RESUMO
Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.
Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas de Transporte/metabolismo , Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Células HeLa , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Domínios de Homologia de srcRESUMO
Clathrin-mediated endocytosis (CME) regulates the uptake of cell-surface receptors as well as their downstream signaling activities. We recently reported that signaling can reciprocally regulate CME in cancer cells and that this crosstalk can contribute to cancer progression. To further explore the nature and extent of the crosstalk between signaling and CME in cancer cell biology, we analyzed a panel of oncogenic signaling kinase inhibitors for their effects on CME across a panel of normal and cancerous cells. Inhibition of several kinases selectively affected CME in cancer cells, including inhibition of ERK1/2, which selectively inhibited CME by decreasing the rate of clathrin-coated pit (CCP) initiation. We identified an ERK1/2 substrate, the FCH/F-BAR and SH3 domain-containing protein FCHSD2, as being essential for the ERK1/2-dependent effects on CME and CCP initiation. Our data suggest that ERK1/2 phosphorylation activates FCHSD2 and regulates EGF receptor (EGFR) endocytic trafficking as well as downstream signaling activities. Loss of FCHSD2 activity in nonsmall cell lung cancer (NSCLC) cells leads to increased cell-surface expression and altered signaling downstream of EGFR, resulting in enhanced cell proliferation and migration. The expression level of FCHSD2 is positively correlated with higher NSCLC patient survival rates, suggesting that FCHSD2 can negatively affect cancer progression. These findings provide insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Transporte/biossíntese , Clatrina/farmacocinética , Endocitose , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Clatrina/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas de Neoplasias/genéticaRESUMO
Nervous wreck (Nwk) is a conserved F-BAR protein that attenuates synaptic growth and promotes synaptic function in Drosophila. In an effort to understand how Nwk carries out its dual roles, we isolated interacting proteins using mass spectrometry. We report a conserved interaction between Nwk proteins and BAR-SH3 sorting nexins, a family of membrane-binding proteins implicated in diverse intracellular trafficking processes. In mammalian cells, BAR-SH3 sorting nexins induce plasma membrane tubules that localize NWK2, consistent with a possible functional interaction during the early stages of endocytic trafficking. To study the role of BAR-SH3 sorting nexins in vivo, we took advantage of the lack of genetic redundancy in Drosophila and employed CRISPR-based genome engineering to generate null and endogenously tagged alleles of SH3PX1. SH3PX1 localizes to neuromuscular junctions where it regulates synaptic ultrastructure, but not synapse number. Consistently, neurotransmitter release was significantly diminished in SH3PX1 mutants. Double-mutant and tissue-specific-rescue experiments indicate that SH3PX1 promotes neurotransmitter release presynaptically, at least in part through functional interactions with Nwk, and might act to distinguish the roles of Nwk in regulating synaptic growth and function.