Journal: Nature Communications

Article Title: Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II

doi: 10.1038/s41467-020-18173-6

Figure Lengend Snippet: a Purified, recombinant Cdk9/cyclin T1 phosphorylates wild-type (WT) GFP-PP1γ, expressed in human cells and recovered by anti-GFP immunoprecipitation, but not a Thr311→Ala (TA) mutant variant. Phosphorylation was detected with antibody specific for the carboxy-terminal phosphorylation site (Thr320) in PP1α isoform, analogous to Thr311 of PP1γ. b Inhibition of Cdk9 or Cdk1 diminishes PP1γ-inhibitory phosphorylation in human cells. HCT116 cells were treated with DMSO, a Cdk1 inhibitor (RO-3306), a Cdk9 inhibitor (NVP-2), or both, as indicated. Extracts were analyzed by direct immunoblotting (lanes 1–4), or anti-PP1γ immunoprecipitation (IP) followed by immunoblotting (lanes 5–8), with the indicated antibodies. c Cdk9 inhibition diminishes phosphorylation of Spt5-Thr806 but not Ser2 of the Pol II CTD. HCT116 cells were treated with the indicated concentrations of NVP-2 for 1 h and extracts were immunoblotted with the indicated antibodies. d HCT116 cells were treated with 250 nM NVP-2 for indicated times and extracts were immunoblotted with antibodies specific for Spt5, Spt5-pThr806, and Spt5-pSer666, as indicated. Immunoblot ( n = 1) signals were quantified with ImageJ software. e Spt5-derived phosphopeptides containing pSer666 or pThr806 or a control histone H3-derived phosphopeptide containing pSer10, as indicated, were incubated with purified PP1 or lambda phosphatase, as indicated, and phosphate release was measured colorimetrically. Individual data points are shown from three biological replicates ( n = 3); error bars indicate ±standard deviation (±s.d.) from mean. f HCT116 cells were transfected with an siRNA cocktail targeting all three PP1 catalytic-subunit isoforms or a scrambled control (C) siRNA, as indicated, and extracts were immunoblotted for the indicated proteins or protein modifications. Experiments were performed twice with similar results ( a – c , f ). Source data are provided as a file. Uncropped blots can be found in the .

Article Snippet: PP1 isoform-specific siRNAs targeting PP1α (sc-36299), PP1β (sc-36295), and PP1γ (sc-36297) were from Santa Cruz Biotechnology.

Techniques: Purification, Recombinant, Immunoprecipitation, Mutagenesis, Variant Assay, Phospho-proteomics, Inhibition, Western Blot, Software, Derivative Assay, Control, Incubation, Standard Deviation, Transfection

Journal: Nature Communications

Article Title: Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II

doi: 10.1038/s41467-020-18173-6

Figure Lengend Snippet: a Schematic of the MYC gene, indicating positions of primer pairs used in ChIP-qPCR analysis. b ChIP-qPCR analysis of PP4C, PP4R2, and PP1γ on the MYC gene in unperturbed HCT116 cells ( n = 2 biological replicates). c ChIP-qPCR analysis of PP4R2 and PP4R2-pThr173 after inhibition of Cdk9 with NVP-2 (250 nM) or mock treatment (DMSO) for 1 h ( n = 2 biological replicates). d Cells transfected with siRNA targeting PP4R2 or nontargeted negative control siRNA were subjected to formaldehyde cross-linking, chromatin isolation, and reversal of cross-linking before analysis by immunoblotting with indicated antibodies to measure the depletion of PP4R2 and phosphorylation of Spt5 at Ser666 and Thr806. e ChIP-qPCR analysis on MYC shows Pol II distribution with or without PP4R2 depletion. Error bars (+s.d.) and p values were calculated by two-sided Student’s t test from four biological replicates ( n = 4); individual data points from biological replicates are shown in the plots ( b , c , and e ). f Two distinct Cdk9-phosphatase switches govern transitions in Spt5-phosphorylation state—a model. At the 5′ pause prior to P-TEFb activation, the PP4 complex is active and both CTR1 and the KOW4–KOW5 loop are unphosphorylated, At the 3′ pause, PP1 becomes active to dephosphorylate CTR1 but not Ser666, distinguishing the two paused complexes. During elongation, Cdk9 phosphorylates Spt5, and inhibits PP4 and PP1. Source data are provided as a file.

Article Snippet: PP1 isoform-specific siRNAs targeting PP1α (sc-36299), PP1β (sc-36295), and PP1γ (sc-36297) were from Santa Cruz Biotechnology.

