Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination

• Published in: Journal of the American Chemical Society Vol. 145; no. 11; pp. 6039 - 6044
• Main Authors: Iwase, Reina, Dempsey, Daniel R., Whedon, Samuel D., Jiang, Hanjie, Palanski, Brad A., Deng, Bedphiny, Cole, Philip A.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.03.2023
• Subjects: Deubiquitinating Enzymes - metabolism; Endosomal Sorting Complexes Required for Transport - metabolism; Lipids; Nedd4 Ubiquitin Protein Ligases - genetics; Nedd4 Ubiquitin Protein Ligases - metabolism; neoplasms; phosphorylation; PTEN Phosphohydrolase - chemistry; tumor suppressor proteins; ubiquitin; Ubiquitin - chemistry; ubiquitin-protein ligase; Ubiquitin-Protein Ligases - metabolism; Ubiquitination
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/jacs.2c13871

Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN’s biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.