Journal: Nature Communications

Article Title: Aberrant expression of CITED2 promotes prostate cancer metastasis by activating the nucleolin-AKT pathway

doi: 10.1038/s41467-018-06606-2

Figure Lengend Snippet: CITED2 forms a multimeric complex with nucleolin, p300, and PRMT5 subunits. a HEK293T cells were transfected with the Flag/SBP-CITED2 plasmid. CITED2-interacting proteins were precipitated using anti-Flag tag antibody or streptavidin, and identified by LC-MS/MS analyses. The proteins identified commonly in two precipitates are listed. b Prostate cancer cell lysates were immunoprecipitated with anti-CITED2 antibody or IgG, and the precipitates were immunoblotted with the indicated antibodies. c HEK293T cells were transfected with pcDNA or CITED2, and the cell lysates were subjected to FPLC. The FPLC elutes were immunoblotted with the indicated antibodies. The red box indicates a bigger-sized complex (600 to 700 kDa) that is formed by CITED2 overexpression. d In vitro binding analysis. Recombinant protein CITED2, PRMT5, NCL, and P300 were put together in a test tube. Proteins in tube were immunoprecipitated with indicated antibodies, and the precipitates were immunoblotted. Input levels were verified by electrophoresis and Coomassie staining. e Representative immunofluorescence images. PC3 cells were grown on coverslips, fixed with methanol, and stained with the indicated antibodies. All samples were stained with DAPI to visualize nuclei. The scale bar represents 20 μm. f The Flag/SBP-CITED2 constructs are shown in the top panel. HEK293T cells were cotransfected with one of the CITED2 constructs and Myc-PRMT5, HA-P300, and Flag/SBP-peptides were immunoprecipitated with anti-Flag and Myc-PRMT5 and HA-P300 were detected by western blotting. g HEK293T cells were transfected with the CITED2 constructs, and cell lysates were immunoprecipitated with anti-NCL and Flag/SBP-CITED2 peptides were detected by western blotting. h The Myc-PRMT5 constructs are shown in the top panel. HEK293T cells were cotransfected with one of the PRMT5 construct and Flag/SBP-CITED2, and Myc-peptides were immunoprecipitated with anti-Myc and Flag/SBP-CITED2 were detected by western blotting. All experiments were carried out at three distinct samples

Article Snippet: Human recombinant proteins of CITED2, PRMT5, P300, and NCL were purchased from Origene.

Techniques: Transfection, Plasmid Preparation, FLAG-tag, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Over Expression, In Vitro, Binding Assay, Recombinant, Electrophoresis, Staining, Immunofluorescence, Construct, Western Blot

Journal: Nature Communications

Article Title: Aberrant expression of CITED2 promotes prostate cancer metastasis by activating the nucleolin-AKT pathway

doi: 10.1038/s41467-018-06606-2

Figure Lengend Snippet: CITED2 is essential for post-translational modifications of NCL. a HEK293T cells were transfected with pcDNA or CITED2 and treated with Leptomycin B (200 nM), and the cell lysates were fractionated to cytosolic and nuclear components. The cell fractions were immunoblotted with the indicated antibodies. b HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2 or si-CITED2. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with the indicated antibodies. c HEK293T cells were transfected with Myc-PRMT5 and/or si-PRMT5. Cell lysates were immunoprecipitated with anti-dimethyl arginine antibody and precipitated NCL was immunoblotted. d HEK293T cells were transfected with HA-p300. Cell lysates were immunoprecipitated with anti-acetyl lysine antibody and precipitated NCL was immunoblotted. e , f HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2 or si-CITED2. Cell lysates were immunoprecipitated with anti-dimethyl arginine or anti-acetyl lysine antibody and precipitated NCL was immunoblotted. g , h HEK293T cells were cotransfected with the indicated plasmids and siRNAs. Cell lysates were immunoprecipitated with anti-dimethyl arginine or anti-acetyl lysine antibody and precipitated NCL was immunoblotted. i HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2, and the cell lysates were fractionated to cytosolic and nuclear components. The cell fractions were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. j The ERG–CITED2–PRMT5/p300–NCL pathway in prostate cancer. ERG is upregulated due to the TMPRSS2–ERG gene fusion and transactivates the CITED2 gene in prostate cancer cells. Overexpressed CITED2 induces the methylation and acetylation of NCL in the nucleus by recruiting PRMT5 and p300, then modified NCL is translocated to the cytoplasm. All experiments were carried out at three distinct samples

Article Snippet: Human recombinant proteins of CITED2, PRMT5, P300, and NCL were purchased from Origene.

