Protein kinase activity of the glycolytic enzyme PGK1 regulates autophagy to promote tumorigenesis

ABSTRACT

Macroautophagy/autophagy is a cellular defense response to stress conditions and is crucial for cell homeostasis maintenance. However, the precise mechanism underlying autophagy initiation, especially in response to glutamine deprivation and hypoxia, is yet to be explored. We recently discovered that PGK1 (phosphoglycerate kinase 1), a glycolytic enzyme, functions as a protein kinase, phosphorylating BECN1/Beclin 1 to initiate autophagy. Under glutamine deprivation or hypoxia stimulation, PGK1 is acetylated at K388 by NAA10/ARD1 in an MTOR-inhibition-dependent manner, leading to the interaction between PGK1 and BECN1 and the subsequent phosphorylation of BECN1 at S30 by PGK1. This phosphorylation enhances ATG14-associated PIK3C3/VPS34-BECN1-PIK3R4/VPS15 complex activity, thereby increasing phosphatidylinositol-3-phosphate (PtdIns3P) generation in the initiation stage of autophagy. Furthermore, NAA10-dependent PGK1 acetylation and PGK1-dependent BECN1 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumor formation. Our work reveals the important dual roles of PGK1 as a glycolytic enzyme and a protein kinase in the mutual regulation of cell metabolism and autophagy in maintaining cell homeostasis.

Most cancer cells predominantly produce energy by a high rate of glycolysis, even in the presence of ample oxygen. This tumor-specific Warburg effect promotes tumor progression. In the glycolytic pathway, there are 2 ATP-generating enzymes, PK (pyruvate kinase) and PGK1. Our work and that of others have demonstrated that PKM/PKM2 (pyruvate kinase, muscle) acts as a protein kinase to regulate critical cellular functions, including metabolism, gene transcription, chromatid segregation, and cytokinesis. Recently, we were the first to report that PGK1 acts as a protein kinase to phosphorylate PDK1 (pyruvate dehydrogenase kinase 1) at T338 in mitochondria, leading to inhibition of mitochondrial pyruvate metabolism and enhancement of glycolysis. Here, we demonstrated that the protein kinase activity of PGK1 is involved in autophagy regulation (Fig. 1).

Figure 1.

A schematic model of PGK1-regulated autophagy initiation under glutamine deprivation and hypoxia. In the presence of sufficient nutrients, active MTOR phosphorylates the acetyltransferase NAA10 at S228 and inhibits the interaction between NAA10 and PGK1. Glutamine deprivation and hypoxia result in inhibition of MTOR-mediated acetyltransferase NAA10 S228 phosphorylation, leading to NAA10-dependent K388 acetylation of approximately 20% of the total PGK1 and subsequent PGK1-mediated BECN1 S30 phosphorylation. This phosphorylation enhances the activity of the ATG14-associated PIK3C3-BECN1-PIK3R4 complex to induce autophagy. Ac, acetylation; P, phosphorylation.

Glutamine deprivation occurs in tumors that have outgrown the existing vasculature and are experiencing ischemia, resulting in autophagy with unclearly defined mechanisms of induction. Autophagy can be initiated by AMPK-mediated BECN1 phosphorylation at S93/96 in response to glucose starvation or ULK1-mediated BECN1 phosphorylation at S15 in response to amino acid deprivation. We found that AMPK- or ULK1-dependent BECN1 phosphorylation is not responsible for glutamine deprivation-induced autophagy initiation. A mass spectrometry analysis revealed that PGK1 is associated with BECN1 after glutamine deprivation and is acetylated at K388 under this condition. In addition, we revealed that the acetyltransferase NAA10 is associated with PGK1 and acetylates PGK1 at K388. Reconstitution with a PGK1^K388R acetylation-dead mutant in endogenous PGK1-depleted cells significantly inhibits the activity of ATG14-associated PIK3C3-BECN1-PIK3R4 in PtdIns3P production and conversion of LC3B-I to LC3B-II, suggesting that PGK1 K388 acetylation is required for glutamine deprivation-induced autophagy.

