Fig 2: Binary interactions of PP5 or N2Bus and role of Hsp90a 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)

Fig 3: PP5 dephosphorylates recombinant and endogenous human N2Busa, 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 32P-ATP autoradiography using N2Bus phosphorylated by cGMP-activated PKG and dephosphorylated by PP5 or PP5c. ‘No PK/PP’ is control in presence of 32P-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

Fig 4: Proposed actions of PP5 in cardiomyocytes. 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, Ca2+/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)

Fig 5: Post-translational Regulation of the Co-chaperone FNIP1CK2-mediated relay phosphorylation of FNIP1 promotes its interaction with the molecular chaperone Hsp90. This leads to gradual activation and enhanced activity and stability of the kinase and non-kinase clients. PP5-mediated dephosphorylation of FNIP1 enables O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) to O-GlcNAcylate S938 on FNIP1. This consequently causes a ubiquitination of K1119 and subsequent degradation of FNIP1 in the proteasome.