Myofilament Tyrosine Phosphorylation By Src Kinase Is Upregulated In Erbb2 Transgenic Mice

Objectives: Tyrosine (Tyr) phosphorylation of the myofilament is an emerging, and potentially important, post-translational modification in cardiomyopathy. ErbB2, a Tyr receptor kinase, was overexpressed in transgenic mice (ErbB2-Tg) resulting in significant cardiac hypertrophy. We hypothesize that the development of cardiac hypertrophy in ErbB2-Tg is associated with increased myofilament Tyr phosphorylation and may implicate myofilament Tyr phosphorylation in cardiac hypertrophy. Methods: Proteins were isolated from ErbB2-Tg and Ntg heart homogenates ( n =4 per group). Reduction/alkylation was followed by trypsinization. Resulting peptides were desalted in C 18 columns and lyophilized. Phosphorylated Tyr (p-Tyr) enrichment was performed on 20 mg of peptides using a p-Tyr Mouse mAb kit (Cell Signaling). Immuno-precipitated and desalted peptides were analyzed by LC-MS/MS (Orbitrap Elite, Thermo). Raw data were searched with Mascot 2.3. Label-free quantification with MS1 extracted ion chromatograms was performed using Skyline. Western blot analysis for total and phosphorylated Src kinase was performed per manufacturer’s protocol (Cell Signaling). Results: We found a total of 286 p-Tyr modified peptides in ErbB2-Tg compared to 226 in control NTg mice. Over 70 p-Tyr sites on myofilament protein were up-regulated in ErbB2-Tg, including troponin I, myosin heavy chain, titin, α-tropomyosin, myosin-binding protein-C3, myosin regulatory light chain-2 and myosin light chain-1. We used PhosphoMotif Finder to search the potential responsible kinase. Most of the p-Tyr sites were consistent with Src kinase motifs. Furthermore, Western blot analysis showed that total, and phospho-Src (Y416) expression was increased in ErbB2-Tg mice. Conclusion: We concluded that these novel p-Tyr sites on myofilament proteins are increased in ErbB2-Tg mice and correlate with up-regulated Src kinase activity. Thus increased tyrosine myofilament phosphorylation may be involved in the development of cardiac hypertrophy. Since ErbB2 is a therapeutic target of trastuzumab therapy this may also have translational implications to ameliorate off target effects of cancer treatment.