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
Review
. 2012 Jan;32(1):2-11.
doi: 10.1128/MCB.06159-11. Epub 2011 Oct 24.

Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks

Sebastian Alers  1 , Antje S Löffler, Sebastian Wesselborg, Björn Stork
Affiliations
Review

Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks

Sebastian Alers et al. Mol Cell Biol. 2012 Jan.

Abstract

Living cells are adaptive self-sustaining systems. They strictly depend on the sufficient supply of oxygen, energy, and nutrients from the outside in order to sustain their internal organization. However, as autonomous entities they are able to monitor and appropriately adapt to any critical fluctuation in their environment. In the case of insufficient external nutrient supply or augmented energy demands, cells start to extensively digest their own interior. This process, known as macroautophagy, comprises the transport of cytosolic portions and entire organelles to the lysosomal compartment via specific double-membrane vesicles, called autophagosomes. Although extensively upregulated under nutrient restriction, a low level of basal autophagy is likewise crucial in order to sustain the cellular homeostasis. On the other hand, cells have to avoid excessive and enduring self-digestion. The delicate balance between external energy and nutrient supply and internal production and consumption is a demanding task. The complex protein network that senses and precisely reacts to environmental changes is thus mainly regulated by rapid and reversible posttranslational modifications such as phosphorylation. This review focuses on the serine/threonine protein kinases AMP-activated protein kinase, mammalian _target of rapamycin (mTOR), and unc-51-like kinase 1/2 (Ulk1/2), three interconnected major junctions within the autophagy regulating signaling network.

