Single Molecule Analysis of PKC Phosphorylation of Titin's PEVK Domain

Abstract

Titin's I-band region contains three spring-like domains that are primarily responsible for the development of passive tension in cardiac muscle. PKC phosphorylation targets one of these I-band domains_the PEVK region, which is rich in proline (P), glutamate (E), valine (V), and lysine (K). It has been shown that two serine residues within the PEVK are targeted by PKC phosphorylation, S26 and S170. We investigate the effects of PKC phosphorylation of these two residues on the single molecule level using force-extension curves generated by atomic force microscopy (AFM). We constructed four recombinant proteins: two PEVK single mutants (S26A and S170A), a PEVK double mutant (S26AS170A), and a wild-type PEVK segment. All constructs are flanked by immunoglobulin-like domains, Ig27 and Ig84, and the unfolding of these domains generates a single molecular “fingerprint”. The force-extension curve leading up to the first unfolding peak describes the force-extension relationship of the PEVK. Preliminary data suggests that mutating either serine residue alters PEVK resistance to extension, which is quantified by the molecule's persistence length (PL). Wild-type PEVK underwent a large decrease in its PL after phosphorylation by PKC (by ∼50%), and both single mutants have PLs similar to that of phosphorylated wild-type PEVK. Furthermore, phosphorylation of both single mutants resulted in a small PL decrease. Phosphorylation decreased PL for the S26A mutation by 16% (from 0.55 ± 0.02 to 0.46 ± 0.02 (mean ± SE)), and the serine-170 mutation PL by 11% (from 0.53 ± 0.04 to 0.47 ± 0.03). The double mutant was not affected by PKC (from 0.51 ± 0.04 to 0.51 ± 0.03). We conclude that both serines play a structural role in determining the relationship between longitudinal force and PEVK extension, and that this role is modulated through phosphorylation by PKC.