Fig. 1: The elastic N2Bus domain of titin interacts with PP5 | Nature Communications

Fig. 1: The elastic N2Bus domain of titin interacts with PP5

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

Fig. 1

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

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