PubMed Disclaimer

Figures

Fig 1
Fig 1
Regulation of autophagy induction by the TOR and AMPK complex in yeast, flies, and mammals. (A) Under nutrient-rich conditions, activated TORC1 inhibits autophagy induction in yeast through direct phosphorylation of Atg13. This hyperphosphorylation of Atg13 prevents binding to Atg1. Inactivation of TORC1, as induced by starvation or rapamycin treatment, results in rapid dephosphorylation of Atg13, leading to the formation of the active Atg1-Atg13-Atg17 kinase complex (50, 51). The Atg13-mediated dimerization of Atg1 has recently been described to be essential for the autophosphorylation and subsequent activation of Atg1 (114). Furthermore, Snf1, a yeast ortholog of mammalian AMPK, has been found to be a positive effector of autophagy, presumably through regulation of Atg1 and Atg13 (107). (B) In contrast to yeast, the Atg1-Atg13 complex is stable under nutrient-rich conditions in Drosophila and dTORC1 phosphorylates both Atg13 and Atg1. In starved animals or when dTORC1 is specifically inhibited, these sites are dephosphorylated, Atg1 kinase activity is elevated, thus leading to autophosphorylation and phosphorylation of Atg13 (10, 11). Snf4aγ, a Drosophila ortholog of the mammalian AMPK gamma subunit, was found to be an inducer of autophagy; nevertheless, the exact mechanism remains elusive (68). (C) In mammals, two orthologs of yeast Atg1, termed Ulk1 and Ulk2, have been linked to starvation induced autophagy. Both are found in a stable complex with Atg13, FIP200 (5, 26, 28, 29, 37, 47), and Atg101, an additional binding partner of Atg13 that has no ortholog in yeast (38, 78). In contrast to yeast, the composition of this complex does not change with the nutrient status. Under fed conditions mTORC1 phosphorylates Ulk1/2 and Atg13, thereby inhibiting the kinase complex (26, 37, 47). In response to starvation, the mTORC1-dependent phosphorylation sites in Ulk1/2 are rapidly dephosphorylated, and Ulk1/2 autophosphorylates and phosphorylates Atg13 and FIP200. Alternatively, Ulk1/2 is phosphorylated by AMPK and thereby activated (20, 54). In addition, AMPK indirectly leads to the induction of autophagy by inhibiting mTORC1 through phosphorylation of raptor (27, 64). Note, however, that this is a schematic overview, and we do not provide any determination regarding the association between AMPK and the Ulk1/2-Atg13-FIP200 complex due to the conflicting results published in the recent past. For further details, see the legend to Fig. 3. (This figure was adopted and modified with permission from reference .)
Fig 2
Fig 2
Fine adjustment of autophagy by the AMPK-mTORC1-Ulk1/2 kinase network. The two protein complexes AMPK and mTORC1 are known to oppositely regulate the autophagy inducing complex Ulk1/2-Atg13-FIP200. Under sufficient supply of growth factors and nutrients, the active mTORC1 stimulates growth related processes such as protein translation, e.g., by phosphorylation of S6K1 and 4E-BP, while simultaneously inhibiting self-consuming processes such as autophagy (94, 110). mTORC1 activity is positively regulated by growth factor signaling via the PI3K-Akt pathway. Akt activates mTORC1 by inhibition of TSC1/2 (41, 71, 72, 119) or PRAS40 (105), two negative regulators of mTORC1 activity that both antagonize the Rheb-mediated activation. Hypoxia counteracts mTORC1 activation via the TSC1/2-Rheb pathway, e.g., by upregulation of REDD1 (7). Amino acids, in contrast, stimulate the Rag-GTPase-dependent recruitment of mTORC1 to lysosomes and its subsequent activation by Rheb-GTPases (54, 89, 90). The catalytic activity of AMPK crucially depends on phosphorylation by upstream kinases, such as the constitutively active LKB1. AMPK activity is further enhanced by decreasing ATP/AMP ratios (30). In addition, the other two known upstream kinases, CaMKKβ and TAK1, have been implicated in AMPK-mediated autophagy induction by intracellular [Ca2+] and TRAIL treatment, respectively (36, 39). Under low-energy conditions, AMPK positively regulates autophagy induction (77) through inhibition of mTORC1. This releases the negative regulation of mTORC1 on the Ulk1/2-Atg13-FIP200 complex, especially on Ulk1/2 kinase activity (26, 37, 47). AMPK inhibits mTORC1 either via the TSC1/2-Rheb pathway (44) or by direct phosphorylation of raptor (27). However, AMPK is also able to bind, phosphorylate, and directly activate Ulk1/2 (5, 21, 55, 64, 88, 95). Again, this interaction is counteracted by mTORC1 (55). For a more detailed discussion see Roach (88) and Fig. 3. Prolonged TORC1 activation, on the other hand, leads to the accumulation of Sestrin (SESN) in Drosophila, a DNA damage-inducible protein that suppresses TOR activity by AMPK activation (63). Furthermore, mTORC1 not only inhibits autophagy by suppressing Ulk1/2 kinase activity, it also simultaneously inhibits DAP1, a negative regulator of autophagy (57). mTORC1 inhibition thus leads to both autophagy induction via Ulk1/2-Atg13-FIP200 and to its restriction via DAP1. Ulk1 kinase activity might be linked to autophagy induction in several ways. Two downstream _targets of Ulk1 have been proposed thus far. First, Ulk1 directly phosphorylates AMBRA1, a Beclin1-interacting protein and regulatory component of the PI3K class III complex (17) and, second, it phosphorylates and activates a distinct myosin light chain kinase (MLCK) in mammals (ZIPK) and Drosophila (Sqa) (100). Two Ulk1-dependent feedback loops additionally help to fine-tune the autophagic response. Ulk1 has been shown to phosphorylate and inhibit both of its upstream regulators AMPK and mTORC1. While phosphorylation of raptor might help to maintain mTORC1 inhibition when nutrients are limited (19, 48), the inhibition of AMPK activity by Ulk1 antagonizes this action and restricts the autophagic response (70). This perplexingly complex network of mutual activation and inhibition will ultimately establish an appropriate response to conflicting demands.
