PTH Receptors

Indeed, AMPK is often discussed as if it were a single entity

Indeed, AMPK is often discussed as if it were a single entity. of the metabolic regulator AMPK.1 Perturbation of NUAK1 has revealed a diverse array of phenotypes, pointing to roles for NUAK1 in regulating cell adhesion,2 directional migration,3, 4 neuronal axon branching,5 glycogen synthesis,6 replicative senescence7 and TAU stabilization.8 Overexpression of NUAK1 is associated with poor prognosis in multiple cancers, including colorectal,9 (Port, double floxed MEFs. Strikingly, phospho-RAPTORS792 was still KRas G12C inhibitor 1 detectable in the same MEFs after CRE recombinase-mediated deletion of AMPK1 and 2, albeit at reduced levels, and NUAK1 inhibition further reduced detection, indicating that NUAK1 can regulate RAPTOR in the absence of functional AMPK (Figure 6d). Accordingly, deletion of NUAK1 in Nuak1FL/FL MEFs also reduced both basal and AMPK-activated RAPTORS792 phosphorylation (Figure 6e). Together these data show that efficient restraint of mTORC1 via inhibitory phosphorylation RAB25 of RAPTOR requires both NUAK1 and AMPK. Open in a separate window Figure 6 NUAK1 regulates RAPTOR via AMPK-dependent and independent mechanisms. (a) Measurement of protein synthesis (methionine incorporation) in HeLa (left panel) and U2OS (right panel) cells transfected with non-targeting (?), NUAK1 and PKC siRNA. Mean and s.d. from three independent experiments shown. Statistical significance was determined by one-tailed unpaired double floxed MEFs, infected overnight with Adeno-LacZ or Adeno-CRE and treated as per (c) with AMPK activators in the presence or absence of 10?m HTH-01-015, blotted with the indicated antibodies. MEFs stably expressing Cre-ER were treated overnight with 100?nm 4-OH-Tamoxifen (+) or vehicle control (?) prior to stimulation as per (d, e) with AMPK activators, then immunoblotted for p-RaptorS792. kinase assays with a one-size-fits-all peptide substrate likely fail to accurately reflect physiological ARK activity in cells. Additionally, several independent groups have definitively shown that AMPK is directly phosphorylated by CamKK2, reflecting an alternative pathway to AMPK activation.25, 26, 41, 42 Activation of AMPK by CamKK2 is particularly important in prostate cancer and in the physiological regulation of skeletal muscle and vascular endothelial cell function.43, 44, 45 Interestingly, the ARK SIK2 was recently shown to be activated by an as-yet unidentified Ca2+-dependent kinase in Ovarian cancer cells.46 Our demonstration that NUAK1 and NUAK2 are similarly regulated by Ca2+-dependent signalling thus fits an emerging pattern of calcium regulating multiple ARKs, either alongside or in the absence of LKB1. This regulation may have particular relevance in LKB1-deficient disease settings. Our data speak to the complexity of signal transduction through AMPK, NUAK1 and the related ARKs. Indeed, AMPK is often discussed as if it were a single entity. Rather, up to 12 different permutations of trimeric AMPK complexes can assemble from the 2 2, 2 and 3-encoded subunits, not accounting for splice variants.22 It is likely that the different AMPK complexes may respond differentially to distinct upstream stimuli, and indeed in terms of their activity towards specific downstream substrates. Our demonstration of a specific requirement for NUAK1 in Ca2+-dependent AMPK activity towards ACC, and a more general requirement for NUAK1 in AMPK activity towards RAPTOR, point towards a highly contextual requirement for NUAK1 and KRas G12C inhibitor 1 may indicate that NUAK1 modulates the activity of a specific subset of AMPK complexes. On top of this, the 11 related ARKs can exhibit both overlapping and private substrate specificities. This is reflected by our demonstration of an AMPK-independent role for NUAK1 in RAPTOR regulation, and by phosphorylation of MYPT1 by NUAK1, NUAK2 and potentially by additional ARKs. Consistent with this, we also find Ca2+-dependent phosphorylation KRas G12C inhibitor 1 of the canonical AMPK substrate ACC even after complete suppression of CamKK-dependent AMPK activity in HeLa cells. Clearly, considerably more work will be needed to disentangle these complex signalling networks. Whereas calcium has long been recognized to drive MYC expression47 and more recently to regulate MYC function,48, 49 the reciprocal regulation of calcium signalling by MYC has not garnered much attention. MYC was shown KRas G12C inhibitor 1 KRas G12C inhibitor 1 to increase calcium signalling during B-cell differentiation by suppressing expression of the calcium exporter PMCA.50 ChIP-SEQ analysis has revealed MYC binding to the promoters of and in diverse cell types,30 consistent with our observation that MYC promotes expression of these genes. The.