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, Staining, Sequencing, 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: 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