Negative regulation of initial steps in skeletal myogenesis by mTOR and other kinases

• Published in: Scientific reports 2016-02, Vol.6 (1), p.20376-20376, Article 20376
• Main Authors: Wilson, Raphael A., Liu, Jing, Xu, Lin, Annis, James, Helmig, Sara, Moore, Gregory, Timmerman, Casey, Grandori, Carla, Zheng, Yanbin, Skapek, Stephen X.
• Format: Article
• Language: English
• Subjects: 13/1; 13/106; 13/109; 13/31; 13/89; 14; 14/34; 14/56; 14/63; 38; 38/47; 38/77; 38/90; 45/41; 631/136/142; 631/136/2091; 631/337/505; 631/532/2439; 631/80/86; Adaptor Proteins, Signal Transducing - antagonists & inhibitors; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Animals; Cell Cycle Checkpoints; Cell Differentiation; Cell Line; Cell Proliferation - drug effects; Cyclin-dependent kinase 4; Gene expression; Humanities and Social Sciences; Kinases; Mice; Microscopy, Fluorescence; multidisciplinary; Muscle Development - drug effects; Myoblasts; Myoblasts - cytology; Myoblasts - metabolism; Myogenesis; Myogenin - metabolism; Piperazines - pharmacology; Progenitor cells; Protein Array Analysis; Protein Kinases - chemistry; Protein Kinases - genetics; Protein Kinases - metabolism; Pyridines - pharmacology; Rapamycin; Real-Time Polymerase Chain Reaction; Regulatory-Associated Protein of mTOR; RNA Interference; RNA, Messenger - metabolism; RNA, Small Interfering - metabolism; RNA-mediated interference; Science; Sequence Analysis, RNA; TOR protein; TOR Serine-Threonine Kinases - antagonists & inhibitors; TOR Serine-Threonine Kinases - genetics; TOR Serine-Threonine Kinases - metabolism; Transcription activation; Transcription factors
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep20376

The transition from a committed progenitor cell to one that is actively differentiating represents a process that is fundamentally important in skeletal myogenesis. Although the expression and functional activation of myogenic regulatory transcription factors (MRFs) are well known to govern lineage commitment and differentiation, exactly how the first steps in differentiation are suppressed in a proliferating myoblast is much less clear. We used cultured mammalian myoblasts and an RNA interference library targeting 571 kinases to identify those that may repress muscle differentiation in proliferating myoblasts in the presence or absence of a sensitizing agent directed toward CDK4/6, a kinase previously established to impede muscle gene expression. We identified 55 kinases whose knockdown promoted myoblast differentiation, either independently or in conjunction with the sensitizer. A number of the hit kinases could be connected to known MRFs, directly or through one interaction node. Focusing on one hit, Mtor , we validated its role to impede differentiation in proliferating myoblasts and carried out mechanistic studies to show that it acts, in part, by a rapamycin-sensitive complex that involves Raptor. Our findings inform our understanding of kinases that can block the transition from lineage commitment to a differentiating state in myoblasts and offer a useful resource for others studying myogenic differentiation.