The effects of PKCalpha phosphorylation on the extensibility of titin's PEVK element.
Post-translational modifications, along with isoform splicing, of titin determine the passive tension development of stretched sarcomeres. It was recently shown that PKCalpha phosphorylates two highly-conserved residues (S26 and S170) of the PEVK region in cardiac titin, resulting in passive tension increase. To determine how each phosphorylated residue affects myocardial stiffness, we generated three recombinant mutant PEVK fragments (S26A, S170A and S170A/S26A), each flanked by Ig domains. Single-molecule force spectroscopy shows that PKCalpha decreases the PEVK persistence length (from 0.99 to 0.68 nm); the majority of this decrease is attributable to phosphorylation of S26. Before PKCalpha, all three mutant PEVK fragments showed at least 40% decrease in persistence length compared to wildtype. Furthermore, Ig domain unfolding force measurements indicate that PEVK's flanking Ig domains are relatively unstable compared to other titin Ig domains. We conclude that phosphorylation of S26 is the primary mechanism through which PKCalpha modulates cardiac stiffness.
Dept. of Physics, University of Arizona, Tucson, AZ 85724, USA.
Brian R
BR
Anderson
Julius
J
Bogomolovas
Siegfried
S
Labeit
Henk
H
Granzier
D000818:Animals; D064211:Connectin; D004548:Elasticity; D006801:Humans; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D064211:Connectin; D009124:Muscle Proteins; D020033:Protein Isoforms; C577285:TTN protein, human; D011494:Protein Kinases; D051571:Protein Kinase C-alpha