Journal: ACS Omega

Article Title: Phosphatase and Pseudo-Phosphatase Functions of Phosphatase of Regenerating Liver 3 (PRL-3) Are Insensitive to Divalent Metals In Vitro

doi: 10.1021/acsomega.3c04095

Figure Lengend Snippet: PRL-3 can dephosphorylate numerous non-protein substrates. A malachite green assay was used to determine if PRL-3 could dephosphorylate the indicated synthetic substrates, amino acids, nucleotides, metabolites, and phospholipids. The amount of inorganic phosphate released after 1.5 h incubation was calculated from a standard curve. Error bars represent the standard deviation. Significance was calculated using an ordinary one-way analysis of variance (ANOVA) analysis. **** p < 0.0001, comparing PRL-3 activity against phospho-tyrosine to phospho-serine and phospho-threonine, *** p < 0.001, comparing PRL-3 activity against PI(4,5)P 2 to the other phosphatidylinositol substrates, ns = no significant difference.

Article Snippet: The following compounds were utilized in this assay: DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate (Thermo Fisher, D6567); pNPP, p -nitrophenyl phosphate, disodium salt (NEB, P0757S); DDAOP, 9H-(1,3-dichloro-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (Bimol, ABD-11629); phospho-serine, O-phospho- L -serine (Cayman Chemical, 16151); phospho-threonine, O-phospho-L-threonine (Cayman Chemical, 35057); phospho-tyrosine, O-phospho- L -tyrosine (Cayman Chemical, 23064); Mg-ATP, equimolar mix of MgCl 2 and ATP; ATP, adenosine 5′-triphosphate (Gold Biotechnology, A-081-1); ADP, adenosine 5′-diphosphate (USBio, 10,490); AMP, adenosine 5-monophosphate (CalBioChem, 118110); cAMP, 3′-5′-cyclic adenosine monophosphate (Sigma, A6885); Mg-GTP equimolar mix of MgCl 2 and GTP; GTP, guanosine 5′-triphosphate (Sigma, G-8877); GDP, guanosine 5′-diphosphate (Sigma, G-7127); GMP, guanosine 5′-monophosphate (Cayman Chemical, 16957); cGMP, 3′-5′-cyclic guanosine monophosphate (Sigma, G6129); CTP, cytosine 5′-triphosphate (Sigma, C-1506); UTP, uridine 5′-triphosphate (Sigma, U-6625); NADH, β-nicotinamide adenine dinucleotide (Sigma-Aldrich, N7004); NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate (Sigma-Aldrich, N1630); G6P, glucose 6-phosphate (Sigma-Aldrich, G7879); F6P, fructose 6-phosphate (Sigma-Aldrich, F3627); F1,6P, fructose 1,6-bisphosphate (Sigma-Aldrich, F6803); GA3P, glyceraldehyde 3-phosphate (Sigma-Aldrich, G5251); 3PG, 3-phosphoglycerate (Sigma, p8877); 16:0 PA, 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (Avanti, 830855); PI, phosphatidylinositol diC8, (Echelon, P-0008); IP6, D-myo-inositol-1,2,3,4,5,6-hexaphosphate (Cayman Chemical, 10008415); PI(3)P, phosphatidylinositol 3-phosphate diC8 (Echelon, P-3008); PI(4)P, phosphatidylinositol 4-phosphate diC8 (Echelon, P-4008); PI(5)P, phosphatidylinositol 5-phosphate diC8 (Echelon, P-5008); PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate diC8 (Echelon, P-3408); PI(3,5)P 2 , phosphatidylinositol 3,5-bisphosphate diC8 (Echelon, P-3508); PI(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate diC8 (Echelon, P-4508); and PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-trisphosphate diC8 (Echelon, P-3908).

