0000
Chen, Zan; Thomas, Stefani N; Bolduc, David M; Jiang, Xuejun; Zhang, Xiangbin; Wolberger, Cynthia; Cole, Philip A
Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases Journal Article
In: Biochemistry, vol. 55, no. 26, pp. 3658-66, 0000, ISSN: 1520-4995.
Abstract | Links | BibTeX | Tags: Chromatography, Endosomal Sorting Complexes Required for Transport, Humans, Immunoprecipitation, Liquid, Nedd4 Ubiquitin Protein Ligases, Phosphorylation, Post-Translational, Protein Processing, PTEN Phosphohydrolase, Tandem Mass Spectrometry, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination, X-Linked Inhibitor of Apoptosis Protein
@article{1299965,
title = {Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases},
author = {Zan Chen and Stefani N Thomas and David M Bolduc and Xuejun Jiang and Xiangbin Zhang and Cynthia Wolberger and Philip A Cole},
doi = {10.1021/acs.biochem.6b00448},
issn = {1520-4995},
journal = {Biochemistry},
volume = {55},
number = {26},
pages = {3658-66},
abstract = {PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN’s C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN’s cellular stability.},
keywords = {Chromatography, Endosomal Sorting Complexes Required for Transport, Humans, Immunoprecipitation, Liquid, Nedd4 Ubiquitin Protein Ligases, Phosphorylation, Post-Translational, Protein Processing, PTEN Phosphohydrolase, Tandem Mass Spectrometry, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination, X-Linked Inhibitor of Apoptosis Protein},
pubstate = {published},
tppubtype = {article}
}
PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN’s C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN’s cellular stability.