Techniques: ChIP-qPCR, Inhibition, Transfection, Negative Control, Isolation, Western Blot, Phospho-proteomics, Activation Assay

Journal: Journal of molecular and cellular cardiology

Article Title: Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations.

doi: 10.1016/j.yjmcc.2015.08.018

Figure Lengend Snippet: Fig. 1. Generation and assessment of cardiac specific PP1 isoform deleted mice. (A) Real- time PCR analysis of expression of PP1 isoforms in adult cardiac myocytes. Rpl27 was used as an internal control. N = 3 for each group. *p b 0.05 vs PP1α. (B) Real-time PCR analysis of expression of PP1 isoforms in the hearts of 2 month-old mice for each of the groups shown, with or without Cre-mediated deletion. N = 4 for each group in the real- time PCR analysis. *p b 0.05 vs fl/flmice. (C) Western blots for PP1 isoforms, I-1, I-2, and gapdh from the hearts of Ppp1c-fl/flmice or Ppp1c-fl/flNkx2.5-Cre mice. Approximately 120 μg of protein was processed to detect I-1 and I-2. (D) Quantification of Western blots shown in Fig. 1C. N = 4 for each of the groups. *p b 0.05 vs control (Con.).

Article Snippet: Protein samples from each fraction were quantified with a Bradford assay (Bio-Rad) and subjected to 10% SDS-PAGE forWestern blot detection of PP1α (Santa Cruz Biotechnology, sc-6104), PP1β (Millipore, 07–1217), PP1γ (Santa Cruz Biotechnology, sc-6108), GAPDH (Fitzgerald, 10–1500), Troponin I (Cell Signaling Technology, 4002), pSer 23/24-Troponin I (Cell Signaling Technology, 4004), Caveolin-3 (BD Biosciences, 610,421), I-1 (Abcam, ab40877), and I-2 (R&D Systems, AF4719).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: Journal of molecular and cellular cardiology

Article Title: Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations.

doi: 10.1016/j.yjmcc.2015.08.018

Figure Lengend Snippet: Fig. 6. Adult deletion of PP1 isoforms by αMHC-MerCreMer and the effects on cardiac function and interstitial fibrosis. (A) Schematic representation of the experimental setup. Eight week- old mice were administered five consecutive days of tamoxifen (0.5 mg/day) and then analyzed at 10 and 14 weeks of age. (B) Western blot for PP1 isoform deletion from cardiac protein extracts in each of the indicated genotypes of mice 6 weeks after the start of tamoxifen treatment. (C) Echocardiographic measurement of cardiac fractional shortening (FS%) of the indi- cated genotypes of mice 6 weeks after tamoxifen administration. *p b 0.05 vs αMHC-MerCreMer. Number of mice used is shown in the bars. (D) Masson's trichrome staining of paraffin- embedded heart sections from αMHC-MerCreMer controls and the indicated Ppp1c-deleted mice. (E) Western blot for PP1 isoform deletion from cardiac protein extracts in each of the indicated genotypes of mice 2 weeks after the start of tamoxifen treatment. (F) Echocardiographic measurement of cardiac fractional shortening (FS%) of the indicated genotypes of mice 2 weeks after tamoxifen administration. *p b 0.05 vs αMHC-MerCreMer. Number of mice used is shown within the bars. Abbreviations, MCM, αMHC-MerCreMer transgene.

Article Snippet: Protein samples from each fraction were quantified with a Bradford assay (Bio-Rad) and subjected to 10% SDS-PAGE forWestern blot detection of PP1α (Santa Cruz Biotechnology, sc-6104), PP1β (Millipore, 07–1217), PP1γ (Santa Cruz Biotechnology, sc-6108), GAPDH (Fitzgerald, 10–1500), Troponin I (Cell Signaling Technology, 4002), pSer 23/24-Troponin I (Cell Signaling Technology, 4004), Caveolin-3 (BD Biosciences, 610,421), I-1 (Abcam, ab40877), and I-2 (R&D Systems, AF4719).

Techniques: Western Blot, Staining

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Effects of PP1α depletion on MEK/ERK activation in B-RafV600E and K-RasG12C/D tumor cells. (A to D) Total cell lysates were analyzed by Western blotting. SK-MEL 28 (A), A375 (B), MiaPaCa-2 (C), and PANC-1 (D) cells were infected with two different lentiviral shRNA systems targeting PP1α (shPP1α#1 and shPP1α#2) for 3 days before harvest. pLKO.1 was the control virus. (E) Densitometry of phospho-MEK1/2 signals normalized to total MEK1/2 levels.