Techniques: Transfection, Immunoprecipitation, Methylation, Modification

Journal: Scientific Reports

Article Title: Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

doi: 10.1038/srep40590

Figure Lengend Snippet: FT-MYPT1 binding proteins of HepG2 nuclear fraction.

Article Snippet: Phosphorylation of recombinant human PRMT5 (Sino Biological Inc.) was initiated by 0.2 mM 32 P-ATP or for LC-MS/MS analysis by 0.5 mM ATP in the absence (control) or in the presence of 0.4 U/ml ROK (Upstate, Millipore), 0.1 μg/ml PKA or 0.1 μg/ml PKC at 30 °C for 120 min. ROK was applied in buffer C and all kinase assay buffers contained 1 μM mycrocystin-LR (MC-LR).

Techniques: Binding Assay, Transduction, Expressing

Journal: Scientific Reports

Article Title: Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

doi: 10.1038/srep40590

Figure Lengend Snippet: ( A ) Immunoprecipitations were carried out using anti-MYPT1 1-296 and anti-PRMT5 antibodies as well as nonimmune rabbit serum as negative control. Immunoprecipitates and HepG2 total lysate were analysed by Western blots using antibodies specific for MYPT1 and PRMT5. ( B ) SPR analysis of the interaction of MYPT1 with PRMT5. Full-length GST-MYPT1 1-1004 was immobilized on anti-GST coupled CM5 sensor chip. FT-PRMT5 was injected over the surfaces in the indicated concentrations. The interaction was monitored using Biacore 3000. The association constant (K a ) value was indicated in the figure.

Article Snippet: Phosphorylation of recombinant human PRMT5 (Sino Biological Inc.) was initiated by 0.2 mM 32 P-ATP or for LC-MS/MS analysis by 0.5 mM ATP in the absence (control) or in the presence of 0.4 U/ml ROK (Upstate, Millipore), 0.1 μg/ml PKA or 0.1 μg/ml PKC at 30 °C for 120 min. ROK was applied in buffer C and all kinase assay buffers contained 1 μM mycrocystin-LR (MC-LR).

Techniques: Negative Control, Western Blot, Injection

Journal: Scientific Reports

Article Title: Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

doi: 10.1038/srep40590

Figure Lengend Snippet: ( A ) Autoradiograms of PRMT5 phosphorylated in the absence or in the presence of 0.1 μg/ml protein kinase A (PKA, left panel), 0.1 μg/ml protein kinase C (PKC, middle panel) or 0.4 U/ml Rho-associated kinase (ROK, right panel) with 32 P-ATP. ( B ) Western blot analysis of ROK-phosphorylated PRMT5 using antibody specific for phospho-Thr. After stripping the membrane anti-PRMT5 antibody was applied to detect PRMT5 as an input control. ( C ) Ion trap collision-induced dissociation (CID) spectra of PRMT5 phosphopeptides. CID of m/z: 656.338 (3+) identified as SDLLLSGRDWNpTLIVGK representing [69–85] of the wild type protein. Thr80 was identified as the modification site (see fragment ion y 11 (phosphorylated)). Peptide fragments are labeled according to the nomenclature by Biemann . ( D ) Effect of ROK inhibitor (10 μM H1152) on the phosphorylation level of PRMT5 during in vitro ROK assay. Control samples were prepared in the absence of ROK, positive control samples were prepared in the presence of ROK without ROK inhibitor. Relative phosphorylation level of Thr80 was judged by Western blot using anti- pPRMT5 T80 antibody and blots for PRMT5 served as loading control. ( E ) Effect of 25 nM FT-MYPT1 and 5 nM rPP1cδ or their combination on the phosphorylation level of PRMT5 at Thr80 80 as judged by Western blot. Data were compared to ROK-phosphorylated PRMT5. ( F , G ) Amount of MEP50 bound to FT-PRMT5 during ROK-phosphorylation ( F ) and dephosphorylation by MP ( G ) compared to unphosphorylated control samples. MEP50 was detected by anti-MEP50 antibody during Western blot and relative amount was normalized to the level of PRMT5. ( H , I ) In vitro arginine methyltransferase assay of unphosphorylated and ROK-phosphorylated PRMT5 measured by the symmetric dimethylation level of histone H2A Arg3 (H2AR3me2s, F) or histone H4 Arg3 (H4R3me2s, G) in the presence of 25 nM FT-MYPT1, 5 nM rPP1cδ or their combinations. Gels have been processed under the same experimental conditions. Values represents mean ± SEM; **p < 0.01, ***p < 0.001, ****p < 0.0001, # p < 0.05, one-way ANOVA followed by Tukey’s multiple comparison test, n = 3.

