Mirk/Dyrk1B mediates survival during the differentiation of C2C12 myoblasts.
The kinase Mirk/dyrk1B is essential for the differentiation of C2C12 myoblasts. Mirk reinforces the G0/G1 arrest state in which differentiation occurs by directly phosphorylating and stabilizing p27(Kip1) and destabilizing cyclin D1. We now demonstrate that Mirk is anti-apoptotic in myoblasts. Knockdown of endogenous Mirk by RNA interference activated caspase 3 and decreased myoblast survival by 75%, whereas transient overexpression of Mirk increased cell survival. Mirk exerts its anti-apoptotic effects during muscle differentiation at least in part through effects on the cell cycle inhibitor and pro-survival molecule p21(Cip1). Overexpression and RNA interference experiments demonstrated that Mirk phosphorylates p21 within its nuclear localization domain at Ser-153 causing a portion of the typically nuclear p21 to localize in the cytoplasm. Phosphomimetic GFP-p21-S153D was pancellular in both cycling C2C12 myoblasts and NIH3T3 cells. Endogenous Mirk in myotubes and overexpressed Mirk in NIH3T3 cells were able to cause the pancellular localization of wild-type GFP-p21 but not the nonphosphorylatable mutant GFP-p21-S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with pro-apoptotic molecules. Phosphomimetic p21-S153D was more effective than wild-type p21 in blocking the activation of caspase 3. Transient expression of p21-S153D also increased myoblast viability in colony forming assays, whereas the p21-S153A mutant had no effect. This Mirk-dependent change in p21 intracellular localization is a natural part of myoblast differentiation. Endogenous p21 localized exclusively to the nuclei of proliferating myoblasts but was also found in the cytoplasm of post-mitotic multinucleated myotubes and adult human skeletal myofibers.
Mercer
Stephen E
SE
Department of Pathology, Upstate Medical University, State University of New York, Syracuse, New York 13210, USA.
Ewton
Daina Z
DZ
Deng
Xiaobing
X
Lim
Seunghwan
S
Mazur
Thomas R
TR
Friedman
Eileen
E
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D032448:Myoblasts, Skeletal / Q000166:cytology* / Q000502:physiology*; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D012038:Regeneration / Q000502:physiology; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection; D000097603:Dyrk Kinases
D053148:Caspase 3; D020169:Caspases; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D020928:Mitogen-Activated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D012694:Serine; D014157:Transcription Factors; D000097603:Dyrk Kinases; C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse