ABSTRACT
AKT is a critical effector kinase downstream of the PI3K pathway that regulates a plethora of cellular processes including cell growth, death, differentiation, and migration. Mechanisms underlying activated phospho-AKT (pAKT) translocation to its action sites remain unclear. Here we show that NEDD4-1 is a novel E3 ligase that specifically regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. NEDD4-1 physically interacts with AKT and promotes HECT domain-dependent ubiquitination of exogenous and endogenous AKT. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in vitro. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4-1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is released into the cytoplasm, imported into the nucleus, or coupled with proteasomal degradation. IGF-1 signaling specifically stimulates NEDD4-1-mediated ubiquitination of pAKT, without altering total AKT ubiquitination. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with wild type AKT. This study reveals a novel mechanism by which a specific E3 ligase is required for ubiquitin-dependent control of pAKT dynamics in a ligand-specific manner.
Subject(s)
Cell Membrane/metabolism , Cell Nucleus/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Insulin-Like Growth Factor I/metabolism , Proto-Oncogene Proteins c-akt/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin/genetics , Animals , Cell Line, Tumor , Cytoplasm/metabolism , Endosomal Sorting Complexes Required for Transport/deficiency , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation , Humans , Insulin-Like Growth Factor I/genetics , Mice , Nedd4 Ubiquitin Protein Ligases , Phosphorylation , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Transport , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Transfection , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/deficiency , UbiquitinationABSTRACT
A series of novel pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives were synthesized by the reaction of ethyl 3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carboxylate and amine in the general heating condition and microwave-assisted condition. The structures of the compounds were determined by IR, (1)H NMR and mass spectroscopy, in addition, representative single-crystal structures were characterized by using X-ray diffraction analysis. Preliminary biological evaluation showed that the compounds could inhibit the growth of A549 cells in dosage- and time-dependent manners. The study on structure-activity relationships showed that compounds with 4-chlorophenyl group at pyrazole moiety, such as 5-benzyl-2-(4-chlorophenyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3o) had much more inhibitory effects. Compound 3o was the most effective small molecule in inhibiting A549 cell growth and might perform its action through modulating autophagy.
Subject(s)
Antineoplastic Agents/chemical synthesis , Lung Neoplasms/drug therapy , Pyrazoles/chemical synthesis , Pyrazoles/toxicity , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/toxicity , Cell Line, Tumor , Crystallography, X-Ray , DNA Damage , Drug Design , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Molecular Structure , Pyrazoles/therapeutic use , Structure-Activity RelationshipABSTRACT
A series of novel 1-(2'-hydroxy-3'-aroxypropyl)-3-aryl-1H-pyrazole-5-carbohydrazide derivatives were synthesized, and the effects of the compounds on A549 cell growth were investigated. The results showed that all of the 1-(2'-hydroxy-3'-aroxypropyl)-3-aryl-1H-pyrazole-5-carbohydrazide derivatives 2 could inhibit the growth of A549 cells in dosage- and time-dependent manners. Typically, compound 2a and 2d induced A549 cells to autophagy but did not cause apoptosis and necrosis in the cells, and 2d had the most autophagy inducing effect in H460 cells. More importantly, 2a and 2d did not inhibit the growth of HUVEC cells.
Subject(s)
Autophagy , Lung Neoplasms/drug therapy , Pyrazoles/chemical synthesis , Apoptosis , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Chemistry, Pharmaceutical/methods , Culture Media/metabolism , Drug Design , Endothelium, Vascular/cytology , Humans , Inhibitory Concentration 50 , Models, Chemical , Pyrazoles/chemistry , Time FactorsABSTRACT
A series of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives were synthesized and the effects of all the compounds on A549 cell growth were investigated. The results showed that all compounds had almost inhibitory effects on the growth of A549 cells. The study on structure-activity relationships and prediction of lipophilicities of compounds showed that compounds with LogP values in the range of 4.12-6.80 had inhibitory effects on the growth of A549 cells, and among of them the hydrazone derived from salicylaldehyde had much more inhibitory effects.
