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  • Tip60 Phosphorylation at Ser 99 Is Essential for Autophagy Induction in Bombyx mori.

Tip60 Phosphorylation at Ser 99 Is Essential for Autophagy Induction in Bombyx mori.

International journal of molecular sciences (2020-09-24)
Wenmei Wu, Kang Li, Haigang Zhao, Xianying Xu, Jing Xu, Man Luo, Yang Xiao, Ling Tian
ABSTRACT

Tip60, a key histone acetyltransferase of the MYST family and member of the nuclear multimeric protein complex (NuA4), regulates the activity and stability of proteins involved in the cell cycle, DNA damage responses, autophagy, etc. However, the function and regulatory mechanism of Tip60 homolog in Bombyx mori are not elucidated. In the present study, Bombyx Tip60 (BmTip60) was functionally identified. Developmental profiles showed that the protein levels and nuclear localization of BmTip60 peaked in fat body during the larval-pupal metamorphosis when autophagy was intensive; simultaneously, the BmTip60 protein migrated to form an upper band as detected by Western blot. Interestingly, the upper band of BmTip60 was reduced by λ-phosphatase treatment, indicating that it was a phosphorylated form of BmTip60. Results showed that BmTip60 was promoted by starvation but not 20-hydroxyecdysone treatment. Transcription factor AMP-activated protein kinase (AMPK) affected by starvation was pivotal for BmTip60 protein migration. In addition, one mammalian phosphorylation site was identified in BmTip60 at Ser99, the constitutive-activation mutation of Ser99 to Asp99 but not its inactive mutation to Ala99 significantly upregulated autophagy, showing the critical role of phosphorylation at Ser99 for BmTip60-mediated autophagy. In conclusion, the starvation-AMPK axis promotes BmTip60 in B. mori, which was requisite for autophagy induction. These results reveal a regulatory mechanism of histone acetyltransferase Tip60 homologs by phosphorylation in insects, and sheds light on further related studies of acetylation regulation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
STX64, ≥98% (HPLC)
Sigma-Aldrich
Dorsomorphin, ≥98% (HPLC)
Sigma-Aldrich
20-Hydroxyecdysone, ≥93% (HPLC), powder
Sigma-Aldrich
Grace′s Insect Medium, With L-glutamine, without sodium bicarbonate, powder, suitable for insect cell culture
Sigma-Aldrich
Lambda protein phosphatase, liquid, Bacteriophage Lambda, recombinant, expressed in E. coli
Sigma-Aldrich
PP2, ≥98% (HPLC)