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: Activation Assay, Western Blot, Infection, shRNA

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Effects of mortalin depletion on MEK/ERK activation and PP1α/PP2A expression in B-RafV600E and K-RasG12C/D tumor cells. Total cell lysates were analyzed by Western blotting. (A) SK-MEL 28 cells were infected with two different lentiviral shRNA systems targeting mortalin (shMort#1 and shMort#2) for the indicated periods before harvest. pLL3.7 was the control virus. (B) A375 cells stably expressing doxycycline-inducible pTRIPZ shRNA targeting mortalin (shMort-mir) were treated with doxycycline for 5 days. (C and D) MiaPaCa-2 (C) and PANC-1 (D) cells were infected with shMort#1 or shMort#2 for 6 days. pPP1 indicates phosphorylation at Thr320 of PP1.

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: Activation Assay, Expressing, Western Blot, Infection, shRNA, Stable Transfection

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Effects of adenosine nucleotides on mortalin interactions with PP1α and MEK2. GST-mortalin (A and B) or GST-PBD (C and D) (0.5 μM) was incubated for 60 min with 0.5 μM recombinant PP1α (A and C) or MEK2 (B and D) in the presence of 1 μM ATP or ADP before GST pulldown using glutathione-Sepharose and Western blotting.

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: Incubation, Recombinant, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Mortalin physically interacts with PP1α and MEK1/2 in vivo. (A to C) Total lysates of SK-MEL 28 cells were subject to IP using the monoclonal antibody specific to MEK1 (A), MEK2 (B), or PP1α (C). Normal IgG corresponding to each antibody was used as the control. IP fractions were immunoblotted (IB) to determine co-IP of the indicated proteins. (D) Total lysates of different human B-RafV600E tumor lines were subject to IP using the anti-MEK2 antibody. Input and IP fractions were immunoblotted for the indicated proteins. (E) Correlation between PP1α-MEK2 interaction and mortalin-MEK2 interaction. For analysis, densitometry data of PP1α and mortalin signals in the IP fractions in panel D were normalized to the corresponding MEK2 pulldown signals. Fold changes of the normalized data are relative to HT29 (P = 0.0254; r = 0.8155). (F) Inverse correlation between PP1α-MEK2 interaction and phospho-MEK1/2 levels. Data for PP1α-MEK2 interactions are from panel E. Data for phospho-MEK1/2 levels were quantitated in the input in panel D by densitometry and normalized to the corresponding MEK1/2 signals. Fold changes of the normalized phospho-MEK1/2 data are relative to A375 (P = 0.0431; r = −0.7694). (G) Inverse correlation between mortalin-MEK2 interaction and phospho-MEK1/2 levels. Data are from panels E and F (P = 0.079; r = −0.7015).

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: In Vivo, Co-Immunoprecipitation Assay

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Mortalin physically interacts with PP1α and MEK2 in vitro. (A) In vitro binding assay using 0.5 μM recombinant PP1α and GST-mortalin. GST was used as the control. (B and C) In vitro binding assay using 0.5 μM recombinant MEK2, PP1α, and GST-mortalin to determine whether mortalin affects the interaction between MEK2 and PP1α.

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: In Vitro, Binding Assay, Recombinant

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: The PBD mutant facilitates PP1α-MEK2 interaction less efficiently than full-length mortalin. (A and B) Dose-dependent effects of mortalin on MEK2-PP1α interaction. Recombinant MEK2 and PP1α (0.5 μM) were incubated for 60 min in the presence of different doses of GST-mortalin (A) or GST-PBD (B) before IP using anti-MEK2 antibody and protein G-agarose. (C) The degree of PP1α-MEK2 interaction affected by GST-mortalin or GST-PBD was determined by densitometry of the PP1α signals normalized by MEK2 signals in panels A and B.

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: Mutagenesis, Recombinant, Incubation

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Mortalin depletion decreases PP1α-MEK1/2 interaction in SK-MEL 28 cells. (A) Total lysates of SK-MEL 28 cells infected with shMort for 4 days were subject to IP using PP1α-specific antibody. Co-IP of the indicated proteins was determined by Western blotting. (B) Endogenous MEK2 was pulled down using an anti-MEK2 antibody. After three stringent washes with the IP buffer, MEK2 IP fractions were incubated with 0.5 μM recombinant PP1α for 60 min before Western blotting. GST was used as the control for recombinant PP1α.