Article Snippet: Phosphorylation of recombinant human PRMT5 (Sino Biological Inc.) was initiated by 0.2 mM 32 P-ATP or for LC-MS/MS analysis by 0.5 mM ATP in the absence (control) or in the presence of 0.4 U/ml ROK (Upstate, Millipore), 0.1 μg/ml PKA or 0.1 μg/ml PKC at 30 °C for 120 min. ROK was applied in buffer C and all kinase assay buffers contained 1 μM mycrocystin-LR (MC-LR).

Techniques: Western Blot, Stripping Membranes, Modification, Labeling, In Vitro, Positive Control, De-Phosphorylation Assay

Journal: Scientific Reports

Article Title: Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

doi: 10.1038/srep40590

Figure Lengend Snippet: Immunofluorescent staining of non-target control (Ctrl) and MYPT1-silenced (siMYPT1) cells using antibodies specific for MYPT1 1-296 ( A ), PRMT5 ( B ), histone H2A symmetric dimethyl Arg3 (H2AR3me2s ( C ) and histone H4 symmetric dimethyl Arg3 (H4R3me2s ( D ) and actin as inducated in the figures. Scale bars: 50 μM. Enlargement of framed regions in merged images are shown on the right on A, B, C and D panels. Nuclear fractions of non-target control (Ctrl) and MYPT1-silenced (siMYPT1) HepG2 cells were prepared and analysed by Western blot using anti-MYPT1 1-296 ( E ), anti-PRMT5 ( F ), anti-pPRMT5 T80 ( G ), anti-histone H2A symmetric dimethyl Arg3 ( H ) and anti-histone H4 symmetric dimethyl Arg3 ( I ) specific antibodies. The relative expression of MYPT1 or PRMT5 and the relative symmetric dimethylation level of H2AR3 or H4R3 were normalized to lamin A/C as internal control. Samples derived from the same experiment and the blots were processed in parallel or assayed after stripping. The relative phosphorylation level of PRMT5 T80 was normalized to the expression level of PRMT5 and then to lamin A/C as internal control by densitometry. Mean ± SEM; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by student t- test.

Article Snippet: Phosphorylation of recombinant human PRMT5 (Sino Biological Inc.) was initiated by 0.2 mM 32 P-ATP or for LC-MS/MS analysis by 0.5 mM ATP in the absence (control) or in the presence of 0.4 U/ml ROK (Upstate, Millipore), 0.1 μg/ml PKA or 0.1 μg/ml PKC at 30 °C for 120 min. ROK was applied in buffer C and all kinase assay buffers contained 1 μM mycrocystin-LR (MC-LR).

