RESUMO
The pentameric WASH complex is best known for its role in regulating receptor trafficking from retromer-rich endosomal subdomains. FAM21 functions to stabilize the WASH complex through its N-terminal head domain and localizes it to endosomes by directly binding the retromer through its extended C-terminal tail. Herein, we used affinity purification combined with mass spectrometry to identify additional FAM21-interacting proteins. Surprisingly, multiple components of the nuclear factor κB (NF-κB) pathway were identified, including the p50 and p65 (RelA) NF-κB subunits. We show that FAM21 interacts with these components and regulates NF-κB-dependent gene transcription at the level of p65 chromatin binding. We further demonstrate that FAM21 contains a functional monopartite nuclear localization signal sequence (NLS) as well as a CRM1/exportin1-dependent nuclear export signal (NES), both of which work jointly with the N-terminal head domain and C-terminal retromer recruitment domain to regulate FAM21 cytosolic and nuclear subcellular localization. Finally, our findings indicate that FAM21 depletion sensitizes pancreatic cancer cells to gemcitabine and 5-fluorouracil. Thus, FAM21 not only functions as an integral component of the cytoplasmic WASH complex, but also modulates NF-κB gene transcription in the nucleus.
Assuntos
Proteínas dos Microfilamentos/metabolismo , NF-kappa B/genética , Neoplasias Pancreáticas/genética , Proteínas/genética , Fator de Transcrição RelA/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Proteínas dos Microfilamentos/genética , NF-kappa B/metabolismo , Sinais de Localização Nuclear/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Proteínas de Ligação a Fosfato , Ligação Proteica/genética , Fator de Transcrição RelA/genética , GencitabinaRESUMO
Cell surface receptors that have been internalized and enter the endocytic pathway have multiple fates including entrance into the multivesicular body pathway on their way to lysosomal degradation, recycling back to the cell surface, or retrograde trafficking out of the endolysosomal system back to the Golgi apparatus. Two ubiquitously expressed protein complexes, WASH and the endosomal coat complex retromer, function together to play a central role in directing the fate of receptors into the latter two pathways. In this chapter, we describe fluorescent- and flow cytometry-based methods for analyzing the recycling and retrograde trafficking of two receptors, α5ß1 and CI-M6PR, whose intracellular fates are regulated by WASH and retromer activity. The guidelines presented in this chapter can be applied to the analysis of any cell surface or intracellular membrane protein to determine the impact of WASH or retromer deregulation on its intracellular trafficking route.
Assuntos
Integrina alfa5beta1/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptor IGF Tipo 2/metabolismo , Endocitose , Endossomos/metabolismo , Citometria de Fluxo , Células HeLa , Humanos , Transporte ProteicoRESUMO
COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.