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: Infection, Co-Immunoprecipitation Assay, Western Blot, Incubation, Recombinant

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Analysis of mortalin mutants for PP1α or MEK2 interaction. (A) Schematic of the mortalin mutants used in this study. AD, ATPase domain; SD2, subdomain 2; PBD, peptide-binding domain; V482F, Val482Phe; V487A/Y, Val487Ala or Val487Tyr; Δtail, tail deletion. (B) Total lysates of SK-MEL 28 cells infected with lentiviral pHAGE expressing N-terminally HA-tagged mortalin mutants for 2 days were analyzed by immunoblotting for co-IP of indicated proteins. (C) In vitro binding assay using 0.5 μM recombinant GST-PBD mutants and HIS-PP1α. (D) In vitro binding assay using 0.5 μM recombinant GST-PBD mutants and HIS-MEK2. (E) Schematic of the peptide aptamers (APT) designed from the groove region of PBD. (F) SK-MEL 28 cells infected with pHAGE expressing N-terminally HA-tagged PBD mutants were treated with 4 μM synthetic peptide aptamers for 2 days. Equal volumes of dimethylformamide were added to untreated cells. Total cell lysates were analyzed by immunoblotting for co-IP of the indicated proteins.

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: Binding Assay, Infection, Expressing, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Recombinant

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Expression of protein phosphatase 1 in human melanoma and pancreatic cancer. (A) Copy number alterations of PPP1C genes encoding PP1α, PP1β, and PP1γ in melanoma and pancreatic ductal adenocarcinoma patient biopsy specimens determined by the copy number analysis algorithms defined by genomic identification of significant targets in cancer (GISTIC) (63) and RAE (64). Deep deletion and shallow deletion are caused mainly by homozygous deletion and heterozygous deletion, respectively. The skin cutaneous melanoma TCGA data set and the pancreatic cancer UTSW data set, originally generated by The Cancer Genome Atlas (TCGA) Research Network (http://cancergenome.nih.gov/) and by Erik Knudsen and coworkers (65), respectively, were obtained from the cBioPortal for Cancer Genomics database (http://www.cBioportal.org). (B) RNA-seq analysis of the melanoma biopsy specimens in panel A. The boxes, whiskers, and asterisks indicate the interquartile range (25 to 75%), the 10 to 90% range, and the minimums, maximums, and outliers, respectively. Fold changes between amplification and diploid were 2.05 (P, <0.0001; t, 6.642) for PPP1CA and 3.41 (P, <0.0001; t, 9.012) for PPP1CB (two-tailed Mann-Whitney test). Fold changes between gain and diploid were 1.222 (P, 0.0043; t, 2.889) for PPP1CA, 1.241 (P, <0.0001; t, 4.016) for PPP1CB, and 1.236 (P, 0.0005; t, 3.542) for PPP1CC (two-tailed Mann-Whitney test). Fold changes between shallow deletion and diploid were 0.732 (P, <0.0001; t, 5.44) for PPP1CA, 0.763 (P, <0.0001; t, 4.111) for PPP1CB, and 0.804 (P, <0.0001; t, 5.353) for PPP1CC (two-tailed Mann-Whitney test). The P value for PPP1CA (amplification, gain, and shallow deletion versus diploid) was <0.05 (with q = 7.344, 3.134, and 4.961, respectively) (one-way ANOVA with Dunnett's multiple-comparison test). (C) Microarray analysis of PPP1C mRNA levels in patient biopsy specimens. Data are from the Talantov melanoma data set from the Oncomine database (www.oncomine.org). Fold changes between benign nevus versus normal skin were 2.517 (P, 0.0052; t, 3.087) for PPP1CA, 0.275 (P, 0.0066; t, 3.002) for PPP1CB, and 0.926 (P, 0.1645; t, 1.436) for PPP1CC (two-tailed t test). Fold changes between cutaneous melanoma versus normal skin were 3.799 (P, <0.0001; t, 13.38) for PPP1CA, 0.19 (P, <0.0001; t, 5.59) for PPP1CB, and 0.832 (P, 0.0006; t, 3.657) for PPP1CC (two-tailed t test). (D) Pearson correlation coefficient analysis of the HSPA9 (encoding mortalin) and PPP1CA mRNA levels in panel C (P < 0.0001; r = 0.6413). (E) Immunohistochemistry of PP1α, PP1β, and PP1γ expression in patient biopsy specimens. Data were obtained from the Human Protein Atlas database (66). PP1α was stained by antibody HPA046833 (www.proteinatlas.org/ENSG00000172531-PPP1CA/cancer), PP1β was stained by HPA065425 (www.proteinatlas.org/ENSG00000213639-PPP1CB/cancer), and PP1γ was stained by HPA013661 (www.proteinatlas.org/ENSG00000186298-PPP1CC/cancer).

