An AMPK-ULK1-PIKFYVE signaling axis for PtdIns5P-dependent autophagy regulation upon glucose starvation

ABSTRACT

Glucose deprivation induces macroautophagy/autophagy primarily through AMPK activation. However, little is known about the exact mechanism of this signaling. A recent study from Dr. David C. Rubinsztein’s lab showed that ULK1 is activated by AMPK upon glucose starvation, resulting in the phosphorylation of the lipid kinase PIKFYVE on S1548. The activated PIKFYVE consequently enhances the formation of phosphatidylinositol-5-phosphate (PtdIns5P)-containing autophagosomes, and therefore drives autophagy upregulation. The novel discovery of how ULK1 regulates the non-canonical autophagy signaling (PtdIns5P-dependent autophagy), not only expands our knowledge of autophagy, but also sheds light on therapeutic strategies for curing human disorders, where glucose-induced starvation can play an important role.

KEYWORDS:

• Lipid kinase
• lysosome
• non-canonical autophagy
• phosphoinositides
• stress

ULK1 is a serine/threonine-specific protein kinase that plays a vital role in autophagy induction [1]. The ULK1 complex, which is composed of ULK1, ATG13, RB1CC1, and ATG101, integrates the upstream nutrient-sensing signals from MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) and AMP-activated protein kinase (AMPK) [2]. In canonical autophagy, the ULK1 complex recruits the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which includes PIK3C3/VPS34, BECN1, PIK3R4/VPS15, NRBF2 and ATG14, to the autophagy initiation site [1]. PIK3C3/VPS34, the lipid kinase, produces phosphatidylinositol-3-phosphate (PtdIns3P), which is critical for autophagosome biogenesis. Karabiyik et al. [3] recently showed a novel role of ULK1 in regulating non-canonical autophagy, which is independent of PtdIns3P formation. The authors also demonstrated that AMPK activates ULK1 upon glucose starvation, resulting in the phosphorylation of PIKFYVE, which plays a role in the generation of PtdIns5P-containing autophagosomes.

A previous report showed that PtdIns5P can replace PtdIns3P as a regulator for autophagosome formation in a PIK3C3/VPS34-independent manner during glucose starvation [4]. In the present study by Karabiyik et al., the authors first repeated and confirmed this finding in HeLa cells. Then, the authors further identified PIKFYVE as the downstream target of AMPK in the process of PtdIns5P-containing autophagosome formation by using either the PIKFYVE inhibitor YM201636 or overexpressing the dominant negative PIKFYVE^K1831E mutant. Notably, PIKFYVE is a lipid kinase that has been extensively explored with regard to its function in phosphatidylinositol-3,5-bisphosphate (PtdIns[3,5]P₂) and PtdIns5P production [5]. PIKFYVE plays an important role in membrane remodeling of the endosomal/endocytic system. AMPK is able to phosphorylate PIKFYVE on S307, therefore affecting PtdIns(3,5)P₂ synthesis [6]. However, the authors found that this direct phosphorylation of PIKFYVE by AMPK is not the reason for the formation of PtdIns5P-containing phagophores.

Karabiyik et al. hypothesized that ULK1, one of the downstream targets of AMPK in canonical autophagy, is involved in PIKFYVE phosphorylation. Therefore, the authors next investigated the role of ULK1 in PtdIns5P-dependent autophagy. They demonstrated that active ULK1 is required for the formation of PtdIns5P-containing phagophores by using the ULK1 inhibitor SBI-0206965 and ULK1 overexpression. The interaction between ULK1 and PIKFYVE increases after glucose starvation based on co-immunoprecipitation and colocalization as revealed by microscopy. Furthermore, co-immunoprecipitation results suggest that PIKFYVE binds to the ULK1 kinase domain at the position of amino acids 1–278. The authors further confirmed that ULK1 phosphorylates PIKFYVE by comparing the gel mobility shift of the latter in cells overexpressing ULK1 and cells with a kinase-dead form of ULK1.

The authors next tested the PIKFYVE lipid kinase activity after ULK1-mediated phosphorylation by measuring PtdIns5P synthesis in vitro. Exploiting the lipid overlay assay, Karabiyik et al. found that S1548 is a critical ULK1-dependent PIKFYVE phosphorylation site. The activation of PIKFYVE by phosphorylation at S1548 specifically leads to an increase in PtdIns5P synthesis.

Last, Karabiyik et al. monitored increased autophagic flux of cells with PIKFYVE activation by using the mRFP-GFP-LC3 assay. In addition, the authors found that aggregates formed from the fusion of EGFP with mutant HTT (huntingtin), which play a role in causing Huntington disease, are dramatically cleared by PIKFYVE^S1548D overexpression in HeLa cells.

Overall, the work from the Rubinsztein lab using multiple cell lines demonstrates a novel non-canonical autophagy pathway dependent on the synthesis of PtdIns5P. The pathway is regulated by the AMPK-ULK1-PIKFYVE axis. The demonstration of the mechanism behind PtdIns5P-containing autophagosome formation enriches our understanding of autophagy upon glucose starvation. For the first time, ULK1 is correlated with PIKFYVE phosphorylation and PtdIns5P-dependent non-canonical autophagy. To note, PIKFYVE has multiple roles in regulating membrane equilibrium, including regulating endolysosome maturation, which is directly affected by AMPK, as well as activating PtdIns5P-containing autophagosome biogenesis that is regulated by ULK1. Moreover, this work shows potential for assisting in the design of therapeutic strategies for treating Hungtington disease. For example, utilizing PtdIns5P-dependent autophagy to remove pathogenic aggregates may be a good strategy for diseases in which PtdIns3P-dependent autophagy is defective.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Institute of General Medical Sciences [GM131919].