title: Negative regulation of initial steps in skeletal myogenesis by mTOR and other kinases.
abstract: 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.
authors: Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Raphael A
authors: RA
authors: Wilson
authors: Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Jing
authors: J
authors: Liu
authors: Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Lin
authors: L
authors: Xu
authors: Quellos High Throughput Screening Core, Department of Pharmacology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA.
authors: James
authors: J
authors: Annis
authors: Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Sara
authors: S
authors: Helmig
authors: Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Gregory
authors: G
authors: Moore
authors: Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Casey
authors: C
authors: Timmerman
authors: Quellos High Throughput Screening Core, Department of Pharmacology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA.
authors: Carla
authors: C
authors: Grandori
authors: Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Yanbin
authors: Y
authors: Zheng
authors: Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Stephen X
authors: SX
authors: Skapek
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D059447:Cell Cycle Checkpoints; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D051379:Mice; D008856:Microscopy, Fluorescence; D024510:Muscle Development / Q000187:drug effects; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D018008:Myogenin / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D040081:Protein Array Analysis; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D000076223:Regulatory-Associated Protein of mTOR; D017423:Sequence Analysis, RNA; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018008:Myogenin; D010879:Piperazines; D011725:Pyridines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000076223:Regulatory-Associated Protein of mTOR; C540162:Rptor protein, mouse; D011494:Protein Kinases; D058570:TOR Serine-Threonine Kinases; C500026:palbociclib