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.

Cysteine and homocysteine can be exploited by GPX4 in ferroptosis inhibition independent of GSH synthesis.

Ferroptosis is inhibited by glutathione peroxidase 4 (GPX4), an antioxidant enzyme that uses reduced glutathione (GSH) as a cofactor to detoxify lipid hydroperoxides. As a selenoprotein, the core function of GPX4 is the thiol-dependent redox reaction. In addition to GSH, other small molecules such as cysteine and homocysteine also contain thiols; yet, whether GPX4 can exploit cysteine and homocysteine to directly detoxify lipid hydroperoxides and inhibit ferroptosis has not been addressed. In this study, we found that cysteine and homocysteine inhibit ferroptosis in a GPX4-dependent manner. However, cysteine inhibits ferroptosis independent of GSH synthesis, and homocysteine inhibits ferroptosis through non-cysteine and non-GSH pathway. Furthermore, we used molecular docking and GPX4 activity analysis to study the binding patterns and affinity between GPX4 and GSH, cysteine, and homocysteine. We found that besides GSH, cysteine and homocysteine are also able to serve as substrates for GPX4 though the affinities of GPX4 with cysteine and homocysteine are lower than that with GSH. Importantly, GPX family and the GSH synthetase pathway might be asynchronously evolved. When GSH synthetase is absent, for example in Flexibacter, the fGPX exhibits higher affinity with cysteine and homocysteine than GSH. Taken together, the present study provided the understanding of the role of thiol-dependent redox systems in protecting cells from ferroptosis and propose that GSH might be a substitute for cysteine or homocysteine to be used as a cofactor for GPX4 during the evolution of aerobic metabolism.