Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser(487) in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr(172). Surprisingly, the equivalent site on AMPK-α2, Ser(491), is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr(172) phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca(2+) ionophore A23187, effects we show to be dependent on Akt activation and Ser(487) phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr(172) phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation

Development of N2O-MTV for Low-Speed Flow and In-Situ Deployment to an Integral Effect Test Facility

A molecular tagging velocity (MTV) technique is developed to non-intrusively measure velocity in an integral effect test (IET) facility simulating a high-temperature helium-cooled nuclear reactor in accident scenarios. In these scenarios, the velocities are expected to be low, on the order of 1 m/s or less, which forces special requirements on the MTV tracer selection. Nitrous oxide (N2O) is identified as a suitable seed gas to generate NO tracers capable of probing the flow over a large range of pressure, temperature, and flow velocity. The performance of N2O-MTV is assessed in the laboratory at temperature and pressure ranging from 295 to 781 K and 1 to 3 atm. MTV signal improves with a temperature increase, but decreases with a pressure increase. Velocity precision down to 0.004 m/s is achieved with a probe time of 40 ms at ambient pressure and temperature. Measurement precision is limited by tracer diffusion, and absorption of the tag laser beam by the seed gas. Processing by cross-correlation of single-shot images with high signal-to-noise ratio reference images improves the precision by about 10% compared to traditional single-shot image correlations. The instrument is then deployed to the IET facility. Challenges associated with heat, vibrations, safety, beam delivery, and imaging are addressed in order to successfully operate this sensitive instrument in-situ. Data are presented for an isothermal depressurized conduction cooldown. Velocity profiles from MTV reveal a complex flow transient driven by buoyancy, diffusion, and instability taking place over short ( 30 min) time scales at sub-meter per second speed. The precision of the in-situ results is estimated at 0.027, 0.0095, and 0.006 m/s for a probe time of 5, 15, and 35 ms, respectively