Techniques: Staining, Western Blot, Expressing, Derivative Assay, Stripping Membranes

Journal: Scientific Reports

Article Title: Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

doi: 10.1038/srep40590

Figure Lengend Snippet: ( A ) Heat map of genes related to MYPT1 silencing in HepG2 cells by microarray analysis. The color code for the signal strength is shown in the box at the bottom in which induced genes are indicated by red and repressed genes are indicated by blue. ( B ) The onthology of the related genes and their classification by GO terms. Detection of retinoblastoma protein ( C ) and c-Myc ( D ) protein expression changes due to MYPT1 silencing from HepG2 whole cell lysates by Western blot. Protein levels were quantified by densitometry normalized to α-tubulin or GAPDH expression level. Samples derived from the same experiment and processed in parallel. RT-PCR analysis of MYPT1, PRMT5 and RAP1A mRNA levels in non-specific siRNA treated and MYPT1 silenced HepG2 cells ( E ). GAPDH was used as an invariant gene. Values are mean ± SEM from three independent experiments; *p < 0.05, **p < 0.01 by student t- test.

Article Snippet: Phosphorylation of recombinant human PRMT5 (Sino Biological Inc.) was initiated by 0.2 mM 32 P-ATP or for LC-MS/MS analysis by 0.5 mM ATP in the absence (control) or in the presence of 0.4 U/ml ROK (Upstate, Millipore), 0.1 μg/ml PKA or 0.1 μg/ml PKC at 30 °C for 120 min. ROK was applied in buffer C and all kinase assay buffers contained 1 μM mycrocystin-LR (MC-LR).

Techniques: Microarray, Expressing, Western Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

doi: 10.1038/srep40590

Figure Lengend Snippet: Reverse phase protein microarray analysis was conducted to study the changes in protein expression and posttranslational modification of PRMT5 ( A , B ), MYPT1 ( C , D ) and histone H2A ( E , F ) proteins in normal and tumor human cell lysates. Human HCC samples (n = 20) were grouped based on their clinically verified stage (middle grey columns) or grade (dark grey columns) classification of tumor. Average of HCC samples irrespectively of grouping and other types of metastatic liver cancer tissues are shown next to each other (light grey columns). Protein microarray contains 15 cancer cell lines and normal tissue lysate of controls of corresponding organs in triplicates (black columns). Value of 1 means average of relative expression, phosphorylation or symmetrical dimethylation of the given protein or residue to the corresponding non-tumor samples. Blots were processed under the same experimental procedure. Datas are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by student t- test.

Article Snippet: Phosphorylation of recombinant human PRMT5 (Sino Biological Inc.) was initiated by 0.2 mM 32 P-ATP or for LC-MS/MS analysis by 0.5 mM ATP in the absence (control) or in the presence of 0.4 U/ml ROK (Upstate, Millipore), 0.1 μg/ml PKA or 0.1 μg/ml PKC at 30 °C for 120 min. ROK was applied in buffer C and all kinase assay buffers contained 1 μM mycrocystin-LR (MC-LR).

Techniques: Microarray, Expressing, Modification

Journal: Scientific Reports

Article Title: Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

doi: 10.1038/srep40590

Figure Lengend Snippet: We hypothesize that MP and ROK are involved in gene expression. Under normal conditions MP modulates symmetrical dimethylation of histone core proteins in cell nucleus via dephosphorylation of PRMT5 at its activatory phosphorylation site (Thr80) causing changes in gene expression. In tumor cells inhibitory phosphorylation of MP on Thr850 is increased leading to higher phosphorylation level of PRMT5 at Thr80 by ROK. Activated PRMT5 provokes gene repression by raising symmetrical dimethylation of histone H4 Arg3 that triggers proto-oncogene activation and tumor formation.

Article Snippet: Phosphorylation of recombinant human PRMT5 (Sino Biological Inc.) was initiated by 0.2 mM 32 P-ATP or for LC-MS/MS analysis by 0.5 mM ATP in the absence (control) or in the presence of 0.4 U/ml ROK (Upstate, Millipore), 0.1 μg/ml PKA or 0.1 μg/ml PKC at 30 °C for 120 min. ROK was applied in buffer C and all kinase assay buffers contained 1 μM mycrocystin-LR (MC-LR).

Techniques: Expressing, De-Phosphorylation Assay, Activation Assay