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:

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: The antibodies used were: rabbit anti-Rpb1 (sc-899; Santa Cruz Biotechnology), rabbit anti-Rpb1 (A304–405A & A304–405A, Bethyl Laboratories), rabbit anti-Rpb1 CTD pSer2 (ab5095, Abcam), rabbit anti-Spt5 (A300-868A, Bethyl Laboratories), mouse anti-Spt5 (sc-133217, Santa Cruz Biotechnology), rabbit anti-Spt5-pSer666 and -pThr806 (Twenty-first Century Century Biochemicals) previously described , rabbit anti-PP4R2 (A300–838A, Bethyl Laboratories), rabbit anti-PPP4C (A300–835A, Bethyl Laboratories), sheep anti-PP4R2-pThr173 (Division of Signal Transduction Therapy, University of Dundee Scotland) , rabbit phospho-PP1α (Thr320) antibody (2581S, Cell Signaling Technology), mouse anti-GFP (sc-9996, Santa Cruz Biotechnology), mouse anti-pan-PP1 (sc-7482, Santa Cruz Biotechnology), goat anti-PP1α (sc-6104, Santa Cruz Biotechnology), mouse anti-PP1β (sc-373782, Santa Cruz Biotechnology), rabbit anti-PP1γ (A300-906A, Bethyl Laboratories), goat anti-PP1γ (sc-6108, Santa Cruz Biotechnology), mouse anti-tubulin (T5168, Sigma-Aldrich), rabbit anti-GST (sc-459, Santa Cruz Biotechnology), mouse anti-FLAG ® M2 (F3165, Sigma-Aldrich), and rabbit anti-FLAG (2368, Cell Signaling).

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: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Domain arrangement in the Z-disk/I-band region of cardiac titin N2B/N2BA isoforms and in PP5. Constructs generated for yeast-two-hybrid (Y2H) screens marked in red, those for GST-pulldown assays in blue, and N2Bus-binding amino acids (AA) of PP5 in green. Ig’s, immunoglobulin-like domains. Inset: Epitope positions of all phospho-titin antibodies used in this study. (m), anti-mouse; (h), anti-human. b Summary of results of GST-pulldown assays probing interaction of N2Bus with full-length PP5, PP5 catalytic subunit (PP5c), or N-terminal PP5 fragments (T; T+). PP5-binding to PEVK or N2A titin domains was also tested. GST, glutathione S transferase (for negative control). Each test was performed a minimum of two times, mostly three times, with identical results. c Demonstration of PP5-N2Bus association by co-immunoprecipitation assay. PP5 (HA-tag) immunoprecipitations (IP) and whole-cell lysates (WCL) from HEK cells analyzed by western blot for N2Bus (myc-tag) and PP5. PP5 -/+indicates absence/presence of PP5 in the assay. This test was performed three times, with identical results. d Binding of PP5 to sarcomeric I-bands is enhanced by phosphorylation. Top: the experimental design for the stretching of single myofibrils and immunofluorescence image of stretched human cardiac myofibril incubated in relaxing buffer with Cy3-conjugated secondary antibodies alone (control), as well as phase-contrast image (PC). Bottom: representative images of myofibrils incubated with exogenous PP5c and stained against PP5c. The myofibril on the right was incubated with catalytic subunit of PKA before PP5c-treatment (arrowheads, I-band localization of PP5). Binding visualized by anti-PP5c primary and Cy3-conjugated secondary antibodies. Similar results were obtained from four other myofibrils per group. Bars, 2 µm. e Results of GST-pulldown assays probing interaction of PP5 with unphosphorylated N2Bus or N2Bus phosphorylated by PKA/PKG, as well as wildtype (WT) and S4185A mutant of C-terminal N2Bus fragment, both phosphorylated by cGMP-activated PKG. Left: representative immunoblots using anti-PP5 antibody. Right: relative signal intensities in ‘Bound’ lane, normalized to the mean intensity of the respective non-phosphorylated/C-Term WT control. Data are mean ± s.e.m., n = 4 assays/condition. * p < 0.05 and ** p < 0.01, by two-tailed Student’s t -test

