Functional Studies

Functional studies.
KU-55993 is a cell-permeable ATP-competitive inhibitor of ATM (half maximal inhibitory concentration (IC50) = 13 nM, Ki = 2.2 nM) with selectivity over other PIKK-family kinases(IC50 = 2.5 μM, IC50 = 9.3 μM and IC50 = 16.6 μM for DNA-PK, mTOR and PI 3-K, respectively; IC50 > 100 μM for PI 4-K and ATR)32. It is reported to have little activity toward a panel of 70conventional serine/threonine kinases. It has been shown to inhibit ATM-dependent cellular protein phosphorylation following ionizing radiation and sensitizes cells that express wildtype ATM (but not mutantATM) to the cytotoxic effects of ionizing radiation and DNA-damaging agents33, 34.
H4IIE cells were cultured in Dulbecco's modified Eagle's medium containing 5% (v/v) FBS to 80% confluence. For AMPKassays, cells were pretreated with 10 μM KU-55933 (for 30 min) before treatment with various concentrations of metformin for 1 h. Lysates were prepared (rapid lysis method35), centrifuged (at 4 °C for10 min and 21,000 × g), and the supernatants were frozen for later analysis. AMPK activity was assayed in immunoprecipitates as described35 using the AMARA peptide36.
To establish that the change inAMPK activity was due to the regulation of phosphorylation of AMPK and that this translated into changes in substrate phosphorylation, we used protein blotting to compare the phosphorylation status ofThr172 of AMPK and Ser79 of ACC (a well characterized marker of AMPK activation). In both cases, metformin-induced phosphorylation was partially reduced (Fig. 3).
For these experiments, H4IIE cellswere cultured to 80% confluence as above, then serum starved for 16 h, pretreated with KU-55933 or vehicle (DMSO) for 1 h and then treated for 3 h with or without metformin. They were then harvestedas described above. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose. The membrane was blocked in TBST containing 5% (wt/v) milk for 1 h and then incubated overnight at 4 °C with...