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Cancerous inhibitor of protein phosphatase 2A (CIP2A) modifies energy metabolism via 5′ AMP-activated protein kinase signalling in malignant cellsAbstract Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an adverse biomarker across many malignancies. Using K562 cells engineered to have high or low CIP2A expression, we show that high CIP2A levels significantly bias cellular energy production towards oxidative phosphorylation (OXPHOS) rather than glycolysis. Mass spectrometric analysis of CIP2A interactors and isobaric tagging for relative and absolute protein quantitation (ITRAQ) experiments identified many associated proteins, several of which co-vary with CIP2A level. Many of these CIP2A associating and co-varying proteins are involved in energy metabolism including OXPHOS, or in 5′ AMP-activated protein kinase (AMPK) signalling, and manipulating AMPK activity mimics the effects of low/high CIP2A on OXPHOS. These effects are dependent on the availability of nutrients, driven by metabolic changes caused by CIP2A. CIP2A level did not affect starvation-induced AMPK phosphorylation of Unc-51 autophagy activating kinase 1 (ULK-1) at Ser555, but autophagy activity correlated with an increase in AMPK activity, to suggest that some AMPK processes are uncoupled by CIP2A, likely via its inhibition of protein phosphatase 2A (PP2A). The data demonstrate that AMPK mediates this novel CIP2A effect on energy generation in malignant cells.
Related flavonoids cause cooperative inhibition of the sarcoplasmic reticulum Ca²⁺ ATPase by multimode mechanisms.Flavonoids are group of plant-derived hydroxylated polycyclic molecules found in fruit and vegetables. They are known to bio-accumulate within humans and are considered to have beneficial health effects, including cancer chemoprotection. One mechanism proposed to explain this is that they are able to induce apoptosis in cancer cells by inhibiting a variety of kinases and also the Ca²⁺ ATPase. An investigation was undertaken with respect to the mechanism of inhibition for three flavonoids: quercetin, galangin and 3,6 dihydroxyflavone (3,6-DHF). Each inhibited the Ca²⁺ ATPase with K(i) values of 8.7, 10.3 and 5.4 μM, respectively, showing cooperative inhibition with n ~ 2. Given their similar structures, the flavonoids showed several differences in their mechanisms of inhibition. All three flavonoids stabilized the ATPase in the E₁ conformation and reduced [³²P]-ATP binding. However, both galangin and 3,6-DHF increased the affinity of Ca²⁺ for the ATPase by decreasing the Ca²⁺-dissociation rate constant, whereas quercetin had little effect. Ca²⁺-induced changes in tryptophan fluorescence levels were reduced in the presence of 3,6-DHF and galangin (but not with quercetin), indicating that Ca²⁺-associated changes within the transmembrane helices are altered. Both galangin and quercetin reduced the rates of ATP-dependent phosphorylation and dephosphorylation, whereas 3,6-DHF did not. Modelling studies suggest that flavonoids could potentially bind to two sites: one directly where nucleotides bind within ATP binding site and the other at a site close by. We hypothesize that interactions of these two neighbouring sites may account for both the cooperative inhibition and the multimode mechanisms of action seen with related flavonoids.