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Construct, Generated, Binding Assay, Negative Control, Co-Immunoprecipitation Assay, Western Blot, Phospho-proteomics, Immunofluorescence, Incubation, Control, Staining, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a PP5 expression by western blot in fetal (E18), newborn (P1) and adult rat heart tissue. PP5 indexed to GAPDH. Data are mean ± s.e.m., n = 5; * p < 0.05, by two-tailed Student’s t -test. b PP5 expression by western blot in neonatal rat ventricular myocyte (NRVM) cultures under baseline conditions (control) and following treatment with arachidonic acid (aa; 200 µM, 2 h) or okadaic acid (oa, 10 nM, 1 h). PP5 indexed to GAPDH. Data are mean ± s.e.m., n = 5 (three different cell culture batches); * p < 0.05 and ** p < 0.01, by Bonferroni adjusted t -test. c PP5 localization in control, aa-treated, and oa-treated NRVM cultures by indirect immunofluorescence. PP5 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with α-actinin antibody (secondary antibody: FITC-conjugated IgG). The merged image also shows staining of nuclei using Hoechst. Bars, 10 µm. d PP5 localization in control, aa-treated, and oa-treated adult rat cardiomyocyte (ARC) cultures by indirect immunofluorescence. The same antibodies as in c were used. Bars, 5 µm. Right bar graph shows proportion of ARC exhibiting clear PP5 striations or no such striated pattern, for each group. Numbers above columns indicate total number of cells included in the analysis. e Total titin phosphorylation in the three ARC groups measured by ProQ Diamond phosphoprotein vs. Sypro Ruby total protein stain. Bar graph shows mean ± s.e.m., n = 9 (from three independent cell preparations); * p < 0.05, by two-tailed Student’s t -test. f Localization of phosphoserine P-S3991 (titin N2Bus) in control, aa-treated, and oa-treated ARC cultures by indirect immunofluorescence, using anti-N2Bus P-S3991 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with α-actinin antibody (secondary antibody: FITC-conjugated IgG). Bars, 5 µm. Right bar graph shows proportion of ARC exhibiting clear N2Bus P-S3991 striations or no such striated pattern, for each group. Numbers above columns indicate total number of cells included in the analysis

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Expressing, Western Blot, Two Tailed Test, Control, Cell Culture, Immunofluorescence, Staining, Phospho-proteomics

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a , b ProQ Diamond phosphoprotein vs. Sypro Ruby total protein stain of human recombinant N2Bus phosphorylated (in presence of ATP) by ERK2 ( a ), catalytic subunit of PKA ( b ), or cGMP-activated PKG ( b ), and effect of human recombinant full-length PP5, PP5 catalytic subunit (PP5c), or enzymatic dead (ED) PP5c mutant H304A on phosphorylation. ‘Control’ is recombinant construct but no ATP/enzyme. n = 5. c 32 P-ATP autoradiography using N2Bus phosphorylated by cGMP-activated PKG and dephosphorylated by PP5 or PP5c. ‘No PK/PP’ is control in presence of 32 P-ATP, but no enzyme. n = 4. d Western blot (WB) of permeabilized human (donor) heart tissue treated with various enzymes, as follows: alkaline phosphatase (AP), cGMP-activated PKG, then PP5 or PP5c. Detection of titin phosphorylation with phospho-specific antibody to P-S4185 in human N2Bus. PVDF was coomassie-stained to reveal loading on gel. WB signals were normalized to PVDF signals. n = 4. e Site-specific phosphorylation of residues within N2Bus and PEVK titin regions by western blot in myocardial tissue from human donor (Don) and end-stage failing (Fail) hearts ( n = 10/group). Detection with phospho-specific antibodies to P-S4010, P-S4099, P-S4185, P-S11878, and P-S12022 in the human titin sequence. WB signals were normalized to PVDF signals. f Mean PP5 expression in human donor vs. end-stage failing hearts ( n = 10/group). In a – f , bar graphs show relative phosphorylation indexed to the respective phosphorylated controls. Data are mean ± s.e.m.; * p < 0.05, ** p < 0.01, and *** p < 0.001, by two-tailed Student’s t -test or Holm-Sidak method