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: Expressing, Generated, RNA Sequencing Assay, Amplification, Two Tailed Test, MANN-WHITNEY, Microarray, Immunohistochemistry, Staining

Journal: Molecular and Cellular Biology

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

doi: 10.1128/MCB.00061-17

Figure Lengend Snippet: Modulation of MEK/ERK activity by mortalin and PP1α in BRAF- or KRAS-mutated cancer cells. (A) Mutations in BRAF or KRAS can induce high-magnitude MEK/ERK activity. (B) Upregulated mortalin and PP1α may have a role in modulating MEK/ERK activity in certain BRAF/KRAS tumor cells because mortalin can facilitate PP1α-mediated MEK1/2 dephosphorylation. This mechanism may be important for determining the physiological outputs of MEK/ERK activity, i.e., proliferation versus growth arrest.

Article Snippet: For in vitro binding assays, equal concentrations (0.5 μM) of N-terminally GST-tagged mortalin (Sigma-Aldrich), N-terminally HIS-tagged PP1α (ProSpec, East Brunswick, NJ), and N-terminally HIS-tagged MEK2 (ProSpec) were incubated in 50 mM Tris (pH 7.4)–100 mM KCl–100 mM NaCl–5 mM MgCl 2 –5% glycerol–10 mM β-mercaptoethanol for 30 min at room temperature.

Techniques: Activity Assay, De-Phosphorylation Assay

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Putative PP1-binding motifs within NCX1. Topology model of Na+/Ca2+ exchanger 1 (NCX1) consisting of 10 transmembrane domains (TMs) and a large cytosolic loop between TM5 and TM6 (11, 12) is shown. Within the TMs are two α repeats (α1 and α2) that face opposite sides of the membrane and catalyze ion translocation (36) (TMs involved in ion translocation are shown in black). The cytosolic loop mediates regulation of the exchanger and contains the two Ca2+-binding domains (CBD1 and CBD2). In silico screening of human, rat, and mouse NCX1 primary sequences identified three putative PP1-binding motifs. The first site, RVFF, is localized within the membrane loop connecting TM3 and -4. The second site, KIFF/KVFF (human and mouse/rat), is localized in CBD1, and the third site, KVLF, is localized at the end of the intracellular loop.

Article Snippet: The recombinant proteins used were as follows: PP1α (14–595, Merck Millipore Billerica); custom-made His 6 -trigger factor (TF)-NCX1 cyt and GST-PP1α (both Genscript Corp.); PP1α (P0754S, New England Biolabs, Ipswich, MA); and PP1 inhibitor 2 (P0755S, New England Biolabs).

Techniques: Binding Assay, Membrane, Translocation Assay, In Silico

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: PP1c binds directly to NCX1. A, epitope mapping was performed by overlaying an array of immobilized overlapping 20-mer rat PP1-α (P62138) peptides with anti-PP1 (E-9) (sc-7482, top panel). Amino acids in bold constitute the core epitope, localized in the PP1 catalytic subunit (PP1c), and are relevant for anti-PP1c binding (n = 2). Immunodetection without anti-PP1c was used as a negative control (bottom panel). B, NCX1 and PP1c were analyzed in cytoplasmic and membrane fractions isolated from rat LV and neonatal cardiomyocytes using anti-NCX1 and anti-PP1c (in triplicate). Vinculin and epidermal growth factor receptor (EGFR) were used as controls for cytoplasmic and membrane fractions, respectively. C, rat LV lysates were subjected to immunoprecipitation (IP) using anti-NCX1 (in triplicate). Immunoprecipitates (right panels) and lysate (input; positive control for the immunoblot, left panels) were immunoblotted with NCX1, PP1c, and PLM antibodies. As a negative control, NCX1 antibody pre-incubated with a specific anti-NCX1 blocking peptide or non-relevant rabbit IgG was used. D, confocal images of in situ proximity ligation assay. NCX1-PP1c co-localization was analyzed in adult cardiomyocytes using anti-NCX1 and anti-PP1c (left panel, interaction indicated by green spots, see “Experimental Procedures” for details). Incubation without primary antibodies (middle panel) and pre-incubating anti-NCX1 with blocking peptide (right panel) were used as negative controls. Nuclei were stained with SYTOX orange. E, schematic figure of recombinant proteins used in this study. The cytosolic loop of NCX1 was purified, and His6 and trigger factor (TF) tags were added to the N terminus (His6-TF-NCX1cyt, top panel). Because of the size of the tags, His6-TF-NCX1cyt migrates as a 150-kDa protein. Commercially available PP1α was used in the initial analysis of the interaction (in F) after which rat PP1-α (P62138) with a GST tag (GST-PP1α) was generated (bottom panel) and used in the remainder of the study. F, recombinant proteins were subjected to immunoprecipitation using anti-NCX1 and anti-His6 (in triplicates). Immunoprecipitates (right panels) and proteins (input panels; positive control for the immunoblot) were immunoblotted with anti-NCX1 and anti-PP1c. As a negative control, non-relevant rabbit and mouse IgG were used. G, SPR analysis was performed by immobilizing recombinant His6-TF-NCX1cyt (ligand) on a CM5 chip and measuring the response when injecting a range of concentrations of GST-PP1α (analyte) (n = 3).

