For example, FGFR1 Talazoparib cost (fibroblast growth factor receptor 1) directly phosphorylates Tyr105 of PKM2, thereby inhibiting the formation of its active tetramer, suggesting that tyrosine phosphorylation of PKM2 may serve as a critical glycolytic switch in cancer cells.24 As shown in Fig. 4A (left), ATP stimulated Tyr105 phosphorylation of PKM2 in both WT and Cd39-null hepatocytes in vitro, albeit with a more dramatic stimulatory effect on null cells. We then examined tyrosine phosphorylation of PKM2 in mouse livers after portal venous administration of ATP in vivo. ATP also promoted Tyr105 phosphorylation of PKM2 in WT livers

and that Cd39-null livers had much higher tyrosine phosphorylation even before ATP infusion (Fig. 4A, right). Furthermore, total PKM2 proteins were increased by ATP in both WT and null cells (Fig. 4B). In parallel, expression of LDH-A mRNA was elevated in quiescent Cd39-null cells as compared to WT cells and was further enhanced by ATP in both cells (Fig. 4C, left). Moreover, similar patterns were also noted with LDH-A proteins (Fig. 4C, right). We next evaluated the impact of Cd39 deletion on mitochondrial function in hepatocytes. As shown in Fig. 4D (top), ATP downregulates expression of cytochrome B mRNA and upregulates expression of mitochondrial

uncoupling protein UCP2 mRNA in WT cells (see also bottom panel for comparison of WT versus null, Fig. 4D; Fig. S4A,B for other genes). Decreased expression of cytochrome B, Cox2 (cyclooxygenase 2), glucagon (a mitochondrial regulator), and increased levels of UCP2 were noted in quiescent Cd39-null livers in contrast to Epigenetics Compound Library intact WT livers

(Fig. S4C), associated with enhanced ribosome biogenesis (indicated by mRNA levels of 18S rRNA) (Fig. S4D). Importantly, decreases in intracellular ATP levels were noted in Cd39-null cells (Fig. 4E; Table S2). In addition, lactate formation, the endproduct of glycolysis, was significantly increased in null cells (Fig. 4F, P = 0.01), as measured in a real time fashion. Collectively, these data show that ATP-initiated purinergic signaling is associated with altered 上海皓元医药股份有限公司 bioenergetic metabolism of hepatocytes that promotes aerobic glycolysis by modifying glycolytic enzyme expression and/or activity as well as disrupting mitochondrial function to favor anabolic pathways and promote cell growth. We first studied the activation of Ras in response to ATP in vitro. Levels of activated Ras in WT cells were increased by ATP (Fig. 5A). Ras was also elevated in quiescent Cd39-null cells and increased following ATP stimulation, in contrast to WT cells (Fig. 5A). We next examined the phosphorylation of downstream components of Ras, PI3K, and mTOR pathways by ATP in hepatocytes. In both WT and null cells, ATP enhanced the phosphorylation of components of many pathways, including ERK1/2, JNK/SAPK, NF-κB in Ras-MAPK signaling, AKT in PI3K signaling, and mTOR, S6K1, S6 in mTOR signaling (Fig. 5B).