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Staining, Recombinant, Mutagenesis, Phospho-proteomics, Control, Construct, Autoradiography, Western Blot, Sequencing, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Expression level of PP5 (left) and phospho-Raf1 S338 (right) in PP5 TG vs. WT mouse hearts by western blot. mean ± s.e.m., n = 4 hearts/group (age 5–6 months); duplicate analysis/group. b Sarcomeric localization of PP5 in PP5 WT and TG hearts by immunogold electron microscopy. Bars, 500 nm. Bar graph shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data are mean ± s.e.m., n = 5 ROIs from 2 hearts/group. c PP5 localization in cardiomyocytes from PP5 TG and WT mouse hearts by indirect immunofluorescence. PP5 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK (titin) antibody (secondary antibody: FITC-conjugated IgG). Bars, 2 µm (main) and 1 µm (insets). d PP5 overexpression specifically decreases titin phosphorylation at N2Bus in PP5 TG vs. WT hearts. Total titin phosphorylation measured by ProQ Diamond/Sypro Ruby staining (upper left), site-specific titin phosphorylation detected by western blot using antibodies to P-S3991, P-S4043, P-S4080 (all N2Bus; right panels), P-S2080 (titin Z/I junction), and P-S12742 (PEVK region). Phospho-titin signals were normalized to total titin signals detected by WB using a panel of sequence-specific antibodies (Pan). Means were indexed to those of control (WT) groups. Data are mean ± s.e.m., n = 4 hearts/group, samples analyzed in triplicate. e Localization of phospho-N2Bus P-S3991 in cardiomyocytes from PP5 TG and WT hearts by indirect immunofluorescence. Anti-N2Bus P-S3991 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK antibody (secondary antibody: FITC-conjugated IgG). Bars, 2 µm (main) and 1 µm (insets). f Sarcomeric localization of phospho-N2Bus P-S3991 in PP5 WT and TG hearts by immunogold electron microscopy. Bars, 500 nm (main) and 100 nm (insets). Bar graph shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data are mean ± s.e.m., n = 5 ROIs from 2 hearts/group. In a and d , bar graphs show relative signal changes indexed to the respective controls. In a , b , d , and f , * p < 0.05 and *** p < 0.001, by two-tailed Student’s t -test

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Expressing, Western Blot, Electron Microscopy, Immunofluorescence, Over Expression, Phospho-proteomics, Staining, Sequencing, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Representative image of permeabilized cardiomyocyte glued at the ends to a force transducer and micromotor, respectively, and experimental protocol. Bar, 20 µm. b Passive tension ( F passive ) vs. SL curves of permeabilized single PP5 TG and WT cardiomyocytes in relaxing solution, before and after treatment with recombinant PP5c. c F passive -SL curves of permeabilized single PP5 TG and WT cardiomyocytes before and after treatment with ERK2. d F passive –SL curves of permeabilized single WT cardiomyocytes before and after treatment with ERK2 and additional exposure to PP5c. Data are mean ± s.e.m., n = 5 cells/condition (2 different hearts/condition). Curves are second-order polynomial fits to the means. * p < 0.05, TG vs. WT; # p < 0.05, WT + PP5c vs. WT in b and WT + ERK2 vs. WT in c and d ; † p < 0.05, TG + ERK2 vs. TG in c and WT + ERK2 + PP5c vs. WT + ERK2 in d ; all by two-tailed Student’s t -test