Techniques: Malachite Green Assay, Incubation, Standard Deviation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P 2 hydrolysis in the plasma membrane

doi: 10.1016/j.jbc.2023.104812

Figure Lengend Snippet: Analysis of OCRL translocation in Jurkat T-cells upon anti-CD3 stimulation. A , confocal microscopy analysis of endogenous OCRL ( green ) and ORP4L ( red ) localization in shNT- or sh ORP4L -transfected Jurkat T-cells. Cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min. Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. B , cells with wildtype OCRL1 or OCRL1 (mut) overexpression, confocal microscopy analysis of OCRL (by Xpress) and ORP4L in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml −1 ). Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL1 distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. C , BiFC analysis between OCRL 1 /pVn-N1 and ORP4L /pVc-C1 or OCRL 1 (mut)/pVn-N1 and ORP4L /pVc-C1 in Jurkat T-cells. After transfected with the fragments for 24 h, cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min and analyzed. Scale bars, 10 μm. The histograms showed the relative BiFC intensity from 10 cells. D , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with ORP4L knockdown. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. The histograms showed the relative OCRL protein levels in PM ( upper ) and Golgi ( lower ) from three independent experiments. E , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with OCRL1 or OCRL1 (mut) overexpression. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. OCRL were detected by Xpress antibody. The histograms showed the relative OCRL protein levels in PM (upper) and Golgi (lower) from three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student's t test. BiFC, bimolecular fluorescence complementation; CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1; OSBP; oxysterol-binding protein, ORP4L; OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PM, plasma membrane.

Article Snippet: Briefly, after centrifugation, 1 μl of suspension was spotted onto nitrocellulose membrane (Bio-Rad Laboratories), probed with PI(4,5)P 2 antibody (1:500, Echelon Biosciences), PI(4)P antibody (1:500, Echelon Biosciences), or PM internal loading control pan-cadherin antibody (Santa Cruz), and detected using an HRP-conjugated secondary antibody and enhanced chemiluminescence.

Techniques: Translocation Assay, Confocal Microscopy, Transfection, Over Expression, Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P 2 hydrolysis in the plasma membrane

doi: 10.1016/j.jbc.2023.104812

Figure Lengend Snippet: OCRL-ORP4L interaction is required for PI(4,5)P 2 consumption in the PM. A , Western blot showing OCRL expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . Cells were subjected to control (shNT), OCRL1 knockdown (sh OCRL1 ) alone, or combined with OCRL1 rescued expression (sh OCRL1 + OCRL1 ) or combined with OCRL1 (Mut) rescued expression (sh OCRL1 + OCRL1 (Mut)). Cells were transduced with lentivirus expressing shNT or sh OCRL1 for 48 h, before transduction with lentivirus expressing OCRL1 or OCRL1 (Mut). After additional 48 h of culture, the cells were used for analysis. B , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( A ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with OCRL1 knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in C . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. D , Western blot showing ORP4L expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . The cells subjected to control (shNT), ORP4L knockdown (sh ORP4L ) alone, or combined with ORP4L rescued expression (sh ORP4L + ORP4L ) or combined with ORP4L (Mut) rescued expression (sh ORP4L + ORP4L (Mut)). Cells were transduced with lentivirus expressing shNT or sh ORP4L for 48 h, before transduction with lentivirus expressing ORP4L or ORP4L (Mut). After additional 48 h of culture, the cells were used for analysis. E , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( D ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with ORP4L knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in F . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. ∗∗∗ p < 0.001; Student's t test. CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1, OSBP, oxysterol-binding protein; ORP4L, OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PLC; phosphoinositide phospholipase C; PM, plasma membrane.

Article Snippet: Briefly, after centrifugation, 1 μl of suspension was spotted onto nitrocellulose membrane (Bio-Rad Laboratories), probed with PI(4,5)P 2 antibody (1:500, Echelon Biosciences), PI(4)P antibody (1:500, Echelon Biosciences), or PM internal loading control pan-cadherin antibody (Santa Cruz), and detected using an HRP-conjugated secondary antibody and enhanced chemiluminescence.

