Oxygen levels are critical for cellular physiology. Under low oxygen (hypoxia), cells increase glycolysis while limiting mitochondrial oxidative metabolism. Such adaptation is largely mediated by the transcription factors HIF, which accumulate under hypoxia 1, 2. Prolyl hydroxylase domain proteins (PHD) function as cellular oxygen sensors and target HIF for subsequent degradation under normoxia 2. The PHD/HIF pathway is conserved in Drosophila, where a single prolyl hydroxylase, Fatiga, has been characterized. We have previously reported that Fatiga regulates cellular growth rates in a HIF-independent manner, yet mechanisms remained elusive 3.

In mammals, four isoenzymes of PK are found: type L in liver, R in erythrocytes, M1 in adult and M2 in embryonic cells. During tumorigenesis, tissue-specific isoforms of PK are replaced by PK-M2, and this isoform plays an important role in the metabolic shift of cancer cells 5, 6. PK-M2 is a tetramer in the presence of FBP (fructose 1,6-bisphosphate), showing a high affinity to its substrate PEP (Km 0.17 mM). In the absence of FBP, PK-M2 exists as a dimer or monomer, and its affinity to PEP is much lower (Km 2.1 mM) 7. In tumor cells, PK-M2 exists mainly as dimer/monomer and is believed to be inactive under the physiological PEP concentration 6. To distinguish different PK-M2 forms, we measured PK activity using different concentrations of PEP. Indeed, activity of GST-PK-M1 was less sensitive to reduction in PEP concentration (from 5 mM to 0.2 mM) than GST-PK-M2 (Supplementary information, Figure S1A). Similarly, overexpressed PK-M2 was more sensitive to decreased PEP concentration than PK-M1 (Supplementary information, Figure S1B and S1C). Furthermore, whereas GST-PK-M1 was insensitive to FBP stimulation, activity of GST-PK-M2 could be stimulated by FBP at low PEP concentration (0.2 mM) but not at high PEP concentration (5 mM; data not shown). These data show that PK activity in response to different PEP concentrations can be used to distinguish between the M1-like behavior of M2 (constant tetramer form) and the M2 behavior (equilibrium between tetramer and dimer/monomer form).

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