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Interactions of PP5 or PP5c by GST-pulldown assay. The PP5-GST assay is a negative control. b Interactions of N2Bus by GST-pulldown assay. c Impact of Hsp90 on PP5-N2Bus binding in GST-pulldown ‘competition’ assays. ‘PP5→Hsp90’: N2Bus immobilized on GSH-beads, incubated with PP5, then washed, and Hsp90 added thereafter. ‘Hsp90→PP5’: N2Bus immobilized on GSH-beads, incubated with Hsp90, then washed, and PP5 added thereafter. Summary data in bar graph are relative values indexed to the ‘PP5→Hsp90’ condition; mean ± s.e.m., n = 5 experiments/condition; * p < 0.05, by two-tailed Student’s t -test. d Localization of Hsp90 in cardiomyocytes from PP5 TG and WT hearts by indirect immunofluorescence. Anti-Hsp90 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK (titin) antibody (secondary antibody: FITC-conjugated IgG). Bars, 5 µm (main) and 1 µm (inset)

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: GST Pulldown Assay, Glutathione S-Transferase Assay, Negative Control, Binding Assay, Incubation, Two Tailed Test, Immunofluorescence

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Total titin phosphorylation measured by ProQ Diamond/Sypro Ruby staining (left) and site-specific titin phosphorylation detected by western blot using antibodies to P-S3991 (N2Bus; middle) or P-S2080 (Z/I junction; right). Site-specific titin phosphorylation levels were normalized to total titin levels detected by WB using sequence-specific antibodies (Pan). Means were indexed to those of control (WT) groups. Data are mean ± s.e.m., n = 3 hearts/group, samples analyzed in triplicate; * p < 0.05, by two-tailed Student’s t -test. b , c , d Localization of PP5 ( b ), phospho-N2Bus P-S3991 ( c ), and phospho-Z/I-junction P-S2080 ( d ) in cardiomyocytes from FHL-1 WT and KO hearts by indirect immunofluorescence. Anti-PP5, anti-N2Bus P-S3991 or anti-Z/I-junction P-S2080 (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK antibody (secondary antibody: FITC-conjugated IgG). Bars, 5 µm (main) and 1 µm (insets). e , f Sarcomeric localization of phospho-N2Bus P-S3991 ( e ) and phospho-Z/I-junction P-S2080 ( f ) in FHL-1 WT and KO hearts by immunogold electron microscopy. Bars, 500 nm (main) and 100 nm (insets). Bar graph in e and f shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data in e and f are mean ± s.e.m., n = 5 ROIs from two hearts/group. In a and e , * p < 0.05 and *** p < 0.001, by two-tailed Student’s t -test

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Phospho-proteomics, Staining, Western Blot, Sequencing, Control, Two Tailed Test, Immunofluorescence, Electron Microscopy

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: Under basal conditions, PP5 activity towards titin N2Bus and MAPK/ERK family member Raf1 is low and the relatively high distensibility of N2Bus results in relatively low titin-based passive tension (left side). The strain-dependent mechanosensor connecting MAPKs to N2Bus via FHL-1 functions normally, as downstream signaling from Raf1 to ERK2 is enabled. When PP5 expression is increased (as in failing hearts) and PP5 becomes activated through interaction with Hsp90, Ca 2+ /S100 protein, arachidonic acid (aa), or long chain fatty acid-CoA esters (LCACE), the phosphatase translocates to the I-band mechanosensor at N2Bus (right side). Thus, N2Bus (previously phosphorylated by ERK2, PKA, PKG, or CaMKII) is dephosphorylated, which reduces its distensibility and increases titin-based passive tension; the mechanosensor is now less sensitive. Raf-1 is also dephosphorylated and signaling to ERK2 is disabled, such that the mechanosensor function is additionally compromised. The process is embedded in signaling pathways activated via G-protein coupled receptor (GPCR) and Ras, and it can be reversed when PP5 is deactivated. (Molecules that have a color code were studied here, those with no color/white background were inferred from the literature)

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Activity Assay, Expressing, Protein-Protein interactions