Semisynthetic Approach to Explore the N-terminal Regulation of Tumor Suppressor PTEN

Abstract

Phosphatase and Tensin Homolog (PTEN) is a tumor suppressor protein that prevents the activation of the proliferative Akt signaling pathway by dephosphorylating the phospholipid second messenger, PI(3,4,5)P3 into PI(4,5)P2. PTEN also regulates cell-cycle progression, DNA repair, and chromosome stability in the nucleus. Post-translational modifications (PTMs) such as phosphorylation and ubiquitination tightly regulate PTEN’s cellular functions. Phosphorylation of the C-terminal tail increases protein stability and inhibits phosphatase activity. Ubiquitination has been reported to influence PTEN’s enzymatic function, stability, and subcellular localization depending on the site of modification and the type of ubiquitin chain. The N-terminal region of PTEN possesses a PI(4,5)P2 binding motif and a ubiquitination site at residue K13. The N-terminus is highly dynamic, with crystal structures of the PTEN N-terminus existing in both disordered and α-helical conformations. This region is also sensitive to mutations, and mutation of the K13 ubiquitination site diminishes phosphatase activity and nuclear localization. However, a detailed understanding of how PTEN is regulated by PTMs is lacking due to difficulties in preparing site-specifically and stoichiometrically modified proteins for biochemical studies. Therefore, we performed segmental 15N labeling of PTEN to probe the crosstalk between the dynamic N-terminus and the C-terminal phosphorylated tail using NMR. We also developed a semisynthetic method employing sequential expressed protein ligations to install ubiquitin at the K13 mimic of PTEN. This approach enabled us to biochemically study the effects of ubiquitination on PTEN. We also tested a variety of ubiquitinated peptide linkages for processing by deubiquitinases. Our studies revealed that the phosphorylated C-terminal tail binding diminishes the presumed α-helical character of the PTEN N-terminus. K13 ubiquitination of PTEN inhibits enzymatic activity towards PI(3,4,5)P3, reduces lipid vesicle binding, and modulates processing by NEDD4-1 E3 ligase. Moreover, the ubiquitinated PTEN protein and peptides with a K13 mimic can be efficiently cleaved by USP7 and other deubiquitinases. We have developed a novel semisynthetic strategy for generating ubiquitinated protein. Our semisynthetic approach would motivate further studies to dissect the effects of PTMs on other complex proteins.