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

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

Characterization of the SUMO-Binding Activity of the Myeloproliferative and Mental Retardation (MYM)-Type Zinc Fingers in ZNF261 and ZNF198

<div><p>SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM)-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs). Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs). In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198.</p></div

MYM-type zinc fingers interact with the α-helix and second β-strand in SUMO-2.

<p><i>In vitro</i> expressed full-length ZNF261 and ZNF198 were incubated with GST or GST-tagged SUMO-2, SUMO-2(QFI), SUMO-2(x3), and SUMO-2(x3)(QFI). Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. All binding assays contained equivalent amounts of GST and GST-tagged SUMO proteins as determined by immunoblot analysis of eluted proteins with an anti-GST antibody.</p

Zinc chelation does not significantly perturb SUMO-binding activity of ZNF261.

<p>A: <i>In vitro</i> expressed full-length ZNF261 proteins were incubated overnight with immobilized GST or GST-tagged SUMO-2(x3) in assay buffer (untreated) or assay buffer containing 50 mM EDTA/100 mM sodium acetate, pH 5.5 (EDTA treated), or 1,10-phenanthroline (1,10-phen), or 1,7-phenanthroline (1,7-phen). Unbound proteins were washed away and bound proteins were visualized by SDS-PAGE followed by autoradiography. <b>B</b>: Immobilized GST or GST-tagged SUMO-2(x3) was incubated with ³⁵S-labeled ZNF261(1-495) wild-type or ZNF261(1-495) containing cysteine to alanine substitutions. Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography.</p

MYM-type zinc fingers in ZNF261 and ZNF198 are involved in SUMO-binding.

<p>A: GST and GST-SUMO-2(x3) were immobilized on glutathione coated plates and incubated with ³⁵S-labeled full-length or N- or C-terminus truncation fragments of ZNF261. Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. <b>B</b>: Pyruvate kinase or pyruvate kinase fused to the MYM-type zinc fingers from ZNF261 or ZNF198 were incubated with immobilized GST (white), GST-SUMO-2 (grey), and GST-SUMO-2(x3) (black). Unbound proteins were removed by washing and bound proteins were determined by liquid scintillation detection of eluted proteins. Plotted values represent the mean +/− S.D. from three independent experiments. <b>C</b>: ZNF261 was run at three concentrations in the analytical ultracentrifuge (10, 20, and 40 µM). Representative sedimentation data from the 20 µM ZNF261(1-495) data set is shown in the left panel. The data was globally fit to a single species model and the residuals between the calculated and experimental absorbance are shown below. The residuals appear randomly scattered around zero indicating that a single species model describes the data. ZNF261(1-495) (20 µM) was run with three concentrations of SUMO-2(x2) (20, 40, and 80 µM) in the analytical ultracentrifuge. Representative sedimentation data of the 20 µM ZNF261 and 80 µM SUMO-2(x2) data set is shown in the right panel. Data were globally fit to an A+B → AB model and the residuals between the calculated and experimental absorbance are shown below. The global reduced chi-squared value was 3.13.</p

MYM-type zinc finger containing proteins ZNF261 and ZNF198 have SUMO-binding activity.

<p>A: Schematic diagrams of MYM-type zinc finger containing proteins. Amino acid number is shown next to each schematic diagram. Locations of the myeloproliferative and mental retardation-type zinc fingers (MYM-type zinc fingers, gold), proline/valine-rich domains (P/V-rich, green), and Cre-like domains (CL domain, blue) are shown. The MYM-type zinc finger consensus motif where X is any amino acid is shown below. <b>B</b>: Full-length ZNF261 or ZNF198 proteins were <i>in vitro</i> transcribed and translated in the presence of [³⁵S] methionine and bound to immobilized GST-tagged SUMO-1, SUMO-2 or SUMO-2(x3). Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. Binding to GST alone was performed as a negative control. All binding assays contained equivalent amounts of GST and GST-tagged SUMO proteins as determined by immunoblot analysis of eluted proteins with an anti-GST antibody.</p

The N-terminus and MYM-type zinc fingers in ZNF198 are required for localization to PML-NBs.

<p>A: U2OS cells were transfected with constructs encoding myc-tagged ZNF198 full-length or truncation fragments. Localization of ZNF198 with PML-NBs was analyzed by fluorescence microscopy. <b>B</b>: Full-length and truncation fragments of myc-tagged ZNF198 were expressed in U2OS cells, immunopurified with anti-myc agarose beads, and detected with anti-myc and anti-SUMO-2 antibodies. Full-length protein bands are marked with an asterisk in each input lane. <b>C</b>: Myc-tagged ZNF198 and FLAG-tagged PML were co-expressed in U2OS cells, immunopurified with anti-myc agarose beads or anti-FLAG agarose beads, and detected with anti-myc and anti-PML antibodies.</p

Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module’s molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p