Major finding: AMPK substrate phosphorylation is required for completion of mitosis and cytokinesis.
Approach: An ATP analogue-specific AMPKα2 mutant allows in vivo labeling of AMPK substrates.
Impact: Adipocytes secrete cytokines to attract tumor cells and provide lipids as an energy source.
AMP-activated protein kinase (AMPK) is activated by cellular stimuli that increase the AMP:ATP ratio, such as low nutrient levels, and is a key regulator of energy homeostasis, although recent evidence has suggested that AMPK may function in other cellular processes. To systematically identify AMPK substrates and gain insight into the cellular function of AMPK, Banko and colleagues performed a chemical genetics screen for proteins directly phosphorylated by AMPK. Briefly, the ATP-binding pocket of one of the catalytic subunits of AMPK (AMPKα2) was mutated such that it could accommodate bulky ATP analogues. These modified ATP nucleotides could then be transferred to AMPKα2 substrates and be recognized by a specific monoclonal antibody. AMPK targets can thus be specifically labeled in vivo, immunoprecipitated, and analyzed by mass spectrometry. Using this approach, several known AMPK substrates were identified, along with 28 proteins not previously known to be targets of AMPK. These proteins were shown to be phosphorylated by endogenous AMPK in an in vitro kinase assay, and AMPK phosphorylation consensus motifs were detected in many of these newly identified substrates. Unexpectedly, these substrates were significantly enriched for proteins involved in mitotic cell division. Consistent with a role of AMPK-dependent phosphorylation in mitosis, AMPK activity, as well as phosphorylation of the newly identified AMPK substrate, PPP1R12C, was increased in mitotic cells. Furthermore, AMPK inhibition and mutation of the AMPK phosphorylation site in PPP1R12C similarly impaired the completion of cell division, as evidenced by an increase in multinucleated cells. These findings provide a mechanism for AMPK activity in mitosis and raise the intriguing possibility that the suspected tumor suppressor function of AMPK may be linked to its ability to coordinate mitotic progression with cellular energy status.
Note: Research Watch is written by Cancer Discovery Science Writers. Readers are encouraged to consult the original articles for full details. For more Research Watch, visit Cancer Discovery online at www.AACR.org/CDnews.
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