To test NF|êB transcriptional effects on GLUT1 localization independent of AKT regulation, we expressed constitutively energetic myristoylated AKT and myrAKT with a S473D mutation in IB4tet|¤NI|êBa and IB4tet|¤NI|êBa-fGLUT1. The activating S473D mutation renders AKT action independent of S473 phosphorylation . myrAKT and myrAKTS473D sustained surface endogenous- or flag-GLUT1 levels right after Wortmannin treatment method, but failed to accomplish so immediately after inhibition of NF|êB transcription . Similarly, glucose import in myrAKT and myrAKTS473D expressing cells was elevated over management cells but nonetheless dependent on NF|êB-mediated transcription . Note that myrAKT and myrAKTS473D expression amounts had been not altered . As constitutive AKT signaling did not overcome the effects of |¤NI|êBa, NF|êB-mediated gene expression is required for surface localization of GLUT1 downstream or independent of AKT action.
NF|êB transcription is vital for AKT-mediated AS160 phosphorylation AKT promotes GLUT4 membrane localization by inhibitory phosphorylation of AKT Substrate of 160kDa . To analyze AS160 impact on GLUT1 localization in lymphocytes, we transfected IB4 or IB4|¤NI|êBa-fGLUT1 with expression hop over to this site vectors for either manage, HA-AS160 or mutant HA-AS160 lacking all AKT phosphorylation web-sites . HA-AS160 expression had no effect on GLUT1 localization, whereas HA-AS160-4p triggered retention of each endogenous-and fGLUT1 .Thus AS160 is an necessary regulator of GLUT1 membrane localization in B-lymphocytes. Steady with constitutive GLUT1 localization on the plasma membrane, AS160 was phosphorylated at AKT websites in IB4tet|¤NI|êBa .
Wortmannin inhibited AS160 PAS-phosphorylation in control uninduced cells, but had little effect in IB4tet|¤NI|êBa stably expressing myrAKT or myrAKTS473D . Rapamycin blocked TORC1-dependent phosphorylation of S6K at T389 but had no impact on AS160 phosphorylation hop over to here and rather very little effect on surface endogenous- or flag-GLUT1 . We found that NF|êB is exclusively demanded to recruit AKT for the phosphorylation of AS160. Inhibition of NF|êB-mediated transcription by |¤NI|êBa resulted in loss of AS160 PAS site phosphorylation in management, myrAKT and myrAKT S473D expressing cells . Importantly, the impact of NF|êB was specified to AS160 as AKT target TSC2 T1462 phosphorylation was unaffected by NF|êB inhibition . In addition the action of AMPKa, which can encourage AS160 phosphorylation , was not altered right after NF|êB inhibition .
Therefore, we have now shown that the NF|êB pathway has two roles in GLUT1 localization. IKKB is required for AKT activation, whereas NF|êB-mediated transcription allows AKT to phosphorylate AS160 .