Article Snippet: The recombinant proteins used were as follows: PP1α (14–595, Merck Millipore Billerica); custom-made His 6 -trigger factor (TF)-NCX1 cyt and GST-PP1α (both Genscript Corp.); PP1α (P0754S, New England Biolabs, Ipswich, MA); and PP1 inhibitor 2 (P0755S, New England Biolabs).

Techniques: Binding Assay, Immunodetection, Negative Control, Membrane, Isolation, Immunoprecipitation, Positive Control, Western Blot, Incubation, Blocking Assay, In Situ, Proximity Ligation Assay, Staining, Recombinant, Purification, Generated

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Mapping of the PP1c interaction site in NCX1. A, schematic figure showing full-length NCX1-GFP and a series of GFP-NCX1 deletion mutants that were generated to map the PP1c interaction site in NCX1. The numbering of NCX1 includes the N-terminal amino acid signal peptide sequence. B, GST-PP1α together with NCX1 deletion variants were subjected to immunoprecipitation (IP) using anti-PP1c (in triplicates). Precipitates were analyzed with immunoblotting. The asterisks in the top panel indicate the NCX1 dimer (64) and missing GFP-NCX1(243–402) fragment. Noteworthy, the GFP-NCX1(243–402) fragment was not visible when we re-ran the samples on a second gel with good separation around 50 kDa and used a light chain-specific antibody (data not shown). The middle panel shows anti-PP1c-precipitated GST-PP1α. In the bottom panel the input lysates were immunoblotted to show the migration and corresponding molecular weight of NCX1-GFP and the deletion mutants. The asterisks in this panel indicate the NCX1 dimer, monomer, and migration of the GFP-NCX1(243–402) deletion fragment. C, identification of PP1 binding by overlaying GST-PP1α on 20-mer overlapping NCX1 peptides synthesized on membrane. Rat NCX1 protein (EDM02743) was used spanning amino acids 243–799. Binding was analyzed using anti-GST HRP (n = 2) top panel. Boxed areas show KVFF and KVLF respectively. Underlined amino acids in the first boxed area represent the RVXF -PP1-binding motif and a putative Φ1Φ2 motif, whereas amino acids in bold indicate the common sequence in the four peptide sequences. Incubation without GST-PP1α was used as a negative control (lower panel). D, one of the peptide sequences identified in C; 402PVSKVFFEQGTYQCLENCGT421, was synthesized with a glycine spacer and was overlaid with GST-PP1α (right panel) to determine the effect on PP1 binding when EN, putative Φ1Φ2 motif, was deleted (n = 2). The peptide sequence without GST-PP1α served as negative control (left panel).

Article Snippet: The recombinant proteins used were as follows: PP1α (14–595, Merck Millipore Billerica); custom-made His 6 -trigger factor (TF)-NCX1 cyt and GST-PP1α (both Genscript Corp.); PP1α (P0754S, New England Biolabs, Ipswich, MA); and PP1 inhibitor 2 (P0755S, New England Biolabs).