Techniques: Western Blot, Expressing, Transduction, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P 2 hydrolysis in the plasma membrane

doi: 10.1016/j.jbc.2023.104812

Figure Lengend Snippet: Analysis of OCRL translocation in Jurkat T-cells upon anti-CD3 stimulation. A , confocal microscopy analysis of endogenous OCRL ( green ) and ORP4L ( red ) localization in shNT- or sh ORP4L -transfected Jurkat T-cells. Cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min. Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. B , cells with wildtype OCRL1 or OCRL1 (mut) overexpression, confocal microscopy analysis of OCRL (by Xpress) and ORP4L in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml −1 ). Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL1 distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. C , BiFC analysis between OCRL 1 /pVn-N1 and ORP4L /pVc-C1 or OCRL 1 (mut)/pVn-N1 and ORP4L /pVc-C1 in Jurkat T-cells. After transfected with the fragments for 24 h, cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min and analyzed. Scale bars, 10 μm. The histograms showed the relative BiFC intensity from 10 cells. D , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with ORP4L knockdown. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. The histograms showed the relative OCRL protein levels in PM ( upper ) and Golgi ( lower ) from three independent experiments. E , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with OCRL1 or OCRL1 (mut) overexpression. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. OCRL were detected by Xpress antibody. The histograms showed the relative OCRL protein levels in PM (upper) and Golgi (lower) from three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student's t test. BiFC, bimolecular fluorescence complementation; CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1; OSBP; oxysterol-binding protein, ORP4L; OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PM, plasma membrane.

Article Snippet: Cells were incubated with 1 μM soluble short-chain diC8-PI(4,5)P 2 (Echelon Biosciences) for 10 min at room temperature in culture medium, then the PM was prepared by using Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies, Inc.) according to the manufacturer’s instructions.

Techniques: Translocation Assay, Confocal Microscopy, Transfection, Over Expression, Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P 2 hydrolysis in the plasma membrane

doi: 10.1016/j.jbc.2023.104812

Figure Lengend Snippet: OCRL-ORP4L interaction is required for PI(4,5)P 2 consumption in the PM. A , Western blot showing OCRL expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . Cells were subjected to control (shNT), OCRL1 knockdown (sh OCRL1 ) alone, or combined with OCRL1 rescued expression (sh OCRL1 + OCRL1 ) or combined with OCRL1 (Mut) rescued expression (sh OCRL1 + OCRL1 (Mut)). Cells were transduced with lentivirus expressing shNT or sh OCRL1 for 48 h, before transduction with lentivirus expressing OCRL1 or OCRL1 (Mut). After additional 48 h of culture, the cells were used for analysis. B , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( A ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with OCRL1 knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in C . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. D , Western blot showing ORP4L expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . The cells subjected to control (shNT), ORP4L knockdown (sh ORP4L ) alone, or combined with ORP4L rescued expression (sh ORP4L + ORP4L ) or combined with ORP4L (Mut) rescued expression (sh ORP4L + ORP4L (Mut)). Cells were transduced with lentivirus expressing shNT or sh ORP4L for 48 h, before transduction with lentivirus expressing ORP4L or ORP4L (Mut). After additional 48 h of culture, the cells were used for analysis. E , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( D ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with ORP4L knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in F . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. ∗∗∗ p < 0.001; Student's t test. CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1, OSBP, oxysterol-binding protein; ORP4L, OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PLC; phosphoinositide phospholipase C; PM, plasma membrane.

Article Snippet: Cells were incubated with 1 μM soluble short-chain diC8-PI(4,5)P 2 (Echelon Biosciences) for 10 min at room temperature in culture medium, then the PM was prepared by using Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies, Inc.) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Transduction, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a PIP Binding Site in the N-Terminal Domain of V-ATPase a4 and Its Role in Plasma Membrane Association

doi: 10.3390/ijms24054867

Figure Lengend Snippet: Mutation within the putative a4 PIP binding motif reduced interactions with PI(4,5)P 2 -enriched liposomes. ( A ) Liposome pull-down assay with PolyPIPosomes (Echelon) enriched with PI(3,4)P 2 , PI(4,5)P 2 and PI(3,4,5)P 3 of the WT and double mutant K234A/K237A. In total, 30 μg proteins (wildtype and mutant) was incubated for 1 h at room temperature with 20 μL of 1 mM PolyPIPosomes containing 5% of the PIP in binding buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Nonidet P-40). A total 5 μg of purified proteins was input as loading control. Quantification: Relative signal intensity of the proteins (WT, double mutants) pulled down with indicated liposomes to input. Data represent mean ± S.D. from three independent experiments. Pair t -tests were run to analyze the significance in mean difference. ** indicates p < 0.01. ( B ) Liposome pull-down assay with PI(4,5)P 2 -enriched PolyPIPosome with WT and K234A/K237A and K237del mutants. Quantification: relative intensity of the proteins pulled down with PI(4,5)P2-enriched liposome to input. Data represent mean ± S.D. from three independent experiments. Statistical significance was analyzed by one-way ANOVA with Dunnett’s multiple comparisons test comparing mutants to WT. ** indicates p < 0.01, *** indicates p < 0.001. ( C ) Liposome pull-down assay with PI(4)P-enriched PolyPIPosome with WT and K237del mutant. Quantification: relative intensity of the proteins pulled down with the liposome to input (30 ng). Data represent mean ± S.D. from five independent experiments.

