphorylation, acetylation, methylation, etc. are site-selectively introduced into the peptide fragment. The resulting peptide fragment selectively reacts with the N-terminal protein thioester fragment to generate the intact native protein kinase with site-specific PTMs.
References: Chu et al. Cell 2018, Chu & Viennet et al. eLife 2020
Approach 2: Enzyme-catalyzed expressed protein ligation
This approach can help to avoid Cysteine at the ligation site for EPL strategy.
Reference: Henager & Chu et al. Nature Methods 2016.