Aims: 

Posttranslational modifications alter ligand-binding characteristics and the mechanical properties of the giant protein titin. The titin N2-B unique sequence (N2-Bus), a cardiac-specific region in the titin-spring segment of the sarcomeric I-band, can be phosphorylated by protein kinase-G (PKG), thus decreasing passive myocyte stiffness. We searched for phosphorylation sites in another unique domain in I-band titin, the N2A-domain, which is expressed in the cardiac titin isoform N2BA (variable Mw, 3.2–3.7 MDa), but not in the N2B-isoform (3.0 MDa). The N2BA isoform (compliant spring) is upregulated at the expense of the N2B isoform (stiffer spring) in failing human hearts. The N2A-domain has interaction sites for proteins of the muscle ankyrin repeat protein family. Moreover, the N2A-domain can be phosphorylated in vitro by PKG. We aimed to detect the amino acid(s) in the N2A-domain phosphorylated by PKG.

Methods: 

Online prediction routines (NetphosK; GPS: Group-based prediction system) pointed at various potential phosphosites in the N2A-domain. Using this information we produced recombinant truncation constructs of the N2A-domain. Site-directed mutagenesis was employed to exchange amino-acids. Constructs were incubated with PKG-1b in the presence of cGMP and gamma-32P ATP and analyzed by autoradiography on SDS-PAGE gels.

Results: 

We confirmed serine 8651 (S8651) [titin accession number, NP_596869.4] in the N2A-domain as a PKG target. Incubation with other kinases (mitogen activated protein kinases, protein kinase-A, protein kinase-Ca, -d, and -e) showed no phosphorylation signal.

Conclusion: 

The N2A-domain of titin contains a novel PKG-dependent phosphorylation site. Phosphorylation at this site could influence titin-ligand interactions.