We next determined how NAA10 senses glutamine deprivation and acetylates PGK1. We showed that NAA10 S228 is phosphorylated by MTOR. Glutamine deprivation results in inhibition of MTOR and MTOR-mediated NAA10 S228 phosphorylation, which induces the binding of NAA10 to PGK1 and subsequent PGK1 acetylation. This acetylation is required for the interaction between PGK1 and BECN1, a conclusion that is supported by the results of an in vitro assay demonstrating that purified PGK1 and BECN1 bind to each other in a PGK1 acetylation-dependent manner. An in vitro kinase assay, followed by a mass spectrometry analysis, revealed that PGK1 functions as a protein kinase and phosphorylates BECN1 at S30. This phosphorylation is clearly increased upon glutamine deprivation and is significantly inhibited by reconstitution with PGK1^K388R in endogenous PGK1-depleted cells, suggesting that PGK1-mediated BECN1 S30 phosphorylation is dependent on PGK1 acetylation by NAA10.

The N terminus of BECN1 is frequently phosphorylated and is involved in the regulation of PIK3C3-BECN1-PIK3R4 complex activity to produce PtdIns3P and initiate autophagy. PtdIns3P production is substantially induced by glutamine deprivation and significantly decreased by reconstituted expression of BECN1^S30A in endogenous BECN1-depleted cells, suggesting that PGK1-mediated BECN1 S30 phosphorylation enhances ATG14-associated PIK3C3-BECN1-PIK3R4 activity. However, this enhanced activity is not due to an alteration in PIK3C3-BECN1-PIK3R4-ATG14 complex formation; instead, it is caused by a conformational change of PIK3C3 induced by BECN1 S30 phosphorylation, thereby enhancing the binding of PtdIns to PIK3C3 to produce more PtdIns3P. These results indicate that PGK1-mediated BECN1 S30 phosphorylation is required for glutamine deprivation-induced PIK3C3 activation and autophagy.

In addition to glutamine deprivation, hypoxia stimulation or amino acid starvation, which inhibit MTOR activity, reduce MTOR-mediated NAA10 S228 phosphorylation, and enhance NAA10-mediated PGK1 K388 acetylation and PGK1-mediated BECN1 S30 phosphorylation. Furthermore, BECN1 S30 phosphorylation is required for hypoxia-induced autophagy. ULK1 and ULK2 are required in the autophagy response to amino acid deprivation. However, GBM cells expressing limited ULK1/2 are dependent on PGK1-mediated BECN1 S30 phosphorylation, but not ULK1/2-regulated BECN1 S15 phosphorylation, to initiate autophagy. In addition, BECN1 S30 phosphorylation is required for glutamine deprivation-induced autophagy in BxPC-3 pancreatic ductal adenocarcinoma cells and MDA-MB-231 breast cancer cells expressing ULK1/2, further supporting the essential role of PGK1-mediated BECN1 S30 phosphorylation in glutamine deprivation-induced autophagy.

Autophagy is activated to support cell proliferation and survival upon stress. Depletion of PGK1 or BECN1 or reconstituted expression of PGK1^K388R or BECN1^S30A in corresponding endogenous PGK1- or BECN1-depleted GBM cells inhibits cell proliferation under hypoxic conditions. Furthermore, an in situ brain tumorigenesis mouse model demonstrated that expression of PGK1^K388R or BECN1^S30A exhibits significant tumor growth inhibition, with suppressed autophagy, as evidenced by reduced MKI67/Ki-67 and enhanced SQSTM1/p62 expression levels. In addition, PGK1 K388 acetylation levels are positively correlated with BECN1 S30 phosphorylation levels in GBM patients, both of which are correlated with a shorter survival duration. These results support a role for PGK1-phosphorylated BECN1 and subsequent autophagy activation in the clinical behavior of human GBM.

Hyperactivation of MTOR has been observed in human cancers, and different MTOR-inhibitory agents have been developed for treatment. However, limited clinical effects have been reported for MTOR inhibitors; this may be partially due to upregulated tumor-protective autophagy. Here, we showed that MTOR inhibition leads to PGK1-dependent autophagy, which in turn promotes tumor survival in response to extracellular stresses. These findings highlight the potential to increase cancer treatment efficacy by inhibiting both MTOR activity and PGK1-regulated autophagy.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by National Cancer Institute grants 2R01 CA109035 and 1R01 CA169603, and National Institute of Neurological Disorders and Stroke grant 1R01 NS089754.