RESUMO
Autophagy is an evolutionarily conserved process that enables catabolic and degradative pathways. These pathways commonly depend on vesicular transport controlled by Rabs, small GTPases inactivated by TBC/RabGAPs. The Rac1 effector TBC/RabGAP Armus (TBC1D2A) is known to inhibit Rab7, a key regulator of lysosomal function. However, the precise coordination of signaling and intracellular trafficking that regulates autophagy is poorly understood. We find that overexpression of Armus induces the accumulation of enlarged autophagosomes, while Armus depletion significantly delays autophagic flux. Upon starvation-induced autophagy, Rab7 is transiently activated. This spatiotemporal regulation of Rab7 guanosine triphosphate/guanosine diphosphate cycling occurs by Armus recruitment to autophagosomes via interaction with LC3, a core autophagy regulator. Interestingly, autophagy potently inactivates Rac1. Active Rac1 competes with LC3 for interaction with Armus and thus prevents its appropriate recruitment to autophagosomes. The precise coordination between Rac1 and Rab7 activities during starvation suggests that Armus integrates autophagy with signaling and endocytic trafficking.
Assuntos
Autofagia , Proteínas Ativadoras de GTPase/metabolismo , Queratinócitos/patologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Meios de Cultura/metabolismo , Ativação Enzimática , Imunofluorescência , Proteínas Ativadoras de GTPase/genética , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Recém-Nascido , Queratinócitos/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Fagossomos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7 , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Cell-cell adhesion and intracellular trafficking are regulated by signaling pathways from small GTPases of the Rho, Arf, and Rab subfamilies. How signaling from distinct small GTPases are integrated in a given process is poorly understood. RESULTS: We find that a TBC/RabGAP protein, Armus, integrates signaling between Arf6, Rac1, and Rab7 during junction disassembly. Armus binds specifically to activated Rac1 and its C-terminal TBC/RabGAP domain inactivates Rab7. Thus, Armus is a novel Rac1 effector and a bona fide GAP for Rab7 in vitro and in vivo, a unique and previously unreported combination. Arf6 activation efficiently disrupts cell-cell contacts and is known to activate Rac1 and Rab7. Arf6-induced E-cadherin degradation is efficiently blocked by expression of Armus C-terminal domain or after Armus RNAi. Coexpression of Arf6 with dominant-negative Rab7 or Rac1 also inhibits junction disassembly. Importantly, Armus RabGAP expression also prevents EGF-induced scattering in keratinocytes, a process shown here to require Arf6, Rac1, and Rab7 function. To our knowledge, this is the first report to demonstrate a molecular and functional link between Rac1 and Rab7. CONCLUSIONS: Our data indicate that active Rac1 recruits Armus to locally inactivate Rab7 and facilitate E-cadherin degradation in lysosomes. Thus, the integration of Rac1 and Rab7 activities by Armus provides an important regulatory node for E-cadherin turnover and stability of cell-cell contacts.
