Fig. 7: LKB1 is a natural sensor for Asn and Asp.
a Protein expression in U2OS cells transfected with Control, ASNS siRNA and/or LKB1 siRNA as indicated. b Protein expression in LKB1 wildtype (WT) and knockout (KO) HCT116 cells treated with siCtrl or siASNS as indicated. c Western blot analysis of lysates from HCT116 cells and A549 cells transfected with control or ASNS siRNA. d In vitro LKB1 kinase activity assay using HEK-293 cell-purified proteins as indicated in the presence or absence of Asn or Asp at 30 °C for 30 min. e HEK-293 cell-Purified HA-LKB1 immobilised on anti-HA agarose beads (LKB1 beads) was stimulated by incubating with Asn or Asp for 30 °C for 30 min, and then unbound Asn/Asp was removed by washing and further incubated with LKB1 beads with purified Flag-AMPK protein for another 30 °C for 30 min. AMPK phosphorylation was determined. f In vitro LKB1 kinase activity was determined using SF21 cell-purified His-LKB1-STRAD-MO25 complex and His-AMPKα in the presence or absence of Asn or Asp at 30 °C for 30 min. g Surface plasmon resonance (BIAcore) measurement of the interaction between purified LKB1 and Asn (left) or Asp (right). Graphs of equilibrium response units (RU) and compound concentrations are shown. The estimated KD values were 45.4 µM and 64.4 µM. h Binding of radiolabelled [3H]Asn and [3H]Asp to HEK-293 cell-purified proteins (see Methods for details). Unlabelled amino acids were added where indicated. i HCT116 cells were treated with ASNS siRNA for 0, 6 and 18 h as indicated before sample preparation for LC/MS-based analysis of the absolute amounts of asparagine. The dissociation constant KD of LKB1 for asparagine is indicated. Data are mean ± s.d., unpaired two-tailed Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant. Source data are provided as a Source Data file.
