Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Oct 16;326(5951):437-40.
doi: 10.1126/science.1172156.

AMPK regulates the circadian clock by cryptochrome phosphorylation and degradation

Katja A Lamia  1 , Uma M Sachdeva, Luciano DiTacchio, Elliot C Williams, Jacqueline G Alvarez, Daniel F Egan, Debbie S Vasquez, Henry Juguilon, Satchidananda Panda, Reuben J Shaw, Craig B Thompson, Ronald M Evans
Affiliations

AMPK regulates the circadian clock by cryptochrome phosphorylation and degradation

Katja A Lamia et al. Science. .

Abstract

Circadian clocks coordinate behavioral and physiological processes with daily light-dark cycles by driving rhythmic transcription of thousands of genes. Whereas the master clock in the brain is set by light, pacemakers in peripheral organs, such as the liver, are reset by food availability, although the setting, or "entrainment," mechanisms remain mysterious. Studying mouse fibroblasts, we demonstrated that the nutrient-responsive adenosine monophosphate-activated protein kinase (AMPK) phosphorylates and destabilizes the clock component cryptochrome 1 (CRY1). In mouse livers, AMPK activity and nuclear localization were rhythmic and inversely correlated with CRY1 nuclear protein abundance. Stimulation of AMPK destabilized cryptochromes and altered circadian rhythms, and mice in which the AMPK pathway was genetically disrupted showed alterations in peripheral clocks. Thus, phosphorylation by AMPK enables cryptochrome to transduce nutrient signals to circadian clocks in mammalian peripheral organs.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Phosphorylation of S71 or S280 by AMPK destabilizes CRY1. (A) Human embryonic kidney (HEK) 293 cells expressing WT or mutant FLAG-CRY1 were treated with cycloheximide (CHX) as indicated. AA denotes CRY1 with both S71A and S280A mutations. (B) FBXL3-v5 and PER2 bound to FLAG-CRY1 were detected by immunoblot (IB) following immunoprecipitation (IP) from HEK 293 cells. (C) (Top) Alignment of the regions surrounding mouse (m) CRY1-S71 and ACC1-S79. (Bottom) In vitro kinase assays using FLAG-CRY1 purified from HEK 293 cells and purified AMPK (6). Phosphorylated CRY1 was detected by radiography (left) or IB using an antibody against phosphoACC1-S79 (right). (D) Phosphorylation of stably expressed FLAG-CRY1 by endogenous AMPK was detected by IB following IP from MG132-treated MEFs. (E) MEFs were treated with vehicle (−) or 2 mMAICAR (+) for 2 hours. (F) MEFs were treated with CHX ± 2 mM AICAR as indicated. CRY1 was detected by IP and IB for the FLAG epitope in (C to F). Graphs in (F) represent the means ± SD of three quantified IB samples per condition.
Fig. 2
Fig. 2
Disruption of AMPK signaling alters circadian rhythms in MEFs. (A) MEFs were synchronized by serum shock and transferred to media containing glucose and AICAR as indicated. Quantitative PCR (QPCR) was performed by using cDNA from samples collected at the indicated times. Data are means ± SD of a representative experiment analyzed in triplicate. (B) Typical results of continuous monitoring of luciferase activity from U2OS cells stably expressing Bmal1-luciferase. (C and D) Quantification of the circadian period (C) and amplitude (D) of Bmal1-driven luciferase from experiments performed as described in (B). Data in (C) and (D) are means ± SD for four samples per condition. Analysis of variance (ANOVA) indicated a significant difference between categories. **P < 0.01 versus samples cultured in 25 mM glucose.
Fig. 3
Fig. 3
AMPK is rhythmic and regulates CRY1 stability in mouse livers. (A) IB for phospho-Raptor-S792 (pRaptor) and Raptor in mouse liver lysates. CT, circadian time. (B) QPCR analysis of cDNA prepared from mouse livers. (C) IB for AMPKα1, AMPKα2, CRY1, PER2, REV-ERBα, and Lamin A in liver nuclei from two mice at each indicated circadian time (CT). WT (α1+/+ or α2+/+) and ampkα1−/− (α1−/−) or ampkα2−/− (α2−/−) samples demonstrate antibody specificity. (D) Mice were injected with saline or AICAR, and liver samples were collected 1 hour later at zeitgeber time (hours after lights on, ZT) ZT18. (Top) CRY1 in liver nuclei (Nuc) and phospho-Raptor in whole-cell lysates (WCL). (Bottom) Means ± SD of the relative amount of CRY1. **P<0.01 versus saline-treated samples. (E) CRY1, PER2, REV-ERBα, and Lamin A detected by IB in liver nuclei from LKB1+/+ and LKB1L/L mice. (F) QPCR for dbp, reverbα, cry1, and per2 in mouse liver. All transcripts were normalized to u36b4.
See this image and copyright information in PMC

Comment in

  • Physiology. Feeding the clock.
    Suter DM, Schibler U. Suter DM, et al. Science. 2009 Oct 16;326(5951):378-9. doi: 10.1126/science.1181278. Science. 2009. PMID: 19833950 No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Stephan FK, Zucker I. Proc. Natl. Acad. Sci. U.S.A. 1972;69:1583. - PMC - PubMed
    1. Damiola F, et al. Genes Dev. 2000;14:2950. - PMC - PubMed
    1. Yamazaki S, et al. Science. 2000;288:682. - PubMed
    1. Stokkan KA, Yamazaki S, Tei H, Sakaki Y, Menaker M. Science. 2001;291:490. - PubMed
    1. Schibler U, Ripperger J, Brown SA. J. Biol. Rhythms. 2003;18:250. - PubMed

Publication types

MeSH terms