Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases.
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.
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
Zan
Z
Chen
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
Stefani N
SN
Thomas
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
David M
DM
Bolduc
Cell Biology Program, Memorial Sloan-Kettering Cancer Center , New York, New York 10065, United States.
Xuejun
X
Jiang
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
Xiangbin
X
Zhang
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
Cynthia
C
Wolberger
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
Philip A
PA
Cole
D002853:Chromatography, Liquid; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D000075702:Nedd4 Ubiquitin Protein Ligases; D051059:PTEN Phosphohydrolase / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D025801:Ubiquitin; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C465283:WWP2 protein, human; D044767:Ubiquitin-Protein Ligases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human