Phosphoproteomics Profiling Defines a Target Landscape of the Basophilic Protein Kinases AKT, S6K, and RSK in Skeletal Myotubes

Phosphorylation-dependent signal transduction plays an
important
role in regulating the functions and fate of skeletal muscle cells.
Central players in the phospho-signaling network are the protein kinases
AKT, S6K, and RSK as part of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK
pathways. However, despite their functional importance, knowledge
about their specific targets is incomplete because these kinases share
the same basophilic substrate motif RxRxxp[ST]. To
address this, we performed a multifaceted quantitative phosphoproteomics
study of skeletal myotubes following kinase inhibition. Our data corroborate
a cross talk between AKT and RAF, a negative feedback loop of RSK
on ERK, and a putative connection between RSK and PI3K signaling.
Altogether, we report a kinase target landscape containing 49 so far
unknown target sites. AKT, S6K, and RSK phosphorylate numerous proteins
involved in muscle development, integrity, and functions, and signaling
converges on factors that are central for the skeletal muscle cytoskeleton.
Whereas AKT controls insulin signaling and impinges on GTPase signaling,
nuclear signaling is characteristic for RSK. Our data further support
a role of RSK in glucose metabolism. Shared targets have functions
in RNA maturation, stability, and translation, which suggests that
these basophilic kinases establish an intricate signaling network
to orchestrate and regulate processes involved in translation.