Identification of the downstream molecules of agrin/Dok-7 signaling in muscle.
FASEB J
; 34(4): 5144-5161, 2020 04.
Article
em En
| MEDLINE
| ID: mdl-32043676
ABSTRACT
The development of the neuromuscular junction depends on signaling processes that involve protein phosphorylation. Motor neuron releases agrin to activate muscle protein Dok-7, a key tyrosine kinase essential for the formation of a mature and functional neuromuscular junction. However, the signaling cascade downstream of Dok-7 remains poorly understood. In this study, we combined the clustered regularly interspaced short palindromic repeats/Cas9 technique and quantitative phosphoproteomics analysis to study the tyrosine phosphorylation events triggered by agrin/Dok-7. We found tyrosine phosphorylation level of 36 proteins increased specifically by agrin stimulation. In Dok-7 mutant myotubes, however, 13 of the 36 proteins failed to be enhanced by agrin stimulation, suggesting that these 13 proteins are Dok-7-dependent tyrosine-phosphorylated proteins, could work as downstream molecules of agrin/Dok-7 signaling. We validated one of the proteins, Anxa3, by in vitro and in vivo assays. Knocking down of Anxa3 in the cultured myotubes inhibited agrin-induced AChR clustering, whereas reduction of Anxa3 in mouse muscles induced abnormal postsynaptic development. Collectively, our phosphoproteomics analysis provides novel insights into the complicated signaling network downstream of agrin/Dok-7.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Agrina
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Músculo Esquelético
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Fibras Musculares Esqueléticas
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Proteínas Musculares
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Junção Neuromuscular
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article