Unbiased phosphoproteomics analysis unveils modulation of insulin signaling by extramitotic CDK1 kinase activity in human myotubes

SUMMARY Sensitivity and plasticity of insulin signaling and glucose uptake in skeletal muscle depends on determinants such as genetic variation and obesity. We collected muscle biopsies and isolated myoblasts from a multi-ethnic cohort of lean South Asians (N=10), lean Chinese (N=10), and obese Chinese (N=10), and analysed the proteome and phosphoproteome dynamics in terminally differentiated myotubes after a low-dose insulin stimulation (10nM at 0, 5, 30 min). The myotubes initially responded with increased abundance and phosphorylation level changes along the PI3K/AKT/mTOR axis, decreased abundance of translation initiation factors, and increased phosphorylation levels on proteins involved in mRNA processing at 5 min. After the acute response, protein abundance returned to baseline at 30 min, while phosphorylation changes persisted in proteins including AKT, RPS6 and AS160 (TBC1D4). A joint kinase-substrate statistical analysis revealed that protein abundance changes of AKT, PAK1 and CDK1 showed concordant phosphorylation changes in their respective substrates upon insulin stimulation. We also observed increased phosphorylation of some substrates uniquely in each group, particularly the substrates of CDKs showing stronger changes in South Asians than in Chinese. Pharmacological inhibition and siRNA knockdown of CDK1, a non-myogenic kinase, in terminally differentiated myotubes reduced glucose uptake and desensitized several phosphorylation-mediated signaling on protein translation initiation factors, IRS1, and AS160. Our data suggest that basal extramitotic activity of CDK1 is required for PI3K/AKT/mTORC1 signaling cascade and glucose uptake in insulin-stimulated myotubes. The data also provide a rich resource for studying the role of other kinases in the mechanism of insulin resistance in human myotubes.