Techniques: Generated, Sequencing, Immunoprecipitation, Western Blot, Migration, Molecular Weight, Binding Assay, Synthesized, Membrane, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Confirmation of the PP1c interaction site in NCX1 by mutagenesis. NCX1(FL)-GFP, containing the KIFF motif (mouse sequence), together with either GFP-NCX1(I406A, F408A) representing KAFA mutant (A), GFP-NCX1(K405A, F407A) representing the AIAF mutant (B), or GFP-NCX1(F407P) representing the proline substitution KIPF and GFP-NCX1(Δ399–424) representing KIFF deletion (C) were generated and expressed in HEK293 cells after which they were used in immunoprecipitation (IP) experiments. The NCX1 antibody was used to precipitate NCX1 followed by immunoblotting with anti-PP1c for determination of PP1 association. D, rat LV lysates were subjected to immunoprecipitation using anti-NCX1, with and without the addition of 3 mm CaCl2. Immunoprecipitates (right panels) and lysate (input panels, positive controls for the immunoblot) were immunoblotted using relevant antibodies. E, peptide sequence 402PVSKVFFEQGTYQCLENCGT421 (identified in Fig. 3C) with KVFF motif was mutated to KAFA and AVAF and overlaid with GST-PP1α to determine the effect of mutations (n = 2). GST-PP1α binding was analyzed by immunodetection with anti-GST HRP. All experiments were run in triplicates (A–D).

Article Snippet: The recombinant proteins used were as follows: PP1α (14–595, Merck Millipore Billerica); custom-made His 6 -trigger factor (TF)-NCX1 cyt and GST-PP1α (both Genscript Corp.); PP1α (P0754S, New England Biolabs, Ipswich, MA); and PP1 inhibitor 2 (P0755S, New England Biolabs).

Techniques: Mutagenesis, Sequencing, Generated, Immunoprecipitation, Western Blot, Binding Assay, Immunodetection

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Independence and isoform specificity of the NCX1-KIFF/KVFF-anchoring site. A, schematic figure of NCX1-biotinylated peptides used in pulldown and PP1 activity assays. B, pulldown assay with the biotinylated NCX1 peptides and GST-PP1α recombinant protein (in triplicates). PP1 binding was analyzed by immunoblotting with anti-PP1c (upper panel). Anti-biotin HRP was used to show the presence of biotinylated peptides. Incubation of the GST-PP1α recombinant protein with only the beads was used as negative control. C, effect of NCX1-PP1 interaction on PP1 activity was assessed. In each experiment, 1 unit of active PP1c was incubated with a range of recombinant His6-TF-NCX1cyt protein concentrations or (D) biotinylated NCX1 peptides for 20 min, after which the activity was determined. Inhibitor 2 (PP1 inhibitor) was used as control for the assay. The KIFF/KVFF-anchoring motif, a putative Φ1Φ2 motif (EN) and a conserved arginine motif (R), are shown in the alignment of human, rat, mouse NCX1 (E) and NCX1–3 isoforms (F) (black boxes indicate similar functional amino acids (DNA Star). G, pulldown assay with the biotinylated NCX1–3 peptides and GST-PP1α recombinant protein (in triplicates). PP1 binding was analyzed by immunoblotting with anti-PP1c (upper panel). Incubation of GST-PP1α recombinant protein with only the beads was used a negative control. GST-PP1α recombinant protein and biotinylated NCX peptides were used as positive controls for the immunoblot in B and G (input panels on left).

Article Snippet: The recombinant proteins used were as follows: PP1α (14–595, Merck Millipore Billerica); custom-made His 6 -trigger factor (TF)-NCX1 cyt and GST-PP1α (both Genscript Corp.); PP1α (P0754S, New England Biolabs, Ipswich, MA); and PP1 inhibitor 2 (P0755S, New England Biolabs).

Techniques: Activity Assay, Recombinant, Binding Assay, Western Blot, Incubation, Negative Control, Control, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Mapping of the NCX1 interaction site in PP1. A, schematic figure showing PP1c and two deletion mutants that were generated to map the NCX1 interaction site in PP1. A FLAG and His6 tags were inserted on the N terminus. The arrow indicates NCX1 binding region. B, His6-TF-NCX1cyt together with the PP1 deletion variants were subjected to immunoprecipitation using anti-FLAG (in triplicates). Precipitates were analyzed with immunoblotting. C, identification of NCX1 binding by overlaying His6-TF-NCX1cyt on 20-mer overlapping rat PP1 (P62138) peptides synthesized on membrane. Binding was analyzed using anti-His6 HRP (n = 2). Boxed area shows His6-TF-NCX1cyt binding to PP1c; amino acids in bold indicate the common sequence in the three peptide sequences, and underlined amino acids form part of the RVXF binding pocket. D, FLAG-His6-PP1c(1–330) together with a variant, where the NCX1 binding region was deleted; FLAG-His6-PP1c(Δ232–263) was used in immunoprecipitation experiments. The anti-FLAG antibody was used to precipitate FLAG-His6-PP1c variants expressed in HEK293 cells followed by immunoblotting with anti-NCX1 for determination of NCX1 association with PP1c (in triplicates). His6-TF-NCX1cyt in lysate from non-transfected HEK293 cells was used as a negative control. E, residues that are important for PP1-binding of target proteins (30) is shown by the stars in the alignment of human, mouse, and rat PP1. The boxed area show His6-TF-NCX1cyt binding to PP1 (identified in C).

