Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKb is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKb's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.

p38 and c-Jun N-terninal kinase (JNK) are activated in response to acute stress and inflammatory signals. Through modification of a plethora of substrates, these kinases profoundly re-shape cellular physiology for the optimal response to a harmful environment and/or an inflammatory state. Here, we utilized phospho-proteomics to identify several hundred substrates for both kinases. Our results indicate that the scale of signaling from p38 and JNK are of a similar magnitude. Among the many new targets, we highlight the regulation of the transcriptional regulators grb10-interacting GYF protein 1 and 2 (GIGYF1/2) by p38-dependent MAP kinase-activated protein kinase 2 (MK2) phosphorylation and 14–3–3 binding. We also show that the Golgi apparatus contains numerous substrates, and is a major target for regulation by p38 and JNK. When activated, these kinases mediate structural rearrangement of the Golgi apparatus, which positively affects protein flux through the secretory system. Our work expands on our knowledge about p38 and JNK signaling with important biological ramifications.

Unreferenced, ~150 words)Quantitative phosphoproteomics has in recent years revolutionized understanding of cell signaling, but it remains a challenge to scale the technology for high-throughput analyses. Here we present a rapid and reproducible phosphoproteomics approach to systematically analyze hundreds of samples by fast liquid chromatography tandem mass spectrometry using data independent acquisition (DIA). To overcome the inherent issue of positional phosphopeptide isomers in DIA-based phosphoproteomics, we developed and employed an accurate site localization scoring algorithm, which is incorporated into the Spectronaut software tool. Using a library of synthetic phosphopeptides spiked-in to a yeast phosphoproteome in different ratios we show that it is on par with the top site localization score for data-dependent acquisition (DDA) based phosphoproteomics. Single-shot DIA-based phosphoproteomics achieved an order of magnitude broader dynamic range, higher reproducibility of identification and improved sensitivity and accuracy of quantification compared to state-of-the-art DDA-based phosphoproteomics. Importantly, direct DIA without the need of spectral libraries performed almost on par with analyses using specific project-specific libraries. Moreover, we implemented and benchmarked an algorithm for globally determining phosphorylation site stoichiometry in DIA. Finally, we demonstrate the scalability of the DIA approach by systematically analyzing the effects of thirty different kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that a large proportion of EGF-dependent phospho-regulation is mediated by a specific set of protein kinases.