A Novel Pathway for TOR-independent Autophagy Regulation by Akt

Akt Pathway-dependent Regulation of Autophagy

Autophagy, which degrades cytoplasmic components to generate recycled nutrients, can have either an oncogenic or tumor suppressive role in cancer. ¹ In contrast, Akt is known to promote cellular transformation and tumorigenesis, and dysregulated Akt activity is observed in many cancer types.² The oncogenic potential of dysregulated Akt is often associated with its role in the positive regulation of cellular growth, proliferation, and survival. However, Akt signaling is also known to suppress autophagy.³ Akt negatively regulates autophagy in response to mitogens via activation of Target of Rapamycin (TOR), which inhibits multiple autophagy-promoting proteins via phosphorylation.⁴ It has also been reported that Akt can regulate autophagy in a TOR-independent manner, but the mechanism has remained elusive.⁵ A recent paper by Wang et al. describes a TOR-independent mechanism by which Akt can suppress autophagy.⁶ This mechanism could be relevant for the development of future cancer therapies.

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Akt activity suppresses autophagy in a TOR-independent manner. Autophagy is a process by which cells degrade long-lived proteins, organelles, and certain types of bacteria in the cytoplasm. Briefly, the Beclin 1 complex is required for the formation of phagophores that subsequently envelope cytoplasmic components, form an autophagosome, and fuse with a lysosome. The cytoplasmic components are then degraded within the resulting autolysosome. Akt directly phosphorylates Beclin 1, which inhibits Beclin 1 complex activity and suppresses autophagy. The article below details this novel mechanism of autophagy suppression and highlights the implications for cancer.

Akt Phosphorylates Beclin 1 to Suppress Autophagy and Promote Cellular Transformation

To confirm that Akt activity could suppress autophagy independently of TOR activity, a constitutively active, myristoylated form of Akt1 (myr-Akt) was utilized. In the presence of Torin 1, a TOR inhibitor, myr-Akt expression resulted in stabilized p62 and a reduced number of LC3 puncta in HeLa cells, both of which are indicative of reduced autophagy.^6,7,8 The observation that TOR activity was dispensable for autophagy suppression suggested that Akt might directly target an autophagy protein. Previous studies and immunoprecipitation experiments indicated that Akt can interact with Beclin 1, a core autophagy protein.^6,9Recombinant Human Akt1 was also able to directly phosphorylate Beclin 1 in an in vitro kinase assay.⁶ In myr-Akt-expressing HeLa cells, Beclin 1 was phosphorylated at Ser234 and Ser295 in the presence of Torin 1. These results suggest that Akt interacts with, and phosphorylates, Beclin 1 in a manner that is independent of TOR activity.

In order to determine the functional significance of Akt-dependent Beclin 1 phosphorylation, a mutant Beclin 1 with both Akt phosphorylation sites converted to alanine was constructed (Beclin 1 AA).⁶ Expression of Beclin 1 AA prevented myr-Akt-dependent autophagy suppression in Rat2 fibroblast cells. In addition, Beclin 1 AA expression partially inhibited myr-Akt-dependent transformation of Rat2 cells as determined by an anchorage-independent growth assay. The tumor growth rate and volume in mice injected with myr-Akt-expressing Rat2 cells was also reduced by expression of Beclin 1 AA. Collectively, these results suggest that Akt-dependent phosphorylation of Beclin 1 suppresses autophagy and may promote cellular transformation and tumor growth.

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Akt directly phosphorylates Beclin 1 to suppress autophagy. Autophagy initiation is regulated by the Unc-51-like Kinase (ULK) kinase complex and a complex of core autophagy proteins that includes Beclin 1, Vps34, and PIK3R4. Under growth promoting conditions, or if Akt is constitutively active, autophagy can be inhibited in an Akt-dependent manner via TORC1 activation. Akt also directly phosphorylates Beclin 1 on Ser234 and Ser295, which creates a phospho-binding site for 14-3-3 proteins. The Beclin 1/14-3-3 complex interacts with Vimentin filaments, which might sequester Beclin 1 from other core autophagy proteins. Regulation of Beclin 1 by Akt suppresses autophagy and partially mediates the oncogenic capability of a constitutively active form of Akt.

Phosphorylated Beclin 1 Binds to Intermediate Filaments

Beclin 1 and Beclin 1 AA were then immunoprecipitated from HeLa cells to identify proteins that only interact with Beclin 1 following phosphorylation by Akt. 14-3-3 proteins, which are known to bind phospho-Ser/Thr, co-immunoprecipitated with Beclin 1, but not Beclin 1 AA.^6,10 Consistent with previous results showing that 14-3-3 proteins bind to cytoskeletal components, Beclin 1 also pulled down the intermediate filament proteins, Vimentin and Cytokeratin 18.¹¹ The significance of Vimentin in this pathway was investigated via siRNA knockdown experiments. Depletion of Vimentin in Rat2 cells suppressed autophagy and promoted cellular transformation, similar to the results obtained from Beclin 1 AA expression. This suggests that Akt-dependent Beclin 1 phosphorylation and the subsequent interaction of phosphorylated Beclin 1 with Vimentin are both part of the same pathway.

The data presented by Wang et al. are consistent with a model in which Akt-phosphorylated Beclin 1 is sequestered by intermediate filaments via its interaction with 14-3-3 proteins, resulting in autophagy suppression and tumor growth. In support of this model, autophagy has been reported to have anti-tumor activities in certain contexts.¹² These results also suggest that blocking the interaction between Akt and Beclin 1 could enhance the efficacy of cancer therapies that activate autophagy.

References

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4. Janku, F. et al. (2011) Nat. Rev. Clin. Oncol. 8:528.
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6. Wang, R.C. et al. (2012) Science 338:956.
7. Komatsu, M. et al. (2007) Cell 131:1149.
8. Sou, Y.S. et al. (2008) Mol. Biol. Cell 19:4762.
9. Carloni, S. et al. (2010) Autophagy 6:366.
10. Lim, G.E. et al. (2013) Diabetologia 56:825.
11. Sivaramakrishnan, S. et al. (2009) Mol. Biol. Cell 20:2755.
12. Chang, S.H. et al. (2012) J. Radiat. Res. 53:422.

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