Article Snippet: The recombinant proteins used were as follows: PP1α (14–595, Merck Millipore Billerica); custom-made His 6 -trigger factor (TF)-NCX1 cyt and GST-PP1α (both Genscript Corp.); PP1α (P0754S, New England Biolabs, Ipswich, MA); and PP1 inhibitor 2 (P0755S, New England Biolabs).

Techniques: Generated, Binding Assay, Immunoprecipitation, Western Blot, Synthesized, Membrane, Sequencing, Variant Assay, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Model of NCX1-KVFF peptide binding to PP1. A, homology model of rat NCX1 peptide motif KVFF binding to human/rat PP1. The KVFF peptide (green stick model) interacts with PP1 (light blue surface and stick model of selected residues) via polar hydrogen bonds (black dashed lines) and hydrophobic contacts (red dashed lines). The directions of extension of the peptide are indicated by two black arrows. B, PP1c (FLAG-His6-PP1c(1–330)) together with FLAG-His6-PP1c single and double mutants (L243G, F257G) were immunoprecipitated with anti-FLAG, followed by immunoblotting with anti-NCX1 to determine co-precipitation of recombinant His6-TF-NCX1cyt (in triplicates).

Article Snippet: The recombinant proteins used were as follows: PP1α (14–595, Merck Millipore Billerica); custom-made His 6 -trigger factor (TF)-NCX1 cyt and GST-PP1α (both Genscript Corp.); PP1α (P0754S, New England Biolabs, Ipswich, MA); and PP1 inhibitor 2 (P0755S, New England Biolabs).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Recombinant

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Differential localizations of protein phosphatase 1 isoforms determine their physiological function in the heart

doi: 10.1093/abbs/gmy171

Figure Lengend Snippet: Deletion of PP1β from mouse heart leads to increased HDAC7 phosphorylation (A–C) Western blot analysis for phospho-HDAC7, HDAC7, PP1α, PP1β, PP1γ, and GAPDH from the hearts of the indicated mice at 2 months of age. Phosphorylation of MLC2 was detected using a ProQ-Diamond staining method . (D) Quantification of western blots shown in A–C. N = 4 for each group. * P < 0.05 vs. NKX-Cre.

Article Snippet: For adenovirus infection, neonatal myocytes were first switched to serum-free medium and then incubated with purified adenoviruses expressing either β-galactosidase [ ] or PP1α (SL112646; SignaGen Laboratories, Rockville, USA), PP1β (SL112778; SignaGen Laboratories), PP1γ (SL112648; SignaGen Laboratories) for 2 h to enhance infection efficiency.

Techniques: Western Blot, Staining

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Differential localizations of protein phosphatase 1 isoforms determine their physiological function in the heart

doi: 10.1093/abbs/gmy171

Figure Lengend Snippet: PP1s are essential for the physiological cardiac function (A) Real-time PCR analysis of PP1 expression in cardiac myocytes and fibroblasts isolated from neonatal and adult mouse hearts. N = 4 for each group. * P < 0.05 vs PP1α from neonatal cells; # P < 0.05 vs PP1α from neonatal cells; $ P < 0.05 vs PP1α from adult cells; % P < 0.05 vs PP1α from adult myocytes. (B) Western blot analysis for each PP1 isoform in the neonatal and adult mouse hearts. (C) Quantification of protein levels of PP1s in B. N = 4 for each group. * P < 0.05 vs neonatal hearts. (D,E) Fractional shortening (FS) and LVIDd of the aging mice. Mouse numbers for four groups were indicated in the bars. * P < 0.05 vs NKX-Cre; # P < 0.05 vs NKX-Cre; & P < 0.05 vs NKX-Cre. (F) Masson’s trichrome staining of paraffin-embedded heart sections from NKX-Cre controls and the indicated PP1-deleted mice at 3 weeks of age. Scale bar, 50 μm.

Article Snippet: For adenovirus infection, neonatal myocytes were first switched to serum-free medium and then incubated with purified adenoviruses expressing either β-galactosidase [ ] or PP1α (SL112646; SignaGen Laboratories, Rockville, USA), PP1β (SL112778; SignaGen Laboratories), PP1γ (SL112648; SignaGen Laboratories) for 2 h to enhance infection efficiency.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Western Blot, Staining