Endogenous BMAL1 was found to associate with several translation initiation factors in the cytosol of mouse embryonic fibroblasts (MEFs) and in cytoplasmic lysates from the liver and brain as well as with the cap-binding complex (CBC). Pulse-labelling experiments in Bmal1−/− MEFs revealed a reduction in de novo protein synthesis in comparison to wild-type cells. Moreover, Bmal1−/− mice kept in 12-hour light–12-hour dark cycles showed a significant loss of circadian protein synthesis patterns, indicating that BMAL1 stimulates translation during the light (active) period. Importantly, in human cells, a transcriptionally inactive BMAL1 mutant protein stimulated the cap-dependent translation of a plasmid-encoded reporter gene to the same degree as wild-type BMAL1, whereas neither protein affected reporter gene transcription. Thus, the translational activity of BMAL1 is independent of its transcriptional activity.
The mTOR pathway, of which ribosomal protein S6 kinase β1 (S6K1) is a major effector, activates translation following anabolic stimuli. The authors found that BMAL1 is phosphorylated at Ser42 by S6K1, downstream of mTOR complex 1, and that this post-translational modification is required for the association of BMAL1 with translation initiation factors and the CBC, and for cap-dependent translation. Importantly, immunoprecipitation of BMAL1 from synchronized MEFs at several time points post-synchronization revealed that cytosolic BMAL1 rhythmically associated with S6K1 and with translation factors in correlation with its phosphorylation at Ser42. In cytoplasmic extracts from mouse liver harvested during the circadian cycle, BMAL1 phosphorylation and association with CBCs peaked at the end of the active period, which is consistent with protein synthesis being relatively high during this period. These data suggest that BMAL1 undergoes circadian phosphorylation by S6K1 in phase with its association with the translational machinery in vivo. Finally, overall protein synthesis in synchronized cells followed a circadian rhythm that correlated with the BMAL1–CBC association, but the rhythm and magnitude of protein synthesis were only partially dependent on BMAL1 expression and phosphorylation.
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