Fig 3
Fig 3
Mutual phosphorylation of Ulk1, mTORC1, and AMPK. (A) Ulk1 is a hyperphosphorylated protein that is massively dephosphorylated upon starvation. Under normal growth conditions, mTORC1 has been shown to directly bind to and negatively regulate Ulk1/2 kinase activity by direct phosphorylation (26, 37, 47). A total of 16 phosphorylation sites in mouse Ulk1, purified from HEK293T cells under fed conditions, were first mapped by Dorsey et al. (18). These authors additionally identified two sites, differentially phosphorylated in wild-type versus kinase-dead Ulk1 (S1043 and S1047). While the one was suggested as direct _target for autophosphorylation, the other might represent a putative PKA site (18). Shang et al. (95) quantitatively analyzed the differential phosphorylation status in human Ulk1 purified from HEK293T cells between fed versus starved conditions (HBSS containing 1% rich medium) using stable isotope labeling with amino acids in cell culture (SILAC). A total of 13 sites were identified, with the strongest dephosphorylation at S638 and S758 (corresponding to S637 and S757 in mouse Ulk1) showing a >10-fold decrease after starvation, although with different kinetics. The same decrease was seen after rapamycin treatment and mTOR knockdown (95). Interestingly, phosphorylation at S638 was affected by the knockdown of AMPKα and AMPKβ (95). Shang et al. found that AMPK was associated with Ulk1 under fed conditions and proposed that the dephosphorylation of S758 seen after starvation is critical for the dissociation of AMPK/Ulk1. In contrast, Lee et al. (64) proposed that the interaction between AMPK and Ulk1 is essential for the induction of autophagy and that AMPK activity both recruits 14-3-3 proteins to the complex and leads to inactivation of mTORC1 activity by AMPK-mediated phosphorylation of raptor at S792 (27, 64). Egan et al. (21) additionally identified Ulk1 both as an AMPK substrate and as a 14-3-3 binding protein. This group found S467, S555, T574, and S637 of Ulk1 to be phosphorylated after phenformin treatment and confirmed these sites in an AMPK in vitro kinase assay (21). Notably, Bach et al. (4) could meanwhile confirm the AMPK-dependent phosphorylation at S555 and that this induces the binding to 14-3-3 proteins. This group additionally identified a critical phosphorylation site in the Ulk1 activation loop (T180), as well as a potential Akt phosphorylation site (S774) whose phosphorylation is increased after insulin treatment. The phosphorylation and differential regulation of Ulk1 by mTORC1 and AMPK has also been reported by Kim et al. (55). This group identified S757 in mouse Ulk1 as a direct mTOR site, the same identified by Shang et al. in human Ulk1 (95). However, Kim et al., in contrast, suggest that phosphorylation at S757 prevents the interaction between Ulk1 and AMPK. mTORC1 inhibition would thus lead to an association of AMPK and Ulk1. In line with that, the data suggest an activating effect of AMPK on Ulk1 kinase activity by direct phosphorylation at S317 and S777. These authors found that both sites are required for Ulk1 activation after glucose starvation (55). Notably, neither of these two sites has been identified by one of the other groups. All phosphorylation sites identified by the five groups (4, 18, 21, 55, 95) are shown in single-letter code and refer to mouse Ulk1. Proposed kinases are either indicated by name or otherwise labeled with question marks (?). (B) Dunlop et al. (19) and Jung et al. (48) identified raptor as a direct substrate of Ulk1 and Ulk1 thereby as a negative regulator of either mTORC1 activity (48) or substrate binding (19). After overexpression of Ulk1, Dunlop et al. observed an increase in the in vivo phosphorylation of raptor at S696, T706, S792, S855, S859, and S863 using phospho-specific antibodies and could subsequently confirm the latter four sites as directly phosphorylated by Ulk1 (19). The strongest phosphorylation was seen on S859. Interestingly, S792 is the AMPK and 14-3-3 binding site identified by Gwinn et al. (27), by which AMPK negatively regulates mTORC1 activity, while S863 is known to be phosphorylated by mTOR and to promote mTORC1 activity (23, 106). (C) Löffler et al. (70) identified all three subunits of AMPK as a direct substrate of Ulk1 and Ulk2 and mapped several Ulk1-dependent in vitro phosphorylation sites in AMPKα1, -β2, and -γ1 (residues refer to rat AMPK, for some peptides the phospho-acceptor sites could not be distinguished: S360/T368, S486/T488, and S260/T262). The Ulk1-dependent phosphorylation of AMPK has been proposed to negatively regulate AMPK kinase activity, thus constituting a negative regulatory feedback loop (70). (This figure was adopted and modified with permission from reference .)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alers S, et al. Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction. Autophagy, in press - PMC - PubMed
    1. Apel A, Zentgraf H, Buchler MW, Herr I. 2009. Autophagy: a double-edged sword in oncology. Int. J. Cancer 125: 991–995 - PubMed
    1. Arsham AM, Howell JJ, Simon MC. 2003. A novel hypoxia-inducible factor-independent hypoxic response regulating mammalian _target of rapamycin and its _targets. J. Biol. Chem. 278: 29655–29660 - PubMed
    1. Axe EL, et al. 2008. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182: 685–701 - PMC - PubMed
    1. Bach M, Larance M, James DE, Ramm G. The serine/threonine kinase ULK1 is a _target of multiple phosphorylation events. Biochem. J., in press - PubMed

Publication types

Substances

  NODES
Association 2
INTERN 4
Note 1
twitter 2