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: Cancer Science

Article Title: Critical roles of T‐LAK cell‐originated protein kinase in cytokinesis

doi: 10.1111/j.1349-7006.2009.01400.x

Figure Lengend Snippet: T‐LAK cell‐originated protein kinase (TOPK) was activated by cyclin‐dependent kinase 1 (CDK1) through inactivation of protein phosphatase 1 alpha (PP1α). (a) FACS analysis of M‐phase arrested cells after treatment with CDK1 inhibitor. T47D cells were treated with nocodazole for 16 h, followed by incubation with 25 nmol/L of CDK1 inhibitor (CGP74514A) from 0 to 4 h before FACS analysis. The population (%) of each cell cycle in the indicated time points was graphed. Grey bar, G1 phase; white bar, S phase; black bar, G2/M phase. (b) Expression of TOPK‐related proteins after treatment with CDK1 inhibitor. Equal amounts of total protein were immunoblotted with anti‐TOPK and anti‐total‐Rb monoclonal antibodies and with anti‐phospho‐PP1α (Thr320), anti‐total‐PP1α, and anti‐phospho‐Rb (Ser807/811) polyclonal antibodies. The arrow indicates the phosphorylated TOPK protein. (c) FACS analysis as mitosis progression. Only mitotic T47D cells were isolated and released from mitosis (see the Materials and Methods), followed by FACS analysis. The population (%) of each cell cycle is graphed. Grey bar, G1 phase; white bar, S phase; black bar, G2/M phase. (d) Expression of TOPK‐related proteins during mitosis progression. Equal amounts of total protein were immunoblotted with monoclonal antibodies of anti‐cyclin B1 and anti‐CDK1. The arrow indicates the phosphorylated TOPK protein.

Article Snippet: Other recombinant proteins, enzymes, antibodies, and chemicals were purchased from commercial sources: active recombinant TOPK protein (Invitrogen, Carlsbad, CA, USA); lambda protein phosphatase and recombinant CDK1/cyclin B1 proteins (New England Biolabs, Ipswich, MA, USA); anti‐TOPK, anti‐CDK1 and anti‐p97 monoclonal antibodies (BD Biosciences, San Jose, CA, USA); anti‐Flag‐tag and anti‐β‐actin monoclonal antibodies (Sigma‐Aldrich, St. Louis, MO, USA); anti‐HA‐tag monoclonal antibody (Roche, Basel, Switzerland); anti‐PP1α and anti‐phospho‐PP1α (T320) polyclonal antibodies (Cell Signaling Technologies, Beverly, MA, USA); anti‐Rb monoclonal antibody (Upstate Biotechnology, Lake Placid, NY, USA); anti‐phospho‐Rb (Ser807/811), anti‐p97, and anti‐PRC1 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti‐cyclin B1, anti‐myc, and anti‐GST‐tag monoclonal antibodies (Santa Cruz Biotechnology); anti‐phosphothreonine or ‐phosphoserine polyclonal antibodies (Invitrogen); okadaic acid (OA), CDK1 inhibitor (CGP74514A) and protease inhibitor cocktail III (Calbiochem, San Diego, CA, USA); and nocodazole (Sigma‐Aldrich).

Techniques: Incubation, Expressing, Isolation

Journal: Cancer Science

Article Title: Critical roles of T‐LAK cell‐originated protein kinase in cytokinesis

doi: 10.1111/j.1349-7006.2009.01400.x

Figure Lengend Snippet: Schema of the roles of T‐LAK cell‐originated protein kinase (TOPK) in cancer cell mitosis. At an early stage of mitosis, TOPK autophosphorylation is induced though the inactivation of protein phosphatase 1 alpha (PP1α) by cyclin‐dependent kinase (CDK1)/cyclin B1 complex. Then, the activated TOPK phosphorylates p97 through its interaction with p47. Finally, the restored PP1α dephosphorylates and inactivates TOPK to the steady level at the exit of mitosis.

Article Snippet: Other recombinant proteins, enzymes, antibodies, and chemicals were purchased from commercial sources: active recombinant TOPK protein (Invitrogen, Carlsbad, CA, USA); lambda protein phosphatase and recombinant CDK1/cyclin B1 proteins (New England Biolabs, Ipswich, MA, USA); anti‐TOPK, anti‐CDK1 and anti‐p97 monoclonal antibodies (BD Biosciences, San Jose, CA, USA); anti‐Flag‐tag and anti‐β‐actin monoclonal antibodies (Sigma‐Aldrich, St. Louis, MO, USA); anti‐HA‐tag monoclonal antibody (Roche, Basel, Switzerland); anti‐PP1α and anti‐phospho‐PP1α (T320) polyclonal antibodies (Cell Signaling Technologies, Beverly, MA, USA); anti‐Rb monoclonal antibody (Upstate Biotechnology, Lake Placid, NY, USA); anti‐phospho‐Rb (Ser807/811), anti‐p97, and anti‐PRC1 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti‐cyclin B1, anti‐myc, and anti‐GST‐tag monoclonal antibodies (Santa Cruz Biotechnology); anti‐phosphothreonine or ‐phosphoserine polyclonal antibodies (Invitrogen); okadaic acid (OA), CDK1 inhibitor (CGP74514A) and protease inhibitor cocktail III (Calbiochem, San Diego, CA, USA); and nocodazole (Sigma‐Aldrich).

Techniques: