Dataset Information

Phosphorylating Titin's Cardiac N2B Element by ERK2 or CaMKII? Lowers the Single Molecule and Cardiac Muscle Force.

ABSTRACT: Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passive-force levels characterize diastolic stiffening in heart-failure patients, and it is critical to understand the underlying molecular mechanisms and identify therapeutic targets. Here, we used single-molecule force spectroscopy to study the mechanical effects of the kinases calcium/calmodulin-dependent protein kinase II delta (CaMKII?) and extracellular signal-regulated kinase 2 (ERK2) on the single-molecule mechanics of the N2B element. Both CaMKII? and ERK2 were found to phosphorylate the N2B element, and single-molecule force spectroscopy revealed an increase in the persistence length (Lp) of the molecule, indicating that the bending rigidity of the molecule was increased. Experiments performed under oxidizing conditions and with a recombinant N2B element that had a simplified domain composition provided evidence that the Lp increase requires the N2B-Us of the N2B element. Mechanical experiments were also performed on skinned myocardium before and after phosphorylation. The results revealed a large (?30%) passive force reduction caused by CaMKII? and a much smaller (?6%) reduction caused by ERK2. These findings support the notion that the important kinases ERK2 and CaMKII? can alter the passive force of myocytes in the heart (although CaMKII? appears to be more potent) during physiological and pathophysiological states.

SUBMITTER: Perkin J

PROVIDER: S-EPMC4701010 | biostudies-other | 2015 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Phosphorylating Titin's Cardiac N2B Element by ERK2 or CaMKIIδ Lowers the Single Molecule and Cardiac Muscle Force.

Perkin John J Slater Rebecca R Del Favero Giorgia G Lanzicher Thomas T Hidalgo Carlos C Anderson Brian B Smith John E JE Sbaizero Orfeo O Labeit Siegfried S Granzier Henk H

Biophysical journal 20151201 12

Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passi ...[more]

PMID: 26682816

Similar Datasets

Project description:Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (Funfold). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling.

Dataset Information

Phosphorylating Titin's Cardiac N2B Element by ERK2 or CaMKII? Lowers the Single Molecule and Cardiac Muscle Force.

Publications

Phosphorylating Titin's Cardiac N2B Element by ERK2 or CaMKIIδ Lowers the Single Molecule and Cardiac Muscle Force.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets