APPL1 regulates basal NF-κB activity by stabilizing NIK.
APPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here, we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-κB under basal but not TNFα-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate canonical NF-κB signaling. APPL1 synergized with TRAF2 to induce NF-κB activation, and both proteins were necessary for this process and function upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-κB pathway. Importantly, in the canonical pathway, APPL1 appeared to regulate the proper spatial distribution of the p65 subunit of NF-κB in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. By analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-κB target genes that encode cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-κB pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-κB activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-κB activation.
International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109 Warsaw,  Poland.
Anna
A
Hupalowska
Beata
B
Pyrzynska
Marta
M
Miaczynska
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D004795:Enzyme Stability; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D000097782:NF-kappaB-Inducing Kinase
D048868:Adaptor Proteins, Signal Transducing; D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D047992:TNF Receptor-Associated Factor 2; D051996:Transcription Factor RelA; D000097782:NF-kappaB-Inducing Kinase; C464801:APPL1 protein, human