Abstract
Serine/threonine protein phosphatase 5 (PP5) is ubiquitously expressed in eukaryotic cells; however, its function in cardiomyocytes is unknown. Under basal conditions, PP5 is autoinhibited, but enzymatic activity rises upon binding of specific factors, such as the chaperone Hsp90. Here we show that PP5 binds and dephosphorylates the elastic N2B-unique sequence (N2Bus) of titin in cardiomyocytes. Using various binding and phosphorylation tests, cell-culture manipulation, and transgenic mouse hearts, we demonstrate that PP5 associates with N2Bus in vitro and in sarcomeres and is antagonistic to several protein kinases, which phosphorylate N2Bus and lower titin-based passive tension. PP5 is pathologically elevated and likely contributes to hypo-phosphorylation of N2Bus in failing human hearts. Furthermore, Hsp90-activated PP5 interacts with components of a sarcomeric, N2Bus-associated, mechanosensor complex, and blocks mitogen-activated protein-kinase signaling in this complex. Our work establishes PP5 as a compartmentalized, well-controlled phosphatase in cardiomyocytes, which regulates titin properties and kinase signaling at the myofilaments.
Highlights
Serine/threonine protein phosphatase 5 (PP5) is ubiquitously expressed in eukaryotic cells; its function in cardiomyocytes is unknown
To narrow down the interaction site for N2B-unique sequence (N2Bus) on PP5, we used two PP5 deletion constructs in the Glutathione S-transferase (GST)-pulldown assays, the tetratricopeptide repeat (TPR) region at the PP5 N-terminus (‘T’-construct, residues 28–129) and the TPR region with additional amino acids reaching into the catalytic domain (‘T+’-construct, residues 28 to 211) (Fig. 1a)
The Tconstruct showed no interaction with N2Bus, whereas T+ did (Fig. 1b), suggesting that the site of interaction involves the Nterminal part of the catalytic domain
Summary
Serine/threonine protein phosphatase 5 (PP5) is ubiquitously expressed in eukaryotic cells; its function in cardiomyocytes is unknown. During the lifetime of a beating heart, the cardiomyocytes must respond dynamically to a multitude of internal and external stresses Such functional flexibility is supported at the level of the contractile units, the sarcomeres, by the expression of cardiac-specific isoforms of structural, contractile, and regulatory proteins. Some of them, such as cardiac troponin-I, myosin-binding protein-C, or titin, contain unique sequence motifs that can be phosphorylated and dephosphorylated by protein kinases and phosphatases, respectively. These selective biochemical events help to quickly adjust the mechanical function of the cardiac sarcomere to altered physiological requirements, e.g., during exercise. Whereas in vitro evidence suggested that FHL-1 blocks phosphorylation of N2Bus by ERK214, phosphorylation of cardiac fibers by ERK2 readily lowered their titin-based passive tension[30]
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