Protein Phosphatase-5 is a Novel Titin Ligand Which Dephosphorylates Cardiac Titin and Reduces Passive Myocyte Stiffness

Abstract

Titin's elastic I-band region is a hotspot for protein-protein interactions and important for myocyte extensibility and passive stiffness. The titin springs can be phosphorylated at the N2B-unique sequence (N2Bus) by protein kinases (PK)A or PKG and at the PEVK-domain by PKCalpha, which affects passive stiffness. We searched for protein phosphatase(s) acting on the N2Bus. A yeast-2-hybrid (Y2H) screen with the human N2Bus (bait) and a human heart cDNA library (prey) detected the catalytic domain of the serine/threonine protein phosphatase-5 (PP5) as a binding partner of the cardiac N2Bus. The interaction was confirmed in forced Y2H screens with the N2Bus and full-length PP5 or PP5 catalytic subunit (PP5c), and also in GST-pulldown assays. In cardiomyocytes, PP5 was mainly in the cytosol but also in the nucleus and at the sarcomeric I-bands. Recombinant PP5 was found by autoradiography to dephosphorylate recombinant, PKG-phosphorylated, N2Bus, and PP5 bound more strongly to (PKA-/PKG-)phosphorylated N2Bus than to non-phosphorylated N2Bus. Phosphorylation of titin could be reduced in human heart tissue treated ex vivo with recombinant PP5c, an effect detected with phospho-N2Bus (S469) specific antibodies. A transgenic mouse model with PP5 overexpression revealed reduced cardiac titin phosphorylation levels compared to wildtype mouse hearts. PP5 expression was elevated in human heart failure, while titin phosphorylation was depressed. PP5c treatment of enzymatically skinned single human cardiomyocytes significantly reduced passive stiffness, which can potentially be explained by dephosphorylation of the PEVK-domain adjacent to the N2Bus. In conclusion, PP5 is a novel binding partner of cardiac titin at the N2Bus and acts to reduce passive myocyte stiffness by dephosphorylating the titin springs. PP5 may participate in mechanical signaling pathways converging on the titin springs.