Article Snippet: A total of 10 μg of purified proteins was incubated with 20 µL 1 mM PI(4,5)P 2 -PolyPIPosome (Echelon) and 200 µL of binding buffer (50 mM Tris pH 8.0, 150 mM NaCl and 0.05% Nonidet P-40) and incubate rotating at 4 °C for 2 h. The protein-bound liposomes were pelleted by centrifugation at 13,000 rpm for 10 min, followed by 3 washes with 200 µL of binding buffer each.

Techniques: Mutagenesis, Binding Assay, Pull Down Assay, Incubation, Purification

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a PIP Binding Site in the N-Terminal Domain of V-ATPase a4 and Its Role in Plasma Membrane Association

doi: 10.3390/ijms24054867

Figure Lengend Snippet: Ionomycin reduces a4NT-membrane association in vivo. ( A ) Plasmids containing FLAG-tagged a4NT wildtype (WT) were transfected in HEK293 cells. Cells were treated with 5 µM of ionomycin at room temperature for 15 min before harvest. Cells were fixed and permeabilized, then stained for membrane marker anti-Na + /K + -ATPase with Alexa Fluor 488 (green), followed by staining for FLAG-tagged proteins (red). White arrows represent regions of plasma membrane localization. Images are representatives of 20 cells each from 4 experiments. The scale bar represents 10 μm (inset at the right panel is enlarged area of colocalization). ( B ) Quantification. Relative intensity of the red signal overlapping with the green signal to the total red signal. Data represent mean value of at least 20 cells assessed ±SEM from 4 independent experiments. A pair t -test was run to analyze the significance in mean difference. ** indicates p < 0.001. ( C ) Treatment with ionomycin causes depletion of membrane PI(4,5)P 2 , resulting in mislocalization of PH-PLC. Plasmids containing GFP-tagged PH-PLC were transfected in HEK293 cells. Cells were treated with 5 µM of ionomycin at room temperature for 15 min before harvest. Cells were fixed and permeabilized, then stained for membrane marker anti-Na + /K + -ATPase with Alexa Fluor 568 (red). Quantification of the resulting immunofluorescent images were performed by measuring the relative intensity of the red signal overlapping with the green signal to the total red signal. Data represent mean value of at least 20 cells assessed ±SEM from 3 independent experiments. A pair t -test was run to analyze the significance in mean difference. * indicates p < 0.05.

Article Snippet: A total of 10 μg of purified proteins was incubated with 20 µL 1 mM PI(4,5)P 2 -PolyPIPosome (Echelon) and 200 µL of binding buffer (50 mM Tris pH 8.0, 150 mM NaCl and 0.05% Nonidet P-40) and incubate rotating at 4 °C for 2 h. The protein-bound liposomes were pelleted by centrifugation at 13,000 rpm for 10 min, followed by 3 washes with 200 µL of binding buffer each.

Techniques: In Vivo, Transfection, Staining, Marker

Journal: PLoS ONE

Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

doi: 10.1371/journal.pone.0033889

Figure Lengend Snippet: ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.

Article Snippet: PI(3,5)P 2 -DIC8 (P4508) was purchased from Echelon Bioscience Inc. (Salt Lake City, USA).

Techniques: Injection, Expressing, Dominant Negative Mutation

Journal: PLoS ONE

Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

doi: 10.1371/journal.pone.0033889

Figure Lengend Snippet: Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.

Article Snippet: PI(3,5)P 2 -DIC8 (P4508) was purchased from Echelon Bioscience Inc. (Salt Lake City, USA).

Techniques: