PKM2 functions as a histidine kinase to phosphorylate PGAM1 and increase glycolysis shunts in cancer.

Phosphoglycerate mutase 1 (PGAM1) is a key node enzyme that diverts the metabolic reactions from glycolysis into its shunts to support macromolecule biosynthesis for rapid and sustainable cell proliferation. It is prevalent that PGAM1 activity is upregulated in various tumors; however, the underlying mechanism remains unclear. Here, we unveil that pyruvate kinase M2 (PKM2) moonlights as a histidine kinase in a phosphoenolpyruvate (PEP)-dependent manner to catalyze PGAM1 H11 phosphorylation, that is essential for PGAM1 activity. Moreover, monomeric and dimeric but not tetrameric PKM2 are efficient to phosphorylate and activate PGAM1. In response to epidermal growth factor signaling, Src-catalyzed PGAM1 Y119 phosphorylation is a prerequisite for PKM2 binding and the subsequent PGAM1 H11 phosphorylation, which constitutes a discrepancy between tumor and normal cells. A PGAM1-derived pY119-containing cell-permeable peptide or Y119 mutation disrupts the interaction of PGAM1 with PKM2 and PGAM1 H11 phosphorylation, dampening the glycolysis shunts and tumor growth. Together, these results identify a function of PKM2 as a histidine kinase, and illustrate the importance of enzyme crosstalk as a regulatory mode during metabolic reprogramming and tumorigenesis.

O-GlcNAcylation of ATP-citrate lyase couples glucose supply to lipogenesis for rapid tumor cell proliferation.

Elevated lipid synthesis is one of the best-characterized metabolic alterations in cancer and crucial for membrane expansion. As a key rate-limiting enzyme in de novo fatty acid synthesis, ATP-citrate lyase (ACLY) is frequently up-regulated in tumors and regulated by posttranslational modifications (PTMs). Despite emerging evidence showing O-GlcNAcylation on ACLY, its biological function still remains unknown. Here, we observed a significant upregulation of ACLY O-GlcNAcylation in various types of human tumor cells and tissues and identified S979 as a major O-GlcNAcylation site. Importantly, S979 O-GlcNAcylation is required for substrate CoA binding and crucial for ACLY enzymatic activity. Moreover, it is sensitive to glucose fluctuation and decisive for fatty acid synthesis as well as tumor cell proliferation. In response to EGF stimulation, both S979 O-GlcNAcylation and previously characterized S455 phosphorylation played indispensable role in the regulation of ACLY activity and cell proliferation; however, they functioned independently from each other. In vivo, streptozocin treatment- and EGFR overexpression-induced growth of xenograft tumors was mitigated once S979 was mutated. Collectively, this work helps comprehend how cells interrogate the nutrient enrichment for proliferation and suggests that although mammalian cell proliferation is controlled by mitogen signaling, the ancient nutrition-sensing mechanism is conserved and still efficacious in the cells of multicellular organisms.

EGF promotes PKM2 O-GlcNAcylation by stimulating O-GlcNAc transferase phosphorylation at Y976 and their subsequent association.

Epidermal growth factor (EGF) is one of the most well-characterized growth factors and plays a crucial role in cell proliferation and differentiation. Its receptor EGFR has been extensively explored as a therapeutic target against multiple types of cancers, such as lung cancer and glioblastoma. Recent studies have established a connection between deregulated EGF signaling and metabolic reprogramming, especially rewiring in aerobic glycolysis, which is also known as the Warburg effect and recognized as a hallmark in cancer. Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme controlling the final step of glycolysis and serves as a major regulator of the Warburg effect. We previously showed that PKM2 T405/S406 O-GlcNAcylation, a critical mark important for PKM2 detetramerization and activity, was markedly upregulated by EGF. However, the mechanism by which EGF regulates PKM2 O-GlcNAcylation still remains uncharacterized. Here, we demonstrated that EGF promoted O-GlcNAc transferase (OGT) binding to PKM2 by stimulating OGT Y976 phosphorylation. As a consequence, we found PKM2 O-GlcNAcylation and detetramerization were upregulated, leading to a significant decrease in PKM2 activity. Moreover, distinct from PKM2, we observed that the association of additional phosphotyrosine-binding proteins with OGT was also enhanced when Y976 was phosphorylated. These proteins included STAT1, STAT3, STAT5, PKCδ, and p85, which are reported to be O-GlcNAcylated. Together, we show EGF-dependent Y976 phosphorylation is critical for OGT-PKM2 interaction and propose that this posttranslational modification might be important for substrate selection by OGT.

Pyruvate Kinase M2 Promotes the Activation of Dendritic Cells by Enhancing IL-12p35 Expression.

Dendritic cells (DCs) play a central role in both innate and adaptive immunity. Emerging evidence has demonstrated metabolic reprogramming during DC activation. However, how DC activation is linked with metabolic reprogramming remains unclear. Here we show that pyruvate kinase M2 (PKM2), the rate-limiting enzyme in the last step of glycolysis, is critical for LPS-induced DC activation. Upon DC activation, JNK signaling stimulated p300 association with PKM2 for the acetylation of lysine 433, a classic posttranslational modification critical for PKM2 destabilization and nuclear re-localization. Subsequently, nuclear PKM2 partnered with c-Rel to enhance Il12p35 expression, which is important for Th1 cell differentiation. Meanwhile, decreased enzymatic activity of PKM2 due to detetramerization facilitated glycolysis and fatty acid synthesis, helping DCs meet their need for biomacromolecules. Together, we provide evidence for metabolic control of DC activation and offer insights into aberrant immune responses due to dysregulated Th1 functions.